帮助我翻译成日语一下谢谢了
在沪江关注日语的沪友yeqiangfeng遇到了一个关于的疑惑，并悬赏200沪元，已有3人提出了自己的看法。
知识点疑惑描述：
我的家乡处于沿海地区，地势平坦，近年来，建立了不少工厂、企业，呈现一派繁荣景象。然而环境污染成了一个很大的问题。工厂排出的烟雾，以及排放的污水严重影响了我们的生活。为此，我很想进入大学学习环保方面的专业知识。
但是，由于中国还处于发展中国家，对环境污染方面没有比较完善的治理方案。而日本国是个科学技术非常发达的国家，日本政府特别注重自然资源的保护来维持生态平衡。在废品的处理上投入大量的人力、物力与财力，以减少对环境的严重污染。日本国在环保方面的经验很值得我们学习和借鉴。因此，我认为到日本国学习这方面的专业知识将比在国内学习更好。
为了赴日留学打下语言基础，我曾在国内学习一些初级日本语，但我知道这还不能达到上大学所需的国际一级水平。因此我计划先到东京国际文化学院学习一年左右的日本语，然后尽一切努力考入东京农业大学地域环境科学部森林综合科学科学习专业知识。
大学毕业后，我将立即回国，利用所学知识，争取在环保事业方面为社会做出一点贡献。
最佳知识点讲解
知识点相关讲解
我的家乡处于沿海地区，地势平坦，近年来，建立了不少工厂、企业，呈现一派繁荣景象。然而环境污染成了一个很大的问题。工厂排出的烟雾，以及排放的污水严重影响了我们的生活。为此，我很想进入大学学习环保方面的专业知识。
但是，由于中国还处于发展中国家，对环境污染方面没有比较完善的治理方案。而日本国是个科学技术非常发达的国家，日本政府特别注重自然资源的保护来维持生态平衡。在废品的处理上投入大量的人力、物力与财力，以减少对环境的严重污染。日本国在环保方面的经验很值得我们学习和借鉴。因此，我认为到日本国学习这方面的专业知识将比在国内学习更好。
为了赴日留学打下语言基础，我曾在国内学习一些初级日本语，但我知道这还不能达到上大学所需的国际一级水平。因此我计划先到东京国际文化学院学习一年左右的日本语，然后尽一切努力考入东京农业大学地域环境科学部森林综合科学科学习专业知识。
大学毕业后，我将立即回国，利用所学知识，争取在环保事业方面为社会做出一点贡献。
私の郷里は沿海地区があって、地形は平坦で、ここ数年来、多くの工場、企業を創立して、全くの繁栄のありさまが現れます。しかし環境汚染は１つのとても大きい問題になった。工場の排除する煙霧、および排出の汚水が深刻で私達の生活に影響しました。このために、私はとても大学に入って環境保護の方面の専門の知識を学びたいです。
しかし、中国がまた発展途上国があるため、環境汚染の方面は比較的に整った管理案ですに対して。日本国は科学技術のとても発達している国家で、日本政府は特に自然資源の保護することを重視して生態のバランスを維持しにきます。不合格品の処理の上で大量の人力、物資と財力に入って、減らすこと環境に対する深刻な汚染で。日本国の環境保護の方面の経験のとても値打ちがある私達は学んで参考にします。そのため、私は日本国まで(へ)この方面の専門の知識を学ぶのが国内の学習にありますよりもっと良いと思っています。
日本へ留学するために言語の基礎を築いて、私はかつて国内の学習のいくつかの初級日本語で、しかし私はこれがまだ大学の必要な国際1級のレベルを達成することができないことを知っています。そのため私は先に東京国際文化の学院に着いて1年ぐらいの日本語を学ぶと計画して、それからすべて努力して試験して東京農業大学の地域の環境の科学部森林習専業の知識に入ることを総合的に科学的です尽くします。
大学の卒業の後で、私は直ちに帰国して、勉強した事の知識を利用して、環境保護事業の方面で社会のために1時(点)の貢献をすることを努力します。
—— slfx777
我的家乡处于沿海地区，地势平坦，近年来，建立了不少工厂、企业，呈现一派繁荣景象。然而环境污染成了一个很大的问题。工厂排出的烟雾，以及排放的污水严重影响了我们的生活。为此，我很想进入大学学习环保方面的专业知识。
私の故郷は沿海地区にあり、地勢は平坦です。そのため、ここ数年、工場や企業は多く建てられ、繁栄な模様ですが、環境問題も深刻になっています。工場の排出している煙、汚水は我々の生活に深く影響しています。ですので、大学に入って、環境保護方面の知識を学びたいと思っています。
但是，由于中国还处于发展中国家，对环境污染方面没有比较完善的治理方案。而日本国是个科学技术非常发达的国家，日本政府特别注重自然资源的保护来维持生态平衡。在废品的处理上投入大量的人力、物力与财力，以减少对环境的严重污染。日本国在环保方面的经验很值得我们学习和借鉴。因此，我认为到日本国学习这方面的专业知识将比在国内学习更好。
