CDEFGHJKLMPQRSTWXYZ>>>>热点专题：||治疗再生障碍性贫血的药物来源：&& | 治疗再生障碍性贫血的药物有哪些？专家表示，目前临床上治疗再生障碍性贫血的药物有多种，下面专家为大家介绍其中常见的几种，希望它们对大家能够有所版主。治疗再生障碍性贫血的药物一、雄性激素雄性激素是在治疗妇女乳腺癌引起红细胞增多及治疗骨维化，难治性贫血取得成效的基础上引伸到的治疗领域的。目前普通认为本药是治疗再障的有效药物。它是一类具有生物活性的甾 体化合物，兼有雄性化和蛋白质同化作用，以丙酸睾丸酮为代表。二、免疫抑制剂再障患者的体液免疫和细胞免疫都有缺陷，故可应用免疫抑制剂治疗，而应用雄激素、改善微循环药、微量元素等药物疗效不佳。三、血管扩张药作用机制此类药物作用机制，主要是通过神经系统抗胆碱作用，解除骨髓微小血管的痉挛与短路，减轻小动脉与小静脉的收缩，调整骨髓微环境的血流灌注，从而改善骨髓造血微环境，使造血组织有丰富的血液供应，恢复其造血功能，重建&种子&与&土壤&的关系，并可提高环磷酸腺苷水平促进造血。如有健康疑问，可到全球医院网公众号（webQQYY）咨询。（责任编辑：璃夕）掌上淘医安卓版
全球医院网公众号
（本文内容/图片转自网络，仅供参考，一切诊断及医疗的依据请遵从医生的指导。）分享到：相关阅读：······上一篇：下一篇：疾病热文阅读推荐········已有条评论，共人参与，点击查看我来说两句血液科编辑推荐····疾病热点排行榜········精品推荐····热点药品全站热点推荐省份地区科室不孕不育呼吸内科脑外科血管外科其他科：精神病科急诊科血液科合作商家
(C) 版权所有
本站常年 ：福建八闽律师事务所 陈学龙律师、 北京大成（福州）律师事务所 陈杰律师特别声明：本网站内容仅供参考，不作为诊断依据公司移动应用掌上淘医安卓版掌上淘医TV版分享到：您现在的位置：&&>&&>&&>&&>&正文
作者：Allen
来源：生物谷
关键词：诺华,贫血,Revolade,血小板减少,免疫抑制,感染
日讯/BIOON/--近日，用于治疗罕见血液疾病――严重再生障碍性的药物Revolade获欧盟批准，成为首个获批的治疗该疾病的药物，主要针对那些对标准疗法不敏感的患者。
严重再生障碍性贫血表现为骨髓无法生成足够的红细胞、白细胞和血小板，这就导致患者面临极大的感染和出血的风险。在欧洲，每年每百万人中可以检出两名患有该疾病的患者。
目前的治疗手段主要是通过免疫抑制疗法或是造血移植来增加正常血细胞的数量。然而高达1/3的患者对免疫抑制疗法无应答，并且有应答的患者中也存在着30%―40%的复发率。确诊后5年内大约有40%的患者会死于感染或是出血，这就使得一种更加有效的替代疗法成为迫切的需求。
此次Revolade的获批主要是基于II期临床试验数据。40%对免疫抑制疗法无应答的患者在使用Revolade (eltrombopag) 后产生了血液学应答，同时该药物的安全性也达到了要求，中(&=20%)主要的副反应表现为恶心（33%）、疲倦（28%）、咳嗽（23%）、腹泻（21%）和头痛（21%）。
目前该药物已经以商品名Promacta在美国上市，主要适用于对免疫抑制疗法无应答的严重再生障碍性贫血患者，用法为每日一次。还适用于对皮质类固醇、免疫球蛋白和脾切除治疗无效的慢性免疫性血小板减少性紫癜患儿。
此外，Revolade已经在100多个国家被批准用于治疗患有慢性免疫性血小板减少性紫癜的成人患者，并且在超过45个国家被批准用于治疗患有慢性丙肝的血小板减少患者，使得他们能够开始和维持干扰素治疗。(生物谷）
(责任编辑：yixin.zhang)
小编提示：87%用户都在上阅读，扫描立刻下载!
您还可以这样阅读微信扫一扫，体验新式阅读
打开微信扫描二维码 生物谷微信账号：bioonnews 我们提供多种阅读途径供您选择，随时随地掌握医药生物领域最新资讯。
立足行业，提供求职招聘，中高端人才搜索，人才培训及对接等服务 Ta的文章
欢迎行业评论、发现、小道消息、官方爆料、采访约稿
订阅我们的资讯
关注我们新浪微博
中国的市场的确存在很大的隐患，干细胞行业是个专业性要求较高的行业
个体化医疗是未来医学研究与应用的趋势，而个体化治疗的关键在基于生物分子标志物的诊疗策略
中国疫苗市场的巨大潜力，吸引了世界排名最领先的跨国疫苗制造巨头前来淘金。Patent CNA - 营养组合物及在制备治疗再生障碍性贫血药物中的应用 Nutritional composition and ... - Google PatentsCN AApplicationCN Nov 16, 2016Aug 25, 2016Aug 25, 2016.1, CN
A, CN A, CN , CN-A-, CN A, CNA, CN, CN.1, , , , , ,
营养组合物及在制备治疗再生障碍性贫血药物中的应用 Nutritional composition and its application in preparing medicine for treating aplastic anemia medicine translated from CN
A 本发明公开一种营养组合物及在制备治疗再生障碍性贫血药物中的应用，是由赖氨酸、甲硫氨酸、苯丙氨酸、苏氨酸、色氨酸、精氨酸、组氨酸、甘氨酸、天冬氨酸、亮氨酸、异亮氨酸、缬氨酸、丝氨酸、谷氨酰胺、牛磺酸、乳清酸、核苷酸、维生素A、维生素D、维生素B2、维生素B6、维生素B12、烟酸、叶酸、维生素C、铁、锌、锰、铜、硒、铬、钾、钙、镁、肌醇及大豆磷脂按比例配制而成，可以全面修复再障机体，从而达到治疗再生障碍性贫血的效果。 Discloses a nutritional composition for the preparation and treatment of aplastic anemia drugs of the present invention, is composed of lysine, methionine, phenylalanine, threonine, tryptophan, arginine, histidine, glycine, aspartic acid, leucine, isoleucine, valine, serine, glutamine, taurine, whey acid, nucleotide, vitamin A, vitamin D, vitamin B2, vitamin B6, vitamin B12, niacin, folic acid, vitamin C, iron, zinc, manganese, copper, selenium, chromium, potassium, calcium, magnesium, inositol and soy lecithin in proportion to the preparation, aplastic anemia can fully repair the body, so as to achieve the treatment of aplastic anemia results.
1. 一种营养组合物，其特征在于各组分质量比如下：赖氨酸200~1200、甲硫氨酸100~ 1600、苯丙氨酸150~1400、苏氨酸50~600、色氨酸50~400、精氨酸200~1500、组氨酸100~ 1000、甘氨酸100~600、天冬氨酸100~600、亮氨酸100~600、异亮氨酸100~600、缬氨酸100~ 800、丝氨酸100~400、谷氨酰胺200~600、牛磺酸100~500、乳清酸100~300、核苷酸200~1200、 维生素A 0.2~ 0.6、维生素D 0.002~0.006、维生素B2 1~4、维生素B6 1~4、维生素B12 0.001~0.006、烟酸5~20、叶酸0.01~1.2、维生素C 50~150、铁2~10、锌2~10、锰2~10、铜0.5~2、硒0.01~0.1、 铬0.01~0.02、钾10~300、钙100~300、镁100~200、肌醇50~60、大豆磷脂100~2500。 A nutrient composition, characterized in that the mass of each component is as follows: lysine 200 to
to 1600 methionine, phenylalanine 150 to 1400, 50 to 600 threonine, Tryptophanyl acids 50 to 400, 200 to 1500 arginine, histidine 100 to 1000, from 100 to 600 glycine, aspartic acid 100 to 600, 100 to 600 leucine, isoleucine 100 to 600, valine 100 to 800, 100 to 400 serine, glutamine 200 to 600, 100 to 500 taurine, orotic acid 100 to 300, 200 to 1200 nucleotides, vitamins A 0.2 ~ 0.6, vitamin D 0.002 ~ 0.006, vitamin B2 1 ~ 4, vitamin B6 1 ~ 4, vitamin B12 0.001 ~ 0.006, niacin 5 to 20, 0.01 to 1.2 folic acid, vitamin C 50 ~ 150, 10 ~ 2 iron, zinc 2 to 10, 2 to 10 manganese, copper 2 ~ 0.5, 0.01 ~ 0.1 selenium, chromium 0.01-0.02, potassium 10 ~ 300, 100 ~ 300 calcium, magnesium, 100 to 200, 50 to 60-inositol, soybean lecithin 100 to 2500.
2. 根据权利要求1所述的营养组合物，其特征在于各组分质量比如下:赖氨酸600、甲硫氨酸800、苯丙氨酸700、苏氨酸300、色氨酸200、精氨酸760、组氨酸500、甘氨酸300、天冬氨酸300、亮氨酸300、异亮氨酸300、缬氨酸400、丝氨酸200、谷氨酰胺300、牛磺酸500、乳清酸300、核苷酸400、维生素A 0.6、维生素D0.006、维生素B22、维生素B62、维生素B12 0.004、烟酸12、叶酸0.9、维生素C100、铁10、锌10、锰8、铜2、硒0.1、铬0.02、钾300、钙300、镁200、肌醇60、大豆磷脂1200。 2. Nutritional composition according to claim 1, characterized in that the mass of each component is as follows: Lysine 600, 800 methionine, phenylalanine 700, threonine 300, Trp 200, 760 arginine, histidine 500, Gly 300, Asp 300, leucine 300, isoleucine 300, Val 400, Ser 200, glutamine 300, 500 taurine, whey acid 300, 400 nucleotides, vitamins A 0.6, vitamin D0.006, vitamin B22, vitamin B62, vitamin B12 0.004, 12 niacin, folic acid 0.9, vitamin C100, 10 iron, zinc 10, Mn 8, 2 copper, selenium 0.1, 0.02 chromium, potassium, 300, 300, calcium, magnesium 200, inositol 60, soy lecithin 1200.
3. -种如权利要求1或2所述营养组合物在制备治疗再生障碍性贫血药物中的应用。 3. - kind of claim 1 or claim 2 in the manufacture of a nutritional composition requires treatment of aplastic anemia medicine applications.
营养组合物及在制备治疗再生障碍性贫血药物中的应用 Nutritional composition and its application in preparing medicine for treating aplastic anemia medicine
技术领域 TECHNICAL FIELD
[0001] 本发明涉及生命科学领域预防医学与治疗医学，尤其是一种营养组合物及在制备治疗再生障碍性贫血药物中的应用。 [0001] The present invention relates to the field of preventive medicine and life sciences medical treatment, in particular to a nutritional composition for the preparation and treatment of aplastic anemia medicine.
背景技术 Background technique
[0002] 再生障碍性贫血(再障）是一组由化学物质、生物因素、放射线或不明原因引起的骨髓造血功能衰竭，以造血干细胞损伤、骨髓脂肪化、外周血全血细胞减少为特征的疾病。 [0002] aplastic anemia (AA) is a group of bone marrow function by the chemical, biological factors, radiation or unknown causes of failure, hematopoietic stem cell damage, bone marrow fat of peripheral blood pancytopenia disease characterized . 再障发病机制极为复杂，目前认为与以下几方面有关： (1)造血干细胞内在增殖缺陷① 再障骨髓中造血干细胞明显减少，造血干细胞集落形成能力显著降低，异常造血干细胞可抑制正常造血干细胞功能； ② SAA患者（重型再障)DNA修复能力明显降低，用抗淋巴细胞球蛋白（ALG)治疗后仍不能纠正； ③ 部分经免疫抑制剂治疗有效的病例，在长期随访过程中演变为克隆性疾病； ④ 再障患者体内均有一定数量的补体敏感细胞，体外实验也证明再障造血干/祖细胞对补体敏感性增强。 Aplastic anemia pathogenesis is very complex, considered the following aspects: (1) hematopoietic stem cell intrinsic proliferation defect ① aplastic bone marrow hematopoietic stem cells significantly reduced hematopoietic stem cell colony formation was significantly decreased abnormal hematopoietic stem cells can inhibit the normal hematopoietic ② SAA patients (SAA) DNA repair capacity was significantly reduced, with anti-lymphocyte globulin (ALG) after treatment
③ part through effective immunosuppressive treatment cases, clonal evolution of long-term follow- ④ aplastic anemia patients have a certain amount of complement-sensitive cells, in vitro experiments also proved aplastic anemia hematopoietic stem / progenitor cells to complement increased sensitivity.
[0003] (2)异常免疫反应损伤造血干细胞再障患者经免疫抑制治疗后其自身造血功能可能得到改善，此为异常免疫反应损伤造血干细胞最直接的证据。 [0003] (2) abnormal immune response to injury of hematopoietic stem cells in patients with aplastic anemia after immunosuppressive therapy in its own hematopoietic function may be improved, this is the abnormal immune response to injury in the most direct evidence of hematopoietic stem cells.
[0004] (3)造血微环境支持功能缺陷造血微环境包括基质细胞及其分泌的细胞因子，起支持造血细胞增殖及促进各种细胞生长发育的作用。 [0004] (3) hematopoietic microenvironment hematopoietic microenvironment supports functional defects including stromal cells and the secretion of cytokines, supportive of hematopoietic cell proliferation and promote the role of various cell growth and development. 虽然造血微环境不是引起AA的始因，但可加重病情。 Although hematopoietic microenvironment is not caused by the beginning of AA, but can aggravate the condition.
[0005] (4)遗传倾向再障常有HLA-DR2型抗原连锁倾向，儿童再障HLA-DPW3型抗原显著增高，患者家属中常有造血祖细胞增殖能力明显降低，并可见家族性再障。 [0005] (4) a genetic predisposition to aplastic anemia often HLA-DR2 antigen chain tendency, children with aplastic anemia was significantly higher HLA-DPW3 antigen, family members of patients often hematopoietic progenitor cell proliferation significantly reduced, and visibility familial aplastic anemia.
[0006] 再障严重危害人体健康，中国医学科学院血液学研究所(1985)分析住院再障患者392例，随访时间1~26年，病死率为42.6%，其中急性再障为91.6%，慢性再障为24.6%，再障的预后与治疗方法密切相关。 [0006] AA serious harm to human health, Institute of Hematology, Chinese Academy of Medical Sciences (1985) analysis of 392 cases of hospitalized patients with aplastic anemia, up 1 to 26 years, mortality was 42.6%, which was 91.6% of acute aplastic anemia, chronic aplastic anemia was 24.6%, closely related to the prognosis and treatment of aplastic anemia.
[0007] 中国专利号专利号为.8的中国发明专利公开了一种&促进造血干细胞增殖与血红蛋白合成的营养组合物&。 [0007] Chinese Patent No. Patent No. .8 China Patent discloses a &promote hematopoietic stem cell proliferation and hemoglobin synthesis nutritional composition.& 该营养组合物的原料及重量比如下：核苷酸90~ 110、精氨酸20~30、赖氨酸20~30、半胱氨酸10~20、甘氨酸25~35、组氨酸20~30、卵磷脂110~ 130、脑磷脂50~70、维生素E 0.4~0.6、维生素C 5~7、叶酸0.02~0.04、维生素B2 0.05~0.07、 维生素B6 0 · 07~0 · 08、维生素Bi2 0 · 0001~0 · 0002、铁0 · 5~1 · 5、锌0 · 5~0 · 7、锰0 · 4~0 · 6、枸杞多糖14~16、葡萄籽提取物14~16。 The nutritional composition of the raw materials and the weight is as follows: 90 to 110 nucleotides, 20 to Arg 30, Lys 20 ~ 30, 10 ~ 20 cysteine, glycine 25 ~ 35, 20 ~ histidine 30, 110-130 lecithin, cephalin 50 to 70, vitamin E 0.4 ~ 0.6, vitamin C 5 ~ 7, ~ 0.02 folic acid 0.04 vitamin B2 0.05 ~ 0.07, vitamin B6 0 · 07 ~ 0 · 08, vitamin Bi2 0 · 0001 to 0 · 0002, iron 0.5 ~ 1.5, zinc 0.5 ~ 0.7 Mn 0.4 ~ 0.6, LBP 14 to 16, grape seed extract 14 to 16. 可明显提高血中血红蛋白（Hb)、红细胞(RBC)、白细胞(WBC) 及血小板(Pit)数量;可刺激骨髓造血干细胞的增殖，使造血干细胞明显增多；明显提高细胞内线粒体数目；对肝、脾的损伤具有明显的恢复作用。 Can significantly improve blood hemoglobin (Hb), red blood cell (RBC), white blood cell (WBC) and platelets (Pit) can stimulate bone marrow stem cell proliferation, hematopoietic stem cells inc the number of mitochondria within cells sig liver, spleen injury significant recovery effect. 专利号为.8的中国发明专利公开了该营养组合物作为药物可为错配修复基因损伤的恢复提供基本营养条件，从而达到恢复错配修复基因损伤的目的。 Patent No. .8 of Chinese invention patents disclose the nutritional composition as a medicament for mismatch repair gene damage restoration provides basic nutritional conditions, so as to achieve recovery mismatch repair gene damage purposes. 专利号为.2的中国发明专利公开了上述营养组合物可对线粒体损伤的恢复提供基本营养条件，进而充分发挥机体对线粒体损伤的自主修复本能，达到恢复线粒体损伤的目的，但是仅局限于对肝脏、脾脏的线粒体增值及修复。 Patent No. .2 of Chinese invention patents disclose the above nutritional composition may restore damaged mitochondria provide basic nutrition, and thus give full play to the body's self-repair damaged mitochondria instinct, to restore mitochondrial injury purposes, but is limited to the liver spleen and repair mitochondrial added. 专利号为.4的中国发明专利还公开了该营养组合物可促进肝脏干细胞增殖。 Patent No. .4 of Chinese invention patent also discloses that the nutritional composition may promote liver stem cell proliferation. 尽管上述营养组合物具有所述的功能，但是由于作用的局限性，该营养组合物仅适用于治疗营养不良性贫血(缺铁性贫血及叶酸与维生素B12营养不良性贫血）、红细胞生成减少所致贫血以及红细胞破坏过多或丢失所致贫血，而不能够作为治疗再生障碍性贫血的药物。 Although the above composition has the nutritional functions, but because of the limitations of the action, only the nutritional composition useful for treating malnutrition, anemia (iron deficiency anemia, and folic acid and vitamin B12 malnutrition, anemia), reduces the erythropoietin cause anemia and excessive red blood cell destruction, or loss caused by anemia, can not as a treatment for aplastic anemia drugs.