しかし、中国はまだ発展途上国ですので、環境汚染を処理するのに、完璧な方案はまだないです。日本の科学技術は発展していて、自然資源への保護を通して生態のバランスを取ることを重視しています。廃棄物の処理にも人力、財力を入れて、汚染問題をコントロールしています。環境保護方面の経験は学ぶ値打ちがあると思いますので、日本で環境保護関係の専攻をしたほうがより多くの知識を学べるでしょうと思っています。
为了赴日留学打下语言基础，我曾在国内学习一些初级日本语，但我知道这还不能达到上大学所需的国际一级水平。因此我计划先到东京国际文化学院学习一年左右的日本语，然后尽一切努力考入东京农业大学地域环境科学部森林综合科学科学习专业知识。
日本に留学するために、中国で初級日本語を勉強していますが、まだ大学の受験レベルの日本語能力試験の１級にはなっていないことが分っています。ですので、東京国際文化学院に１年間の日本語を勉強して、それから、力を尽くして、東京農業大学の地域環境科学部の森林総合学科の受験をしたいです。
大学毕业后，我将立即回国，利用所学知识，争取在环保事业方面为社会做出一点贡献。
大学卒業後、帰国し、学んだ知識で社会の環境保護事業に貢献したいと思います。
—— syunnii
私の家は、沿岸地域で、うそをついてフラット近年では、多くの工場、企業所、 1つのグループの繁栄を示しています。しかし、環境汚染が大きな問題となる。たばこの煙から工場や汚水排出私たちの生活に深刻な影響を与える。そのためには、私が大学を入力するような環境側面の専門知識を勉強します。
しかし、中国はまだ発展途上国、環境汚染にはまだ完全なガバナンスのプログラムです。の独立行政法人科学技術は非常に先進国、日本政府は特別な天然資源の保護に重点を置いて、生態系のバランスを維持する。廃棄物の処理は、かなりの人間の、素材や金融のリソースを削減するために環境への深刻な公害です。日本では、環境面での経験は相当学習と描画します。したがって、日本を勉強すると思うが、この種の専門知識を学ぶ改善している。
日本を勉強するの言語の基礎を築くために、いくつかの主要な国内の日本語を学ぶが、私も知っているこの上で成し遂げることはできない、国際レベルの大学レベルです。したがって、私は計画の第一回東京国際文化研究所約1年の学習日本語、と入力し、最大限の努力を認めては、東京都大学の農業と地域環境科学部森林総合科学研究の専門知識です。
大学を卒業した後、私はすぐに帰国、知識を使用しては、環境保護のために戦うのが原因で発生するため、コミュニティを作ることに貢献します。
—— aicathy
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BioMed&Central
Page&1&of&12
(page&number&not&for&citation&purposes)
BMC&Genomics
Research&article&Open&Access
Differential&gene&expression&profile&in&pig&adipose&tissue&treated
with/without&clenbuterol
Jin&Zhang1,2,&Qiang&He1,&Qiu&Y&Liu1,&Wei&Guo1,&Xue&M&Deng3,&Wei&W&Zhang1,
Xiao&X&Hu*1&and&Ning&Li*1
Address:&1State&Key&Laboratory&for&Agrobiotechnology,&China&Agricultural&University,&Beijing&100094,&China,&2Life&Science&and&Biotechnology
Department,&HeBei&Normal&University&of&Science&&&Technology,&Qinhuangdao,&066600,&China&and&3College&of&Animal&Science&and&Technology,
China&Agricultural&University,&Beijing&100094,&China
Email:&Jin&Zhang&-&;&Qiang&He&-&;&Qiu&Y&Liu&-&;&Wei&Guo&-&wguo2@wisc.
Xue&M&Deng&-&Deng@;&Wei&W&Zhang&-&;&Xiao&X&Hu*&-&huxx@;
Ning&Li*&-&ninglbau@public3.
*&Corresponding&authors
Abstract
Background:&Clenbuterol,&a&beta-agonist,&can&dramatically&reduce&pig&adipose&accumulation&at&high&dosages.&However,
it&has&been&banned&in&pig&production&because&people&who&eat&pig&products&treated&with&clenbuterol&can&be&poisoned&by
the&clenbuterol&residues.&To&understand&the&molecular&mechanism&for&this&fat&reduction,&cDNA&microarray,&real-time
PCR,&two-dimensional&electrophoresis&and&mass&spectra&were&used&to&study&the&differential&gene&expression&profiles&of
pig&adipose&tissues&treated&with/without&clenbuterol.&The&objective&of&this&research&is&to&identify&novel&genes&and
physiological&pathways&that&potentially&facilitate&clenbuterol&induced&reduction&of&adipose&accumulation.
Results:&Clenbuterol&was&found&to&improve&the&lean&meat&percentage&about&10&percent&(P&&&0.05).&The&adipose&cells
became&smaller&and&the&muscle&fibers&became&thicker&with&the&administration&of&clenbuterol.&The&mRNA&abundance
levels&of&82&genes&(ESTs)&were&found&to&be&statistically&differentially&expressed&based&on&the&Student&t-test&(P&&&0.05)&in
the&microarray&analyses&which&contained&3358&genes&(ESTs).&These&82&genes&(ESTs)&were&divided&into&four&groups
according&to&their&Gene&Ontology&Biological&Process&descriptions.&16&genes&were&cellular&metabolism&related&genes
(including&five&related&to&lipid&metabolism&such&as&apolipoprotein&D&and&apolipoprotein&R),&10&were&signal&transduction
related&genes,&45&were&expressed&sequence&tags&(ESTs)&and&11&others&were&of&various&categories.&Eleven&of&the&82&genes
(ESTs)&were&chosen&for&real-time&PCR&analysis,&with&eight&genes&showing&similar&induction&magnitude&as&that&seen&in&the
microarray&data.&Apolipoprotein&R&was&also&found&to&be&up-regulated&by&the&proteomic&analysis.
Conclusion:&Pig&fat&accumulation&was&reduced&dramatically&with&clenbuterol&treatment.&Histological&sections&and&global
evaluation&of&gene&expression&after&administration&of&clenbuterol&in&pigs&identified&profound&changes&in&adipose&cells.&With
clenbuterol&stimulation,&adipose&cell&volumes&decreased&and&their&gene&expression&profile&changed,&which&indicate&some
metabolism&processes&have&been&also&altered.&Although&the&biological&functions&of&the&differentially&expressed&genes&are
快试试吧，可以对自己使用挽尊卡咯~◆◆
not&completely&known,&higher&expressions&of&these&molecules&in&adipose&tissue&might&contribute&to&the&reduction&of&fat
accumulation.&Among&these&genes,&five&lipid&metabolism&related&genes&were&of&special&interest&for&further&study,&including
apoD&and&apoR.&The&apoR&expression&was&increased&at&both&the&RNA&and&protein&levels.&The&apoR&may&be&one&of&the
critical&molecules&through&which&clenbuterol&reduces&fat&accumulation.
Published:&26&November&2007
BMC&Genomics&&doi:10.64-8-433
Received:&8&February&2007
Accepted:&26&November&2007
This&article&is&available&from:&
&&2007&Zhang&et&&licensee&BioMed&Central&Ltd.