发明内容 SUMMARY
[0008] 本发明是为了解决现有技术所存在的上述技术问题，提供一种营养组合物及在制备治疗获得性再生障碍性贫血药物中的应用， 本发明的技术解决方案是：一种营养组合物，其特征在于各组分质量比如下：赖氨酸200~1200、甲硫氨酸100~1600、苯丙氨酸150~1400、苏氨酸50~600、色氨酸50~400、精氨酸200~1500、组氨酸100~1000、甘氨酸100~600、天冬氨酸100~600、亮氨酸100~600、异亮氨酸100~600、缬氨酸100~800、丝氨酸100~400、谷氨酰胺200~600、牛磺酸100~500、乳清酸100~ 300、核苷酸200~1200、维生素A 0.2~0.6、维生素D 0.002~0.006、维生素B2 1~4、维生素B6 1 ~4、维生素B12 0.001~0.006、烟酸5~20、叶酸0.01~1.2、维生素C 50~150、铁2~10、锌2~10、 锰2~10、铜0.5~2、硒0.01~0.1、铬0.01~0.02、钾10~300、钙100~300、镁100~200、肌醇50~ 60、大豆磷脂100~2500。 [0008] The present invention is to solve the above problems of the prior art exists to provide a nutritional composition and acquired aplastic anemia drugs used in the treatment of preparation, the technical solutions of the present invention is: a nutrient composition, characterized in that the mass of each component is as follows: lysine 200 to
to 1600 methionine, phenylalanine 150 to 1400, 50 to 600 threonine, tryptophan, 50 to 400, 200-1500 arginine, histidine 100 to
to 600 glycine, aspartic acid 100 to 600, 100 to 600 leucine, isoleucine 100 to 600, 100 to 800 of valine, serine 100 to 400, 200 to 600 glutamine, taurine, 100 to 500, 100 to 300 orotic acid, nucleotide 200 to 1200, vitamin A 0.2 ~ 0.6, vitamin D 0.002 ~ 0.006, vitamin B2 1 ~ 4, vitamin B6 1 ~ 4, vitamin B12 0.001 ~ 0.006, niacin 5 to 20, 0.01 to 1.2 folic acid, vitamin C 50 ~ 150, 10 ~ 2 iron, zinc 2 to 10, 2 to 10 manganese, copper 0.5 to 2, Se 0.01 to 0.1, 0.01 to 0.02 chromium, 10 to 300 K, Ca 100 to 300 100 to 200 Mg, 50 - 60 inositol, soy lecithin 100 to 2500.
[0009] 各组分最佳质量比如下:赖氨酸600、甲硫氨酸800、苯丙氨酸700、苏氨酸300、色氨酸200、精氨酸760、组氨酸500、甘氨酸300、天冬氨酸300、亮氨酸300、异亮氨酸300、缬氨酸400、丝氨酸200、谷氨酰胺300、牛磺酸500、乳清酸300、核苷酸400、维生素A 0.6、维生素00.006、维生素822、维生素故2、维生素出2 0.004、烟酸12、叶酸0.9、维生素(：100、铁10、锌10、 锰8、铜2、硒0.1、铬0.02、钾300、钙300、镁200、肌醇60、大豆磷脂1200。 [0009] For example, the components of the best quality under: lysine 600, 800 methionine, phenylalanine 700, 300 threonine, tryptophan 200, 760 arginine, histidine 500, glycine 300, 300 aspartic acid, leucine 300, 300 isoleucine, valine 400, serine 200, 300 glutamine, taurine 500, acid whey 300, 400 nucleotides, vitamins A 0.6 , 00.006 vitamins, vitamin 822, so the two vitamins, the vitamins 2 0.004 niacin 12, 0.9 folic acid, vitamin (: 100, 10 iron, 10 zinc, manganese 8, Cu 2 Se 0.1, Cr 0.02, 300 K, Ca 300, 200 magnesium, inositol 60, soy lecithin 1200.
[0010] 上述营养组合物在制备治疗再生障碍性贫血药物中的应用。 [0010] said nutritional composition in preparing treatment of aplastic anemia medicine applications.
[0011]本发明的营养组合物对DNA切除修复基因损伤、DNA错配修复基因损伤、DNA同源重组修复基因损伤及DNA错误倾向修复基因损伤等均有恢复作用；可使肝细胞、脾脏B细胞、 肾小管上皮细胞及脑海马部神经元细胞线粒体增殖，对肝细胞、脾脏B细胞线粒体基因损伤进行修复;具有促进造血干细胞、肝脏干细胞、间充质干细胞、肾脏干细胞增殖及胃粘膜干细胞增殖的作用;对肝脏损伤、脾脏损伤、肾脏损伤、大脑损伤及骨骼损伤等具有修复作用； 对再障大鼠高表达的多个蛋白分子有明显的抑制作用。 [0011] The nutritional composition of the present invention of DNA excision repair gene damage, damage DNA mismatch repair genes, DNA repair genes homologous recombination and DNA damage repair gene damage and other erroneous tendencies
make liver cells, splenic B cells, renal tubular epithelial cells and brain hippocampal neurons in mitochondrial proliferation of liver cells, splenic B cells mitochondrial DNA promote hematopoietic stem cells, liver stem cells, mesenchymal stem cells, kidney stem cell proliferation and gastri liver injury, spleen injury, kidney damage, brain damage and bone damage, etc. a plurality of protein molecules in rats with aplastic anemia high expression significantly inhibited. 即可以全面修复再障机体，从而达到治疗再生障碍性贫血的效果。 That can be fully restored aplastic body, so as to achieve the treatment of aplastic anemia results.
附图说明 BRIEF DESCRIPTION
[0012] 图1是本发明实施例对再障大鼠体重的影响示意图。 [0012] FIG. 1 is a schematic view of the impact on patients with aplastic anemia rats weighing embodiment of the present invention.
[0013] 图2是本发明实施例对再障大鼠肝、骨髓、脑核苷酸切除修复基因的影响示意图。 [0013] FIG. 2 is a diagram of aplastic anemia liver, bone marrow, brain and rat nucleotide excision repair genes in a schematic view of the present invention.
[0014] 图3是本发明实施例对再障大鼠肝、骨髓、脑碱基切除修复基因的影响示意图。 [0014] FIG. 3 is a diagram of aplastic anemia in rat liver, bone marrow, brain base excision repair genes affect a schematic diagram of the present invention.
[0015] 图4是本发明实施例对再障大鼠肝脏、骨髓以及脑组织中错配修复基因的影响示意图。 [0015] FIG. 4 is an embodiment of the invention on rat liver aplastic anemia, bone marrow and brain tissue mismatch repair genes in a schematic view.
[0016] 图5是本发明实施例对再障大鼠肝、骨髓、脑同源重组修复基因的影响示意图。 [0016] FIG. 5 is an embodiment of aplastic anemia in rat liver, bone marrow, brain homologous recombination repair gene affect a schematic diagram of the present invention.
[0017] 图6是本发明实施例对再障大鼠肝、骨髓、脑S0S修复基因的影响示意图。 [0017] FIG. 6 is an embodiment of the invention on rat liver aplastic anemia, bone marrow, brain S0S repair genes in FIG.
[0018] 图7是本发明实施例对再障大鼠肝脏细胞线粒体的影响示意图（电镜）。 [0018] FIG. 7 is a diagram of aplastic anemia rat liver mitochondria influence schematic (SEM) embodiment of the present invention.
[0019] 图8是本发明实施例对再障大鼠脾脏细胞线粒体的影响示意图（电镜）。 [0019] FIG. 8 is a diagram of aplastic anemia affect mitochondria in rat spleen schematic (SEM) embodiment of the present invention.
[0020] 图9是本发明实施例对再障大鼠肾小管上皮细胞线粒体的影响示意图（电镜）。 [0020] FIG. 9 is a diagram of aplastic anemia in renal tubular epithelial cells mitochondria of rats schematic (SEM) embodiment of the present invention.
[0021] 图10是本发明实施例对再障大鼠脑海马部的影响示意图（电镜）。 [0021] FIG. 10 is an embodiment of the invention of the Aplastic Anemia schematic (SEM) in hippocampus portion.
[0022] 图11是本发明实施例对再障大鼠线粒体膜电位的影响示意图。 [0022] FIG. 11 is an embodiment of the invention aplastic mitochondrial membrane potential in Rats FIG.
[0023]图12是本发明实施例对再障大鼠线粒体DNA含量的影响。 [0023] FIG. 12 is an example of the impact of mitochondrial DNA content in aplastic anemia rats embodiment of the present invention.
[0024] 图13是本发明实施例对再障大鼠骨髓造血干细胞的影响示意图。 [0024] FIG. 13 is a schematic view of the impact on patients with aplastic bone marrow hematopoietic stem cells in rats embodiment of the present invention.
[0025] 图14是本发明实施例对再障大鼠肝脏干细胞的影响示意图。 [0025] FIG. 14 is a schematic view of the impact on patients with aplastic anemia rat liver stem cell embodiment of the present invention.
[0026] 图15是本发明实施例对再障大鼠肾脏干细胞的影响示意图。 [0026] FIG. 15 is a schematic view of the impact on patients with aplastic anemia rat kidney stem cells in the present invention.
[0027] 图16是本发明实施例对再障大鼠胃干细胞的影响示意图。 [0027] FIG. 16 is an embodiment of the invention on AA rat stomach stem cells in Fig.
[0028] 图17是本发明实施例对再障大鼠肠粘膜干细胞的影响示意图。 [0028] FIG. 17 is a schematic diagram of aplastic anemia rat intestinal stem cells affected by the present invention.
[0029] 图18是本发明实施例对再障大鼠神经干细胞的影响示意图。 [0029] FIG. 18 is a schematic view of the impact on patients with aplastic anemia rat neural stem cells of the embodiment of the present invention.
[0030] 图19是本发明实施例对再障大鼠胰腺干细胞的影响示意图。 [0030] FIG. 19 is a schematic view of the impact on patients with aplastic anemia rat pancreatic stem cells in the present invention.
[0031] 图20是本发明实施例对再障大鼠肌肉干细胞的影响示意图。 [0031] FIG. 20 is a schematic view of the impact on patients with aplastic anemia rat muscle stem cells of the embodiment of the present invention.