This&is&an&Open&Access&article&distributed&under&the&terms&of&the&Creative&Commons&Attribution&License&(,
which&permits&unrestricted&use,&distribution,&and&reproduction&in&any&medium,&provided&the&original&work&is&properly&cited.
BMC&Genomics&&
Page&2&of&12
(page&number&not&for&citation&purposes)
Background
The&β2-agonist&clenbuterol&(0.8–3.2&μg/kg&body&weight
twice&daily)&is&used&as&a&bronchodilator&for&the&treatment
of&asthma&in&humans&and&as&a&bronchodilator&as&well&as&a
tocolytic&agent&in&veterinary&medicine&[1].&In&the&past&decade,
high&dosages&of&clenbuterol&(ten&to&one&hundred
times&the&clinically&active&dose)&have&been&fed&to&livestock
to&improve&feed&conversion,&reduce&body&fat&and&increase
muscle&mass&[2,3].&However,&people&who&eat&the&animal
products&can&be&poisoned&by&the&clenbuterol&residues&[4-
6].&Therefore,&the&use&of&clenbuterol&for&growth&promotion
in&food-producing&animals&is&not&approved&within&China,
the&European&community,&the&United&States,&and&most
other&countries&[2,7].
Clenbuterol&influences&cell&metabolism&by&combining
with&β2-adrenergic&receptors&and&by&increasing&the&cAMP
concentration&in&cells.&In&adipocytes,&stimulation&of&β-
adrenergic&receptors&(by&hormones)&increases&cyclic&AMP
levels&and&activates&protein&kinase&A&(PKA),&which&stimulates
lipolysis&by&phosphorylating&hormone-sensitive
lipase&and&perilipin&[8-11].&However&the&molecular&level
mechanism&by&which&clenbuterol&influences&adipose
accumulation&is&still&not&understood.
Recently,&global&gene/protein&expression&analysis&techniques
using&DNA&microarray/2-D&gel&analyses&have&been
widely&used&to&define&the&characteristics&and&specific&patterns
of&gene&expressions&elicited&by&various&toxicants&[12-
14].&In&this&study,&the&molecular&level&mechanism&by
which&clenbuterol&reduces&fat&accumulation&was&studied
with&cDNA&microarray&and&proteomics&techniques&to&analyze
the&fat&tissue&of&Chinese&miniature&pigs&treated&with/
without&clenbuterol.
Results
Adipose&accumulation&decreased&dramatically&by
clenbuterol&administration
HPLC&analyses&of&blood&samples&showed&that&the&clenbuterol
concentrations&in&the&test&pigs&fed&with&clenbuterol
were&about&20&ng/ml&in&the&3&month-old&pigs&and
about&100&ng/ml&in&the&4&month-old&pigs.&Clenbuterol
快试试吧，可以对自己使用挽尊卡咯~◆◆
could&not&be&detected&in&the&control&pigs&fed&without&clenbuterol
(Additional&file&1&Table&S1).&The&test&pigs&and&control
pigs&did&not&exhibit&different&weights&(Additional&file
1&Table&S2),&but&did&exhibit&different&body&compositions
(Table&1).&The&effect&of&clenbuterol&on&body&composition
became&more&dramatic&with&advancing&age.&In&the&3
month-old&group,&the&lean&meat&percentage&was&increased
by&2%,&the&back&fat&thickness&was&reduced&by&~0.2&cm,&and
the&loin&muscle&area&was&reduced&by&4.7&cm2.&This&difference
was&statistically&significant&(P&&&0.05).&In&the&4
month-old&group,&the&lean&meat&percentage&was&increased
by&10.99%,&the&back&fat&thickness&was&reduced&by&0.38&cm
and&the&loin&muscle&area&was&reduced&by&2.18&cm2.&The
changes&of&the&lean&meat&percentage&and&the&loin&muscle
area&were&strongly&statistically&significant&(P&&&0.01)&while
the&changes&of&the&back&fat&thickness&were&statistically&significant
(P&&&0.05).&These&data&indicate&that&clenbuterol
plays&a&role&in&pig&adipose&reduction.&The&sample&quality
was&sufficient&for&further&analysis&to&identify&molecules
with&changed&expression&levels&and&molecules&which
impact&adipose&accumulation.