[0032] 图21是本发明实施例对再障大鼠骨髓间充质干细胞的影响不意图。 [0032] FIG. 21 is an example of the impact of aplastic anemia rat bone marrow mesenchymal stem cells of the embodiment of the present invention is not intended.
[0033]图22是本发明实施例对再障大鼠肝、脾、肾、脑的影响示意图(HE染色）。 [0033] FIG. 22 is a diagram of aplastic anemia rat liver, affecting schematic spleen, kidney, brain (HE staining) embodiment of the present invention.
[0034]图23是本发明实施例对再障大鼠肠、肌肉的影响示意图（HE染色）。 [0034] FIG. 23 is a diagram of aplastic anemia intestinal muscles influence schematic (HE staining) embodiment of the present invention.
[0035]图24是本发明各实验组的肾切片透射电镜图。 [0035] FIG. 24 is the present invention in each experimental group of kidney sections under TEM.
[0036] 图25是本发明实施例对再障大鼠骨髓间充质干细胞生长的影响示意图。 [0036] FIG. 25 is an embodiment of the invention for aplastic anemia rat bone marrow mesenchymal stem cells. Fig.
[0037] 图26是本发明各实验组脂肪细胞的诱导及成脂细胞相关基因的表达示意图。 [0037] FIG. 26 is a schematic diagram of expression induced by fat cells in each experimental group of the present invention and related genes into adipocytes.
[0038] 图27是本发明各实验组成骨细胞的诱导及成骨细胞相关基因的表达示意图。 [0038] FIG. 27 is a schematic view of inducing the expression of the experimental composition of the present invention osteoblasts and osteoblast-related genes.
[0039]图28是本发明实施例对再障大鼠骨髓造血细胞的影响示意图。 [0039] FIG. 28 is a schematic diagram of aplastic anemia rat bone marrow hematopoietic cells affect the implementation of the present invention.
[0040]图29-1、图29-2、图29-3是本发明实施例对再障大鼠间充质干细胞蛋白表达的影响示意图（蛋白芯片谱图）。 [0040] FIG. 29-1, Figure 29-2, Figure 29-3 is an example of the expression of mesenchymal stem cells in rats with aplastic schematic (protein chip spectrum) embodiment of the present invention.
[0041 ]图30-1、图30-2、图30-3是本发明实施例对再障大鼠骨髓细胞蛋白表达的影响示意图（蛋白芯片谱图）。 [0041] FIG. 30-1, Figure 30-2, Figure 30-3 is the impact on patients with aplastic bone marrow protein expression in rat cells schematic (protein chip spectrum) embodiment of the present invention.
[0042]图31-1、图31-2、图31-3是本发明实施例对再障大鼠肝脏细胞蛋白表达的影响示意图（蛋白芯片谱图）。 [0042] FIG. 31-1, Figure 31-2, Figure 31-3 is an example of protein expression in rat liver cells in aplastic influence diagram (protein chip spectrum) embodiment of the present invention.
具体实施方式 detailed description
[0043]实施例1:本发明的营养组合物各组分质量(mg)比如下：赖氨酸200~1200、甲硫氨酸100~1600、苯丙氨酸150~1400、苏氨酸50~600、色氨酸50~400、精氨酸200~1500、组氨酸100~1000、甘氨酸100~600、天冬氨酸100~600、亮氨酸100~600、异亮氨酸100~600、缬氨酸100~800、丝氨酸100~400、谷氨酰胺200~600、牛磺酸100~500、乳清酸100~300、核苷酸200~ 1200、维生素A 0.2~0.6、维生素D 0.002~0.006、维生素B2 1~4、维生素B6 1~4、维生素B12 0.001~0.006、烟酸5~20、叶酸0.01~1.2、维生素C 50~150、铁2~10、锌2~10、锰2~10、铜0.5 ~2、硒0.01~0.1、铬0.01~0.02、钾10~300、钙100~300、镁100~200、肌醇50~60、大豆磷脂100~ 2500〇 [0043] Example 1: the quality of the components of the nutritional composition of the present invention (mg) is as follows: 200 to 1,200 lysine, methionine 100 to 1600, from 150 to 1400 phenylalanine, threonine 50 ~ 600, 50 ~ 400 tryptophan, arginine, 200-00 histidine, glycine 100 to 600, 100 to 600 aspartic acid, leucine 100 to 600, 100 to isoleucine 600, 100-800 valine, serine 100 to 400, 200 to 600 glutamine, taurine, 100 to 500, 100 to 300 orotic acid, nucleotide 200 to 1200, vitamin A 0.2 ~ 0.6, vitamin D 0.002 to 0.006, vitamin B2 1 ~ 4, vitamin B6 1 ~ 4, vitamin B12 0.001 ~ 0.006, niacin 5 to 20, 0.01 to 1.2 folic acid, vitamin C 50 ~ 150, 10 ~ 2 iron, 2 to 10 zinc, manganese 2 to 10, from 0.5 to 2 copper, selenium, 0.01 to 0.1, 0.01 to 0.02 chromium, 10 to 300 K, Ca 100 to 300 100 to 200 Mg, 50 - 60 inositol, soy lecithin 100 ~ 2500〇
[0044] 实施例2:各组分质量(mg)比如下:赖氨酸600、甲硫氨酸800、苯丙氨酸700、苏氨酸300、色氨酸200、精氨酸760、组氨酸500、甘氨酸300、天冬氨酸300、亮氨酸300、异亮氨酸300、缬氨酸400、丝氨酸200、谷氨酰胺300、牛磺酸500、乳清酸300、核苷酸400、维生素A 0.6、维生素00.006、维生素出2、维生素故2、维生素也2 0.004、烟酸12、叶酸0.9、维生素(：100、 铁10、锌10、锰8、铜2、硒0.1、铬0.02、钾300、钙300、镁200、肌醇60、大豆磷脂1200。 [0044] Example 2: The quality of the components (mg) is as follows: 600 lysine, methionine 800, 700 phenylalanine, threonine 300, 200 tryptophan, arginine 760, group acid 500, glycine 300, Asp 300, leucine 300, 300 isoleucine, valine 400, serine 200, 300 glutamine, taurine 500, orotate 300 nucleotides 400, vitamin a 0.6, 00.006 vitamins, vitamin a, vitamin therefore 2, also 2 0.004 vitamin, niacin 12, 0.9 folic acid, vitamin (: 100, 10 iron, 10 zinc, manganese 8, Cu 2 Se 0.1, Cr 0.02, 300 K, 300 calcium, magnesium 200, inositol 60, soy lecithin 1200.
[0045] 所述营养组合物可在制备治疗再生障碍性贫血药物中应用。 [0045] The nutritional composition may be used in the preparation of the treatment of aplastic anemia drugs.
[0046] 原料来源如表1: 表1 [0046] The raw materials shown in Table 1: Table 1
实验： 一.再生障碍性贫血大鼠模型的建立本实验采用SD大鼠。 Experiment: a rat model of aplastic anemia establish SD rats used in this experiment. 实验步骤:第一天X射线2.5Gy照射后，于第4、6、8天分别给予环磷酰胺35mg/kg，氯霉素45 mg/kg腹腔注射。 Experimental procedure: The first day after X-ray irradiation 2.5Gy, 4,6,8 on the first day were given cyclophosphamide 35mg / kg, chloramphenicol 45 mg / kg intraperitoneally. 第15天重复以上步骤。 Repeat these steps for the first 15 days. 以单纯等量生理盐水相应部位注射为正常对照组。 In the corresponding parts of pure saline injection as normal control group.
[0047]实验分组:根据动物药理学的药品剂量和综合试验的设计分析，分为:①正常对照组;②再障模型组;③实施例2营养组合物高剂量组，以2266.95mg/kg. d对大鼠灌胃；④实施例2营养组合物中剂量组，以1511.3mg/kg. d对大鼠灌胃；⑤实施例2营养组合物低剂量组， 以1057.91mg/kg.d对大鼠灌胃。 [0047] Experimental groups: According to the design and analysis of animal pharmacology and integrated drug dose tested, divided into: ① ② AA ③ Example 2, the nutritional composition of high-dose group implemented to 2266.95mg / kg . Example 2 nutritional composition dose group ④ implemented to 1511.3mg / Example 2 nutritional composition low dose group ⑤ implemented to 1057.91mg / kg.d rats fed.
[0048]试验流程:首先，按前述再障大鼠模型的建立方法建立模型，从第5天开始，营养组合物高、中、低剂量组分别以相应剂量对大鼠进行灌胃，每天一次，直至第60天，拉颈处死大鼠，采样进行检测。 [0048] test process: First, according to the aforementioned method of establishing rat aplastic anemia model to model, from the fifth day, the nutritional composition of high, medium and low dose groups respectively corresponding dose rats were fed once a day until the 60th day, the rats were killed by cervical sampling for testing. 同时每日进行整体观察，测量体重。 While overall daily observation, measurement of body weight.
[0049] 二.实验方法1.外周血检测各实验组第60天，称重动物，经眼眶取血，采用全自动血细胞分析仪测定外周血血红蛋白含量、红细胞、白细胞及血小板数;显微镜下观察外周血涂片。 [0049] 1. The two experimental methods in peripheral blood in each experimental group on day 60, the animals were weighed, dried orbital blood using automated hematology analyzer peripheral blood hemoglobin, red blood cells, white blood cel. Under the microscope peripheral blood smear.