Muscle&fibers&became&thicker&and&adipose&cells&became
smaller&with&the&administration&of&clenbuterol
Clenbuterol&produces&specific&protein&anabolic&effects&in
skeletal&muscle&in&addition&to&lipolysis&in&adipose&tissue&of
various&vertebrates&[15-18].&However,&there&has&not&yet
been&any&research&explaining&how&these&two&types&of&tissues
are&changed&by&the&administration&of&clenbuterol.
Muscle&and&adipose&histological&sections&were&analyzed&to
understand&how&these&two&tissue&types&are&changed&(Figure
1A&&1B).
Paraffin&histological&section&analyses&were&done&using
samples&from&the&4&month-old&pig's&biceps&femoris.&Four
histological&section&slides&were&prepared&for&each&pig,&two
transverse&muscle&fiber&slides&and&two&longitudinal&muscle
fiber&slides,&for&analysis&of&the&cross-sectional&area&of
the&fibers&with&a&TD2000&real-color&pathology&image&analysis
system&(Beijing&Tiandibainian&Scientific&Company,
Ltd.).&The&cross-sectional&areas&of&the&muscle&fibers&of&the
test&pigs&and&the&control&pigs&were&significantly&different
Table&1:&Body&composition&of&pigs&treated&with/without&clenbuterol
Age&3&month-old&3&month-old&4&month-old&4&month-old
Clenbuterol&dosage*&0&25&0&50
Pig&Hog&2&Sow&2&Hog&1&Sow&1&Hog&4&Sow&4&Hog&3&Sow&3
Lean&meat&percentage&of&carcass&(%)&52.34&52.46&55.09&55.39&41.38&41.52&52.36&52.52
Thickness&of&back&fat&(cm)&2.361&2.311&2.132&2.104&3.229&3.171&2.824&2.812
Loin&muscle&area&(cm2)&19.460&19.516&24.086&24.312&23.063&23.103&25.228&25.312
*twice&daily&with&the&unit&of&(mg/kg&body&weight)
The&carcass&lean&meat&percentage,&the&back&fat&thickness&and&the&loin&muscle&area&were&all&changed&statistically&in&both&the&3&month-old&group&and
the&4&month-old&group&after&clenbuterol&treatment&(P&&&.05).
BMC&Genomics&&
Page&3&of&12
(page&number&not&for&citation&purposes)
with&a&student&t-test&P&value&of&0.0224&(Additional&file&1
快试试吧，可以对自己使用挽尊卡咯~◆◆
Table&S3).&The&results&show&that&the&administration&of
clenbuterol&increased&the&muscle&fiber&thicknesses&in&the
pigs.
The&4&month-old&pig's&back&fat&tissue&(at&the&fifth&lumbar
vertebra&level)&was&also&analyzed&using&paraffin&histological
section.&Five&random&areas&on&the&slides&were&chosen
for&the&adipose&cell&size&analyses.&The&cell&sizes&were&analyzed
by&counting&all&the&cells&on&the&slides&visible&through
the&microscope&eyepiece&at&the&10&×&40&magnification
(Additional&file&1&Table&S4).&Numbers&of&cells&in&the&test
group&were&strongly&statistically&more&than&in&the&control
group&(P&&&0.01).&Thus,&clenbuterol&caused&a&reduction&in
adipocyte&size.
The&clenbuterol&thickened&the&pig&muscle&fibers&and
reduced&the&sizes&of&the&pig&adipocyte&cells&in&the&back&fat
tissues.&Clenbuterol&is&known&to&increase&muscle&mass&and
reduce&body&fat.&We&suggest&that&clenbuterol&increases&the
muscle&mass&by&thickening&the&muscle&fibers&and&reduces
the&body&fat&by&shrinking&the&adipose&cells.&The&size&of&the
adipose&cells&depends&on&the&sizes&of&the&lipid&droplets&in
the&cells.&Therefore,&the&adipose&cells&become&smaller&as
the&clenbuterol&reduces&the&lipid&droplets&in&the&adipose
cells.