[0050] 2.促红细胞生成素(Epo)检测酶标板每孔加入标准液，对照液及预先用样品稀释液稀释的待测样品l〇〇ul，封板，37° C孵育90分钟。 [0050] 2. erythropoietin (Epo) was added to each well microtiter plates to detect standard solution, control solution and sample pre-dilution of the diluted sample to be tested l〇〇ul, sealing plate, 37 ° C incubated for 90 minutes. 反应后甩干孔内液体后，将准备好的生物素抗小鼠ΕΡ0抗体工作液按每孔100ul依次加入(TMB空白显色孔除外），封板后37°C反应60分钟，0.01M PBS洗涤3次，每次浸泡1分钟左右。 After the reaction liquid after drying hole, the prepared biotinylated anti-mouse antibody ΕΡ0 working solution 100ul per well were added sequentially (TMB was blank except for hole color), after the closure plate 37 ° C for 60 minutes, 0.01M PBS washed three times, each time for about 1 minute soak. 加入准备好的亲和素一过氧化物酶复合物工作液按每孔l〇〇ul依次加入(TMB 空白显色孔除外），37°C反应30分钟。 Add prepared avidin-peroxidase complex fluid per well l〇〇ul added sequentially (TMB was blank except for color hole), 37 ° C for 30 minutes. 按每孔90μ1依次加入已在37°C中平衡30分钟TMB显色液，37°C避光反应，每孔100μ1依次加入TMB终止液。 90μ1 successively added per well equilibrated 30 minutes TMB color reagent in the 37 ° C, 37 ° C in the dark reaction, are then added to each well 100μ1 TMB Stop Solution. 用酶标仪于450nm波长处读取吸光度(0D 值），根据标准曲线测得样本Epo浓度(pg/ml)。 Read the absorbance (0D value) using a microplate reader at 450nm wavelength, measured according to the standard curve samples Epo concentration (pg / ml).
[0051] 3.骨髓检测颈椎脱白处死大鼠，分离双侧股骨打开骨髓腔进行骨髓涂片、骨髓单个核细胞计数、 骨髓病理检查、骨髓间充质干细胞增殖及分化趋势的分析。 [0051] 3. Detection of cervical marrow white rats were killed off, isolated open bilateral femur bone marrow cavity smear analysis of bone marrow mononuclear cell counts, bone marrow biopsy, bone marrow proliferation and differentiation of mesenchymal stem cells trend.
[0052] 4.骨髓间充质干细胞(BMSCs)的分离提取SD大鼠腹腔注射麻醉药后脱颈处死，75%乙醇浸泡大鼠20无菌取双后肢，放入75%的乙醇中浸泡5清除下肢肌肉，从膝关节断离股骨，剪开股骨两端；用10 mL注射器吸取10%胎牛血清的F-12K培养液冲洗骨髓腔，收集骨髓冲洗液，制成单细胞悬液，200目筛网滤过后以IX 1〇8接种于培养瓶中。 [0052] After the separation between 4. marrow mesenchymal stem cells (BMSCs) extraction by intraperitoneal injection of anesthetic SD rats were sacrificed, 75% ethanol for rats 20 sterile of hind limbs, add 75% ethanol soak for 5 remove leg muscles from breaking away from the femur of the knee, cut t 10 mL syringe with 10% fetal bovine serum F-12K medium flushing the marrow cavity, bone marrow fluid was collected, made into single cell The suspension, after filtering with a 200 mesh sieve IX 1〇8 seeded in culture flasks. 置于37° C、5% C02培养箱中培养。 Placed in 37 ° C, 5% C02 incubator. 72小时首次更换培养液，去除未贴壁细胞，以后每隔2天换液1次。 72 hours medium was changed for the first time, remove non-adherent cells, after the medium was changed once every two days. 当细胞铺满整个培养瓶底面积的80%~90% 时，传代培养。 When the cells covered the entire culture 80% to 90% of the bottom area of the subculture.
[0053] 5.间充质干细胞(BMSCs)的成脂诱导分化成脂诱导法:将分离纯化的骨髓间充质干细胞常规培养，消化传代，以1 X 1〇5/孔的密度，400ul/孔接种于预先放置有盖玻片的6孔培养板中，制备细胞爬片，当细胞生长达80% 融合时，加入10%的胎牛血清、lymol/L地塞米松、0.5 mmol/L IBMX、10 mg/L牛胰岛素的H-DMEM诱导3天，再用10%的胎牛血清、10 mg/L牛胰岛素的H-DMEM处理1天，如此循环3次后， 用10%的胎牛血清、10 mg/L牛胰岛素的H-DMEM处理7天，每隔三四天换液1次。 [0053] 5. The mesenchymal stem cells (BMSCs) of adipogenic differentiation adipogenic law: the separation-purified mesenchymal stem cells were cultured, digestion and passage, at a density of 1 X 1〇5 / well, 400ul / pre-placed seeded in 6-well plate coverslip prepare climbing film cells, when cell growth by 80% confluence, 10% fetal bovine serum, lymol / L dexamethasone, 0.5 mmol / L IBMX , 10 mg / L bovine insulin induced H-DMEM for 3 days with 10% fetal bovine serum, 10 mg / L bovine insulin H-DMEM one day treatment, so after three cycles, with 10% fetal bovine serum, 10 mg / L bovine insulin H-DMEM for 7 days, the medium was changed every thirty-four days 1 times. 对照组始终加入含体积分数为1 〇%的胎牛血清、1 〇mg/L牛胰岛素的H-DMEM，每隔三四天换液1次。 The control group is always added volume fraction of 1 billion% fetal calf serum, 1 〇mg / L bovine insulin H-DMEM, the medium was changed every thirty-four days 1 times. 诱导后细胞用PBS洗涤2~3次，10%甲醛室温固定40min JBS洗2~3次，饱和油红0染液用一蒸水以3: 2稀释，室温染色30min，PBS洗数次直至无肉眼观察到沉渣，光镜观察。 After induction, cells were washed with PBS 2 ~ 3 times, fixed in 10% formalin at room temperature 40min JBS washed 2 to 3 times, saturated Oil Red O dye with distilled water in a 3: 2 dilution at room temperature dyeing 30min, PBS washed several times until no visually observed sediment, light microscope.
[0054] 6.间充质干细胞(BMSCs)成骨细胞培养及鉴定取P2代骨髓间充质干细胞，按2X105个/孔接种于12孔板，当细胞贴壁生长达80%融合时，吸除孔中培养液，换为成骨细胞诱导液（含高糖DMEM、体积分数为10%胎牛血清、10-8 mol/L地塞米松、1(T2 mol/L β-甘油磷酸钠、50 mg/L抗坏血酸），每3天更换诱导液1次。培养14天后，吸除培养液，PBS洗2次，10%甲醛室温固定40 min，PBS洗3次，加入0.1%茜红素(3:2 稀释），室温染色l〇min，去除染液，PBS洗3次后，显微镜下观察。 [0054] 6. The mesenchymal stem cells (BMSCs) osteoblast cultures and identification take between P2-generation bone marrow mesenchymal stem cells, according to 2X105 cells / well were seeded in 12-well plates when the cells adherent growth by 80% confluence, suck In addition to well of nutrient solution, for the osteogenic solution (containing high glucose DMEM, the volume fraction of 10% fetal bovine serum, 10-8 mol / L dexamethasone, 1 (T2 mol / L β- glycerophosphate, 50 mg / L ascorbic acid), changed every 3 days induced liquid 1 times. 14 days of culture, sucked media, PBS, washed twice in 10% formalin fixed at room temperature 40 min, PBS washed 3 times, 0.1% Akane red pigment ( 3: 2 dilution) at room temperature l〇min dyeing, dye removal, the PBS washed 3 times, observed under a microscope.
[0055] 7. RNA提取和RT-PCR分析常规应用Trizol法提取各标本的总RNA，紫外分光光度计测定A26Q和A28Q，以检测RNA含量和纯度，并置放于-70° C保存。 [0055] 7. RNA extraction and RT-PCR analysis of routine applications Trizol extraction of total RNA for each sample, and UV spectrophotometer A26Q A28Q, to detect the RNA content and purity, and placed at -70 ° C to save.
[0056]反转录体系20μ1，包含样品RNA ΙμL，按照TaKaRa的反转录试剂盒提供的说明书进行操作。 [0056] Reverse transcription system 20μ1, samples containing RNA ΙμL, in accordance with the instructions TaKaRa reverse transcription kit instructions. 反转录反应条件为:65°C lmin，30°C 5min，65°C 15min，98°C 5min，5°C 5min。 Reverse transcription reaction conditions: 65 ° C lmin, 30 ° C 5min, 65 ° C 15min, 98 ° C 5min, 5 ° C 5min.
[0057] PCR反应体系50 μL，包含反转录液10 yLPCR反应条件：94°C预变性97°C 变性208,64°(：退火208,72°0 208延伸，循环30次；72°(：延伸°(：恒定。取5以1?0財广增产物经1%琼脂糖凝胶电泳，紫外灯下凝胶成像。 [0057] PCR reaction was 50 μL, containing liquid 10 yLPCR reverse transcription reaction conditions: 94 ° C initi 97 ° C denaturation 208,64 ° (: 208,72 ° 0 208 extends annealing, 30 72 ° (: extending 5111 Shu 11,4 ° (:.? constant take 5 to 10 wide fiscal stimulation was subjected to 1% agarose gel electrophoresis, UV light gel imaging.
[0058] 8.透射电镜观察将大鼠部分肝、脾、脑、肾组织标本上取不同部位的3张切片进行透射电镜(JEM-100CXE 型）超微结构的观察，每张切片分别随机观察5-6个部位，并按相同放大倍数摄片，每组随机抽取10张照片，采用Epson微机和Summa Sketch Plus数字化仪，并使用Sigmascan软件， 对各组照片作电镜图像的形态进行分析，同时计数线粒体的平均数目。 [0058] 8. The TEM TEM (JEM-100CXE type) will observe the ultrastructure in rats with liver, spleen, brain, taken in different parts of the kidney tissue samples three slices, each slice were observed respectively 5-6 parts, according to the same magnification radiography, each group randomly selected 10 photos using Epson microcomputers and Summa Sketch Plus digitizer and use Sigmascan software, for each group of photos as a form of electron microscope images were analyzed at the same time the average count of the number of mitochondria.
[0059] 9.线粒体膜电位测定细胞(5X105个)接种于25cm2培养瓶中，37°C培养24 h后，加入不同浓度的姜黄素分别作用1 h和6 h后，收获细胞，PBS洗两遍，重悬于2ml含1.0 μΜ罗丹明123的新鲜培养液中，37°C水浴振摇孵育10 min，离心去掉细胞，荧光比色计测定培养液中罗丹明123的强度，激发波长490 nm，发射波长520 nm，结果以细胞吸收的罗丹明123的荧光强度表示。 After the [0059] 9. The mitochondrial membrane potential assay cells (5X105 months) were seeded in 25cm2 flasks, 37 ° C cultured 24 h, respectively, with different concentrations of curcumin for 1 h and 6 h, the cells were harvested, PBS washed two times, resuspended in 2ml containing 1.0 μΜ rhodamine 123 in fresh medium, 37 ° C water bath shaking incubated 10 min, centrifuged to remove the cells, the fluorescence intensity colorimetric assay culture medium rhodamine 123, excitation wavelength of 490 nm emission wavelength 520 nm, results in cellular uptake of rhodamine 123 fluorescence intensity indicates. [0060] 10.线粒体DNA含量测定将骨髓单个核细胞(5X107)、新鲜组织肝脏、脾、脑、肾匀浆、裂解、离心，获取线粒体。 [0060] 10. Determination of the mitochondrial DNA of bone marrow mononuclear cells (5X107), fresh tissue liver, spleen, brain, kidney homogenates, lysis, centrifugation, to obtain mitochondria. 按照DNA提取试剂盒说明提取线粒体中的DNA，采用紫外分光光度计测量其含量。 DNA extraction kit according to the instructions to extract mitochondrial DNA, UV spectrophotometer its content. 计算公式如下： DNA浓度(yg/yl)= A· X 50μg/ml X 稀释倍数X 10-3 11.病理学观察将大鼠部分肝、脾、脑、肾、肠、肌肉置于4 %多聚甲醛中固定，依次经过常规脱水、石蜡浸泡包埋、切片、HE染色，显微镜观察各组织细胞的形态结构。 Calculated as follows: DNA concentrations (yg / yl) = A · X 50μg / ml X dilution factor X 10-3 11. Pathological observation of the rats were part of the liver, spleen, brain, kidney, intestine, muscle in 4% paraformaldehyde fixed, passes through the conventional dehydration, soaked in paraffin-embedded sections, HE staining, microscopy morphological structures of the tissue cells.
[0061 ] 12.免疫组化染色石蜡切片脱蜡、水化;PBS洗2-3次各5分钟；3% H2〇2(80%甲醇)滴加在玻片上，室温静置10分钟;PBS洗2-3次各5分钟;抗原修复;PBS洗2-3次各5分钟；滴加正常山羊血清封闭液，室温20分钟。 [0061] 12. The immunohistochemical staining of paraffin sections were deparaffinized, PBS washed 3 times for 5 3% H2〇2 (80% methanol) was dropped on a glass slide at room temperature for 10 PBS wash 2-3 times for 5 PBS washed 3 times for 5 dropping normal goat serum blocking solution at room temperature for 20 minutes. 甩去多余液体，滴加一抗50μ1，4° C过夜。 Shake off the excess liquid, a solution of anti-50μ1,4 ° C overnight. 过夜后需在37° C复温45分钟。 After overnight at 37 ° C for an rewarming 45 minutes. PBS洗3次各5分钟；滴加二抗40-50μ1，室温静置，或37° C 1小时;PBS洗3次各5分钟;DAB显色5-10分钟，在显微镜下掌握染色程度;PBS或自来水冲洗10分钟;苏木精复染2分钟，盐酸酒精分化； 自来水冲洗10-15分钟;脱水、透明、封片、镜检。 PBS washed 3 times for 5 dropping two anti 40-50μ1, allowed to stand at room temperature or 37 ° C 1 X PBS washed 3 times for 5 DAB color for 5-10 minutes under a microscope to grasp th PBS or tap water 10 hematoxylin for 2 minutes and differentiation a tap water for 10-15 dehydrated, cleared, were mounted microscopy.
[0062] 13.流式细胞仪分析取大鼠左胫骨，去除表面肌肉组织，修剪骨垢，用18号针插入胫骨踝端，用PBS 5mL冲洗入试管内，以200目筛网过滤。 [0062] 13. Flow cytometry analysis The rat left tibia, remove surface muscle tissue, bone dirt trim with 18-gauge needle inserted into the ankle end of the tibia, rinsed with PBS 5mL into the test tube, to a 200 mesh screen filter. 1000 r/min离心5min，弃上清液。 1000 r / min centrifugal 5min, the supernatant was discarded. 将细胞加入5mL ?61^〇11分离液(相对比重1.0738凡），200〇1'/111；[11，离心25111；[11，取中间单个核细胞层。 The cells were added to 5mL 61 ^ 〇11 separation medium (where the relative weight of 1.0738), 200〇1 '/ 111;? [11, centrifugal 25111; [11, take the middle of the mononuclear cell layer. ？ ? 133洗涤后调整单个核细胞数为1 X 106/管，每样本3管，测定管加入FITC标记的⑶34或⑶45抗体， 37° C孵育2小时。 After washing adjustment 133 mononuclear cells of 1 X 106 / tube, three per sample, measuring tube FITC-labeled antibody ⑶34 or ⑶45, 37 ° C for 2 hours. 然后PBS洗涤一次。 Then washed with PBS once. 用流式细胞仪检测表达CD34/45阳性细胞数量变化，以标记抗体呈阳性的细胞百分率作为表达CD34/45蛋白（骨髓造血干细胞标志物）的计量标准。 Expression of CD34 / 45 positive cells by flow cytometry changes to the labeled antibody-positive cell percentage as an expression of CD34 / 45 protein (bone marrow hematopoietic stem cell marker) measurement standard. 同时以只加标记荧光素的羊抗兔IgG和不加抗体的细胞作阴性对照。 While only fluorescein tagged goat anti-rabbit IgG antibody-producing cells and without as negative control.
[0063] 14.蛋白芯片分析将大鼠部分骨髓单个核细胞、骨髓间充质干细胞、肝脏组织进行蛋白芯片分析，此部分工作由上海康城公司完成。 [0063] 14. A protein microarray analysis will be part of the rat bone marrow mononuclear cells, mesenchymal stem cells, protein microarray analysis of liver tissue, this part of the work done by the Shanghai Cannes company.
[0064] 15.统计学分析所有数据均用SPSS 17.0软件进行统计分析。 [0064] 15. All data were statistically analyzed using SPSS 17.0 software for statistical analysis. 每一项分析至少有三次结果。 There are at least three times each analysis result. 数值用均数土SD表示，采用t检验。 With a mean ± SD values, using t test. P〈0.05认为有显著性差异，并在统计学上有意义。 P &0.05 was considered significant difference, and statistically meaningful.
[0065]三、实验结果(一)营养组合物对再障大鼠全身情况的影响与正常对照组相比，再障模型组大鼠自6天起陆续出现皮毛松弛蓬乱，光泽度降低，部分大鼠有脱毛及皮损。 [0065] Third, the results affect (a) the nutritional composition of the general condition of aplastic anemia rats compared with normal control group, the AA model rats from 6 days onwards appear unkempt fur relaxation, reduce gloss, and some rat hair removal and skin lesions. 同时大鼠消瘦、精神渐显不佳，唇色眼睑苍白、活动减少、少食、体重下降。 Meanwhile rat weight loss, poor mental fade, lip pale eyelids, reduced activity, deprivation, weight loss. 其中再障模型组大鼠体重(218.23±33.52 g)与正常对照组(366.54±49.68 g，*p〈 0.05)相比下降明显。 Wherein the body weight of rats in AA model group (218.23 ± 33.52 g) and the normal control group (366.54 ± 49.68 g, * p &0.05) decreased significantly in comparison.
[0066] 不同剂量的营养组合物组与再障模型组比较，大鼠精神状态渐佳，进食量增大，体重增加(见图1)。 [0066] The nutritional composition of groups of different doses compared with the AA model group, the rats getting good mental state, increased food intake, weight gain (see Figure 1). 表明营养组合物对再生障碍性贫血小鼠全身有显著恢复作用。 It showed that the nutritional composition of aplastic anemia in mice have significant systemic effect recovery.
[0067] (二)营养组合物对再障大鼠外周血的影响与正常对照组相比，再障模型组外周血中红细胞（RBC)、白细胞（WBC)、血小板（PLT) 及血红蛋白（Hb)均显著下降（表1，#ρ〈0.05);不同剂量营养组合物组与再障模型组比较，其外周血中各项指标均显著升高（表2，*p〈0.05)，并呈量效依赖性。 [0067] (ii) the nutritional composition of the impact of aplastic anemia peripheral blood compared with normal control group, the AA model group in peripheral blood erythrocytes (RBC), white blood cell (WBC), platelet (PLT) and hemoglobin (Hb ) decreased significantly (table 1, # ρ &0.05); different doses of the nutritional composition group and AA model group, the peripheral blood of the indicators increased significantly (table 2, * p &0.05), and showed dose-dependent manner.