cDNA&microarray&identified&82&genes&with&changed&mRNA
abundance&in&adipose&tissue&with&stimulation&by
clenbuterol
Eight&microarray&slides&were&used&(four&for&the&3&monthold
group&and&four&for&the&4&month-old&group)&for&global
evaluation&of&the&gene&expression&in&the&adipose&tissue
after&administration&of&clenbuterol.&8335&spots&representing
2770&genes&(ESTs)&in&the&3&month-old&group&and&8740
spots&representing&2862&genes&(ESTs)&in&the&4&month-old
group&passed&the&spots&quality&filter&and&were&analyzed
with&the&Student&t&test.&507&genes&in&the&3&month-old
1A.&Skeleton&muscle&Figure&1&(biceps&femoris)&histological&section&of&pigs&with/without&the&administration&of&clenbuterol
1A.&Skeleton&muscle&(biceps&femoris)&histological&section&of&pigs&with/without&the&administration&of&clenbuterol.
1.&transverse,&test&pigs.&2.&transverse,&control&pigs.&3.&longitudinal,&test&pigs.&4.&longitudinal,&control&pigs.&Vertical&fiber
sections&analyzed&with&TD2000&real-color&pathology&image&analysis&system&(Beijing&Tiandibainian&Scientific&Company&Ltd.).&The
muscle&fibers&of&pigs&become&thicker&when&treated&with&clenbuterol.&1B.&Subcutaneous&back&fat&(at&the&fifth&lumbar&vertebra
level)&histological&section&of&pigs&with/without&the&administration&of&clenbuterol.&1.&amplified10&×&10&of&test
pigs.&2.&amplified&10&×&10&of&control&pigs.&3.&amplified&10&×&40&of&test&pigs.&4.&amplified&10&×&40&of&control&pigs.&The&sizes&of&the
pig&adipose&cells&decreased&when&treated&with&clenbuterol.&The&cell&size&was&analyzed&by&counting&cells&on&the&slide&visible
through&the&microscope&eyepiece.
1&2&3&4
10&#&10&#&10&#&10&#
1&2&3&4
10&#&10&#&10&#&10&#
BMC&Genomics&&
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快试试吧，可以对自己使用挽尊卡咯~◆◆
group&(P&&&0.05&in&four&microarray&slides)&and&336&genes
in&the&4&month-old&group&(P&&&0.05&in&four&microarray
slides)&were&differentially&expressed&(data&not&shown).&The
goal&of&this&study&was&to&identify&gene&expression&profiles
affected&by&clenbuterol.&Therefore,&genes&that&were&differentially
expressed&in&both&groups&(82&genes&in&total)&were
selected&for&further&study&as&being&differentially&expressed
in&adipose&tissue&with&stimulation&by&clenbuterol&(Fig.&2).
These&82&genes&(ESTs)&were&divided&into&4&groups&according
to&their&Gene&Ontology&Biological&Process&descriptions.
16&genes&were&cellular&metabolism&related&genes
(Table&2),&10&were&signal&transduction&related&genes
(Table&3),&45&were&expressed&sequence&tags&(ESTs)&(Table
4)&(no&homologous&sequences&were&found&in&the&NCBI
nucleotide&database&for&17&ESTs,&while&the&other&28&ESTs
hit&only&some&EST&sequences&without&any&functional
annotations)&and&the&other&11&genes&were&of&various&categories
(Table&5).&45&EST&sequences&were&deposited&at
dbEST&of&NCBI&[19].
73%&cDNA&microarray&results&confirmed&by&real-time&PCR
Seven&differentially&expressed&genes&(positive&genes)&were
chosen&for&real-time&PCR&analysis&with&the&4&month-old
group&samples&used&to&validate&the&microarray&data&(Table
6).&Clone&rpfat_18309&and&rpfat_19990&representing&proalpha-
1&type&3&collagen&were&not&detected&by&the&real-time
PCR.&The&other&five&clones,&for&genes&of&apoD&(apolipoprotein
D),&PRKAR1A&(cAMP&dependent&protein&kinase
type&I&regulatory),&COL1A2&(pro-alpha-2&chain&of&type&I
procollagen)&and&COL1A1&(prepro-alpha1(I)&collagen),
showed&a&statistically&significant&increase&in&mRNA&abundance
with&administration&of&clenbuterol&(P&&&0.05).