[0068]与正常对照组相比，再障模型组大鼠促红细胞生成素(ΕΡ0)含量明显升高，且ΕΡ0 与RBC、Hb的降低呈负相关，r=-0.91及r=-0.93(*/7〈0.05)，表明ΕΡ0水平与再障骨髓红系生成能力降低，ΕΡ0反应性能力减弱，ΕΡ0呈代偿性升高有关。 [0068] Compared with normal control group, the AA model rats erythropoietin (ΕΡ0) were significantly increased, and ΕΡ0 with RBC, Hb decrease negative correlation, r = -0.91 and r = -0.93 ( * / 7 &0.05), indicating ΕΡ0 levels and aplastic anemia bone marrow erythroid reduce generating capacity, reduced sexual response ΕΡ0, ΕΡ0 was compensatory elevated. （表1，*/7〈0 · 01) ΑΡ0是一种重要的造血调控因子，具有促进红系祖细胞增殖、分化和成熟，维持外周血中红细胞，血红蛋白恒定的作用，血液中ΕΡ0水平能反映机体中的红细胞生成能力。 (Table 1, * / 7 &0 · 01) ΑΡ0 hematopoietic regulation is an important factor, with the promotion of erythroid progenitor cell proliferation, differentiation and maturation of red blood cells in peripheral blood to maintain hemoglobin constant action, blood levels can ΕΡ0 reflect the body of the ability to produce red blood cells. 而不同剂量的营养组合物组与再障模型组比较，促红细胞生成素(Epo)由于代偿作用含量降低〈0.01);并呈剂量-效应关系。 The nutritional composition group with different doses of AA model group, erythropoietin (Epo) due to the compensatory effect of decreased &0.01); and a dose - response relationship.
[0069] 以上结果提示，营养组合物对再障大鼠外周血各种血细胞有促进升高作用。 [0069] These results suggest that the nutritional composition of aplastic anemia peripheral blood of various blood cells to promote increased role.
[0070] 表2营养组合物对再障大鼠外周血的影响 [0070] Table 2 of the nutritional composition of the peripheral blood of aplastic anemia affect
# P〈〇.05再障组与正常组;*P〈〇.05营养组合物组与再障组(三)本发明实施例营养组合物对再障大鼠基因损伤恢复的影响1.切除修复基因1.1核苷酸切除修复基因核苷酸切除修复（NER)通路被认为是机体内最主要及最重要的损伤修复通路，主要修复外源性物质导致的DNA损伤，例如嘧啶二聚体、光化合物及其它大的化合物及交联所导致的损伤。 # P &〇.05 aplastic anem * P &〇.05 nutritional composition group and AA group (iii) embodiment of the invention the nutritional composition of aplastic anemia gene Injury Recovery 1. Effect of removal 1.1 repair gene nucleotide excision repair gene nucleotide excision repair (NER) pathway is considered to be the most important and the most important body damage repair pathways, mainly foreign repair DNA damage caused by exogenous substances, such as pyrimidine dimers, light compounds and other large compound and cross-linking resulting damage. 本实验选取了3个NER基因（Erccl，Ercc2, Xpc)，从转录水平检测了其在营养组合物组中肝脏、骨髓以及脑中的表达变化(见图2)。 In this study, we selected three NER genes (Erccl, Ercc2, Xpc), to detect the level of transcription from its liver, bone marrow and expression in the nutritional composition group in the brain (see Figure 2).
[0071]在肝脏组织中，与正常对照组相比，再障模型组大鼠肝脏组织中核苷酸切除修复基因Xpc表达量下调；营养组合物处理再障大鼠后，大鼠肝脏组织中Xpc的表达量随营养组合物剂量增加而增加。 [0071] In the liver tissue compared with normal control group, the AA model group rat liver tissue of nucleotide excision repair gene expression X nutritional composition treatment of aplastic anemia rats, rat liver tissue Xpc the expression of the nutritional composition with dose increases. 核苷酸切除修复基因Erccl、ErCC2无明显变化。 Nucleotide excision repair gene Erccl, ErCC2 no significant change.
[0072]在骨髓中，与正常对照组相比，再障模型组大鼠骨髓造血细胞核苷酸切除修复基因Ercc2表达量下调；营养组合物处理再障大鼠后，大鼠骨髓造血细胞Ercc2的表达量随营养组合物剂量增加而增加。 [0072] in the bone marrow, compared with normal control group, the AA model group rat bone marrow hematopoietic cells in nucleotide excision repair gene expression Ercc2 nutritional composition treatment of aplastic anemia rats, rat bone marrow hematopoietic cells of Ercc2 The expression of the nutritional composition with dose increases. 核苷酸切除修复基因Erccl、XpC无明显变化。 Nucleotide excision repair gene Erccl, XpC no significant change.
[0073]在脑组织中，与正常对照组相比，再障模型组大鼠脑组织中核苷酸切除修复基因Ercc2表达量下调；营养组合物处理再障大鼠后，大鼠脑组织中Erccl的表达量随营养组合物剂量增加而增加。 [0073] in the brain, compared with normal control group, the AA model group rat brain tissue of nucleotide excision repair gene expression Ercc2 after the nutritional composition of aplastic anemia treated rats, rat brain Erccl the expression of the nutritional composition with dose increases. 核苷酸切除修复基因Erccl、Xpc无明显变化。 Nucleotide excision repair gene Erccl, Xpc no significant change.
[0074]结果表明本发明实施例营养组合物对再障大鼠核苷酸切除修复基因损伤具有一定的恢复作用。 [0074] The results show that the embodiment of the present invention, the nutritional composition of aplastic anemia rats nucleotide excision repair genetic damage have a certain role in the recovery.
[碱基切除修复基因碱基切除修复（base excision repair, BER)基因主要参与修复内源性烧化剂和外源性致癌剂如亚硝氨引起的DNA碱基的烷化以及细胞内自发的和由于电离辐射及紫外线辐射引起的DNA碱基的氧化。 [ base excision repair gene nucleotide excision repair (base excision repair, BER) genes mainly involved in the alkylation and burnt restored endogenous and exogenous agents carcinogens such as nitrosamines induced DNA base cell Since the spontaneous oxidation and ionizing radiation and ultraviolet radiation-induced DNA bases. 本实验选取了4个MR基因（Apexl，Oggl，Polb， MTH1)，从转录水平检测了其在营养组合物组中的表达变化(见图3)。 In this study, we selected four MR gene (Apexl, Oggl, Polb, MTH1), to detect the transcription level expression changes in the nutritional composition group (see Figure 3).
[0076]在肝脏组织中，各组碱基切除修复基因无明显变化。 [0076] In the liver, the base excision repair gene in each group had no significant change.
[0077]在骨髓中，与正常对照组相比，再障模型组大鼠骨髓造血细胞碱基切除修复基因0ggl、MTHl表达量下调；营养组合物处理再障大鼠后，大鼠骨髓造血细胞碱基切除修复基因0ggl、MTHl的表达量随营养组合物剂量增加而增加。 [0077] in the bone marrow, compared with normal control group, the AA model group rat bone marrow hematopoietic cells in base excision repair gene 0ggl, MTH nutritional composition treatment of aplastic anemia rats, rat bone marrow cells base excision repair gene 0ggl, with the expression level of the nutritional composition MTHl dose increases. 碱基切除修复基因Apexl、P〇lb无明显变化。 Base excision repair gene Apexl, P〇lb no significant change.
[0078]在脑组织中，与正常对照组相比，再障模型组大鼠骨髓造血细胞碱基切除修复基因0ggl、MTHl表达量下调；营养组合物处理再障大鼠后，大鼠骨髓造血细胞中碱基切除修复基因0ggl、MTHl的表达量随营养组合物剂量增加而增加。 [0078] in the brain, compared with normal control group, the AA model group rat bone marrow hematopoietic cells in base excision repair gene 0ggl, MTH after the nutritional composition of aplastic anemia treated rats, rat bone marrow cell base excision repair gene 0ggl, with the expression level of the nutritional composition MTHl dose increases. 碱基切除修复基因Apexl、P〇lb无明显变化。 Base excision repair gene Apexl, P〇lb no significant change.
[0079]结果表明本发明实施例营养组合物对再障大鼠碱基切除修复基因损伤具有一定的恢复作用。 [0079] The results show that the embodiment of the present invention, the nutritional composition of aplastic anemia rats base excision repair gene damage has a certain role in the recovery.
[0080] 2.错配修复基因DNA错配修复系统（MMR)是人体细胞的一种能修复DNA碱基错配的安全保障体系，由一系列特异性修复DNA碱基错配的酶分子（错配修复基因产物）组成。 [0080] 2. The mismatch repair gene DNA mismatch repair (MMR) is a kind of human cells can repair DNA base mismatch security system consists of a series of specific DNA base mismatch repair enzyme molecule ( mismatch repair gene product) components. 该系统能消除DNA合成错误，保持遗传物质的完整性和稳定性，避免遗传物质发生突变，保证DNA复制的忠实性。 The system can eliminate DNA synthesis error, to maintain the integrity and stability of the genetic material, to avoid the occurrence of mutations in the genetic material, to ensure the fidelity of DNA replication. 本实验选用最常用的4个错配修复基因MSH2、MSH3、MLH1、PMS2。 In this experiment, the most commonly used four mismatch repair genes MSH2, MSH3, MLH1, PMS2.
[0081] 在肝脏，骨髓以及脑组织中，与正常对照组相比，再障模型组大鼠中4个错配修复基因的蛋白表达量均下调；营养组合物处理再障大鼠后，大鼠肝脏，骨髓以及脑组织中错配修复基因的表达量随营养组合物剂量增加而增加(见图4)。 [0081] In the liver, bone marrow and brain tissue compared with normal control group, the AA model rats in four mismatch repair gene protein ex nutritional composition for the treatment of aplastic anemia rats, large rat liver, bone marrow and brain tissue in the wrong expression of mismatch repair gene is increased (see Fig. 4) with the nutritional composition dose is increased.
[0082] 结果表明本发明营养组合物对再障大鼠错配修复基因的损伤具有恢复作用。 [0082] The results showed that the nutritional composition of the present invention on rats with aplastic mismatch repair gene damage has restored effect.
[0083] 3.同源重组修复基因（双链断裂修复） 当细胞暴露在电离辐射、DNA交联剂及缺氧等各种致DNA损伤的条件下时，常出现染色体DNA双链断裂。 [0083] 3. homologous recombination repair genes (double-strand break repair) when cells are exposed to a variety of DNA damage induced by ionizing radiation conditions, DNA cross-linking agent and hypoxia, often chromosomal DNA double-strand breaks. 在真核生物细胞中，已知存在两种修复染色体DNA双链断裂的主要途径，即同源重组和非同源DNA末端连接。 In eukaryotic cells, known mainly two ways of chromosomal DNA double-strand break repair exists, namely homologous recombination and non-homologous DNA end joining. 准确的DNA修复是维持染色体完整性的前提，同源重组则在DNA修复过程中发挥关键作用。 Accurate chromosome DNA repair is to maintain the integrity of the premise, the homologous recombination plays a key role in the DNA repair process. 本实验选用最常用的4个同源重组修复基因Rad51、Rad52、Xrccl、Xrcc2〇在肝脏，骨髓以及脑组织中，与正常对照组相比，再障模型组大鼠同源重组修复基因Rad51、Rad52、Xrccl表达量均下调；营养组合物处理再障大鼠后，大鼠重组修复基因Rad51、 Rad52、Xrccl的表达量随营养组合物剂量增加而增加，而Xrcc2无明显变化(见图5)。 In this experiment, the most commonly used four homologous recombination repair gene Rad51, Rad52, Xrccl, Xrcc2〇 in the liver, bone marrow and brain tissue compared with normal control group, the AA model rats homologous recombination repair gene Rad51, Rad52, Xrccl ex nutritional composition treatment of aplastic anemia rats, rat recombination repair gene Rad51, Rad52, expression Xrccl increased with increasing dose of the nutritional composition, while Xrcc2 no significant change (see Figure 5) .
[0084]结果表明本发明实施例营养组合物对再障大鼠同源重组修复基因的损伤具有恢复作用。 [0084] The results show that the embodiment of the present invention, the nutritional composition of aplastic anemia rats homologous recombination repair gene damage has restored effect.
[0085] 4.错误倾向修复(S0S修复)基因当DNA两条链都有损伤并且损伤位点邻近时，损伤不能被切除修复或重组修复，这时在Restriction Endoneuclease和Exoneuclease的作用下造成损伤处的DNA链空缺，再由损伤诱导产生的一整套的特殊DNA聚合酶一S0S修复酶类，催化空缺部位DNA的合成，这时补上去的核苷酸几乎是随机的，仍然终于保持了DNA双链的完整性，使细胞得以生存。 When [0085] 4. The error-prone repair (S0S repair) gene when both strands of DNA damage and have near the site of injury, damage can not be repaired or recombination excision repair damage at this time in the role of Restriction Endoneuclease and Exoneuclease DNA strand vacancy, then the damage induced by a set of specific DNA polymerase a S0S repair enzymes catalyze the synthesis of DNA vacant site, then fill up almost random nucleotides, still holding the DNA double finally the integrity of the chain of the cells to survive. 本实验选用最常用的两个S0S修复基因RecA、LexA。 In this experiment, the most commonly used two S0S repair genes RecA, LexA.
[0086]在肝脏，骨髓组织中，与正常对照组相比，再障模型组大鼠SOS修复基因RecA表达量均下调；营养组合物处理再障大鼠后，大鼠S0S修复基因RecA的表达量随营养组合物剂量增加而增加。 [0086] In the liver, bone marrow tissues compared with normal control group, the AA model group SOS repair RecA gene ex nutritional composition after treatment of aplastic anemia rats, rat S0S repair gene expression of RecA nutritional composition increases with dose increases. S0S修复基因LexA无明显变化(见图6)。 S0S repair gene LexA no significant changes (see Figure 6).
[0087] 在脑组织中，各组之间S0S修复基因无明显变化。 [0087] in the brain, between the groups S0S repair genes did not change.
[0088] 结果表明本发明实施例营养组合物对再障大鼠S0S修复基因的损伤具有恢复作用。 [0088] The results show that the embodiment of the present invention, the nutritional composition of aplastic anemia rats S0S repair damaged genes have recovery effect.
[0089] (四）营养组合物对再障大鼠线粒体的影响1.线粒体形态观察肝组织(汇管区）透射电镜显示（图7)，与正常对照组相比，再障模型组大鼠肝细胞内线粒体数目明显减少，嵴不清楚或消失，细胞浆疏松;不同剂量的营养组合物组与再障模型组比较，大鼠肝细胞内线粒体数目明显增多，胞浆逐渐恢复正常。 Effect [0089] (d) the nutritional composition of aplastic mitochondrial mitochondrial morphology of liver tissue (periportal) Transmission electron microscopy revealed (Fig. 7), compared with normal control group, the AA rat liver model group the number of mitochondria within cells significantly reduced crest unclear or disappeared, the nutritional composition group with different doses of AA model group, the number of mitochondria in rat liver cells increased significantly, the cytoplasm gradually returned to normal.
[0090] 脾透射电镜显示（图8)，与正常对照组相比，再障模型组大鼠脾脏B细胞内线粒体数目明显减少;不同剂量的营养组合物组与再障模型组比较，细胞内线粒体数目明显增多。 [0090] Transmission electron microscopy revealed the spleen (Fig. 8), compared with normal control group, the number of B cells in the spleen cells of rats AA model group dec different doses of nutritional composition group was AA model group, intracellular The number of mitochondria increased significantly.
[0091] 肾小管上皮细胞透射电镜显示（图9)，与正常对照组相比，再障模型组大鼠肾小管上皮细胞内线粒体数目明显减少，排列紊乱;不同剂量的营养组合物组与再障模型组比较， 肾小管基底部上皮细胞线粒体数目明显增多，排列整齐。 [0091] Transmission electron microscopy revealed renal tubular epithelial cells (Fig. 9), compared with normal control group, the rat renal tubular epithelial cells in AA model group significantly reduced the number of mitochondria, different doses of nutritional composition and re-composition group AA model group, the bottom of the tubular base epithelial cells increased the number of mitochondria, arranged in neat rows.
[0092] 脑透射电镜显示（图10)，与正常对照组相比，再障模型组大鼠脑海马部神经元细胞内线粒体数目明显减少;不同剂量的营养组合物组与再障模型组比较，大鼠脑细胞内线粒体数目略有增多。 [0092] Transmission electron microscopy revealed the brain (Figure 10), compared with normal control group, the AA model rats brain hippocampal neurons unit significantly reduce the nu the nutritional composition group with different doses of AA model group , rat brain cells slightly increased the number of mitochondria.
[0093]结果表明本发明实施例营养组合物可促进再障大鼠肝细胞线粒体、脾脏B细胞线粒体、肾小管上皮细胞线粒体及脑海马部神经元细胞线粒体的增加。 [0093] The results show that the embodiment of the invention the nutritional composition may promote mitochondrial aplastic anemia, splenic B cells mitochondria, increased renal tubular epithelial cell mitochondria and hippocampus neurons in rat liver mitochondria.
[0094] 2.线粒体膜电位测定线粒体膜电位是观察线粒体功能的重要指标，线粒体功能的改变多表现为膜电位降低和膜的不稳定。 [0094] 2. Determination of mitochondrial membrane potential mitochondrial membrane potential is an important indicator to observe mitochondrial function, altered mitochondrial function showed more membrane potential and membrane instability. 采用荧光探针罗丹明123(Rhl23)测定营养组合物对线粒体膜电位的影响。 Fluorescent probes rhodamine 123 (Rhl23) affect the nutritional composition of the mitochondrial membrane potential was measured. Rhl23为亲脂性阳离子荧光素，能顺利通过活细胞的细胞膜和线粒体膜，进入线粒体后，由于线粒体膜的负电位差使Rhl23选择性富集在线粒体上，胞浆中结合的非常少。 Rhl23 lipophilic cationic fluorescein, can go through the cell membrane and mitochondrial membrane of living cells into the mitochondria, due to the negative potential of the mitochondrial membrane errand Rhl23 selective enrichment in mitochondria, cytoplasm bound very little. 线粒体对Rhl23的摄入量取决于其膜电位的高低，一定的膜电位形成一定的荧光强度，成正比，强度的大小反映线粒体膜受损程度。 For Rhl23 intake depends on the level of mitochondrial membrane potential, membrane potential in certain a certain fluorescence intensity is proportional to the intensity of the size of the mitochondrial membrane to reflect the extent of damage.
[0095]与正常对照组相比，再障模型组大鼠骨髓造血细胞、肝细胞及脾细胞内线粒体膜电位呈明显下降趋势，表明线粒体功能缺失(表2);不同剂量的营养组合物组与再障模型组比较，大鼠骨髓造血细胞、肝细胞及脾细胞内线粒体膜电位明显增高，呈量效关系(表3)。 [0095] Compared with normal control group, model group aplastic bone marrow hematopoietic cells, liver cells and spleen cells mitochondrial membrane potential was significantly decreased, suggesting that mitochondrial deficits (Table 2); nutritional composition group was different doses compared with the AA model group, rat bone marrow cells, liver cells and spleen cells mitochondrial membrane potential was significantly increased in a dose dependent (table 3).
[0096] 表3 [0096] Table 3
# p〈0.05再障组与正常组;*p〈0.05营养组合物组与再障组与正常对照组相比，再障模型组大鼠脑、肾细胞线粒体膜电位减少（图11)，但无显著差异;不同剂量的营养组合物组与再障模型组比较，大鼠脑、肾细胞线粒体膜电位略有增加， 但无显著差异（图11)。 # P &0.05 aplastic anem * p &0.05 and the nutritional composition group AA group compared with the control group, the model group rat brain aplastic anemia, renal cell mitochondrial membrane potential decrease (Fig. 11), but There was no si the nutritional composition group with different doses of AA model group, cerebral, renal cell mitochondrial membrane potent