The&SCD&(stearoyl-CoA&desaturase)&and&HSL&(hormonesensitive
lipase)&genes&are&very&important&in&lipid&metabolism.
The&mRNA&abundance&of&stearoyl-CoA&desaturase
(SCD)&decreased&in&the&3&month-old&group&and&increased
in&the&4&month-old&group,&while&HSL&was&not&significantly
differentially&expressed&by&stimulation&with&clenbuterol&in
the&microarray&analysis.&PMP22&(peripheral&myelin&protein
22)&and&PHPT1&(phosphohistidine&phosphatase&1)
were&also&not&significantly&differentially&expressed&by
stimulation&with&clenbuterol&in&the&microarray&analysis.
These&four&genes&(negative&genes)&were&not&significantly
differentially&expressed&by&the&real-time&PCR&analysis&with
8&pig&samples&(P&&&0.05)&(Table&7).&In&total,&11&genes&were
analyzed&by&the&real-time&PCR.&Eight&genes&showed&similar
induction&magnitude&as&that&seen&in&the&microarray
&two&could&not&be&detected,&and&one&was&inconsistent
with&the&microarray&results.&Thus,&these&results&provide
strong&biological&validation&of&the&results&from&the&microarray
experiment.
Apolipoprotein&R&protein&highly&presented&in&adipose&with
the&administration&of&clenbuterol
After&spot&detection,&background&subtraction&and&volume
normalization,&600&±&50&protein&spots&were&detected&in
adipose&cells&using&two-dimensional&electrophoresis
methods.&Two&spots&found&to&be&only&expressed&in&the&test
快试试吧，可以对自己使用挽尊卡咯~◆◆
group&were&chosen&for&digestion&in-gel&for&peptide&mass
fingerprint&(PMF)&analysis&with&a&mass&spectrograph&(Fig.
3A&&3B).&A&Mascot&search&using&the&PMF&data&matched
seven&of&the&peptides&with&peptides&from&apolipoprotein
R,&with&a&sequence&coverage&of&36%&and&an&expectation&of
0.00043&[20].&The&other&differentially&expressed&protein
did&not&give&any&positive&results&in&the&database&search.
Discussion
To&identify&the&genes&responding&to&clenbuterol&treatment
in&adipose&tissue,&a&cDNA&microarray,&real-time&PCR&and
2-dimensional&protein&gel&analysis&were&used.&82&genes
were&identified&as&being&differentially&expressed&by&the
microarray&analysis&with&student&t&test&(p&&&0.05).&These
were&categorized&into&to&four&groups.
Lipid&metabolism&related&genes&in&group&I
16&differentially&expressed&genes&are&involved&in&cellular
metabolism&(Table&2).&Five&of&them,&including&apoD
(apolipoproteinD),&apoR&(apolipoprotein&R),&HAD&(L-3-
hydroxyacyl-CoA&dehydrogenase&precursor),&PAP&type&2A
isoform&1&(phosphatidic&acid&phosphatase&type&2A&isoform
1)&and&SCD&(stearoyl-CoA&desaturase),&directly&participate
in&lipid&metabolism.
r8&Fa2&iy.g&0&gua5enr)n&aeieln&ys2&s&bwiso&eitnrhe&pg&dirgoi&fffuaeptr&setinsstiuaell&yw&eitxhp&crleesnsbeudt&ienr&othl&ea&dDmNinAis&tmraictiroona&r(P-
82&genes&were&differentially&expressed&in&the&DNA
microarray&analysis&in&pig&fat&tissue&with&clenbuterol
administration&(P&&&.05)&in&both&groups.&A.&The&black
circle&indicates&the&507&differentially&expressed&genes&in&the&3
month-old&group&(P&&&.05&in&four&microarray&slides).&B.&The
cross-hatched&circle&indicates&the&336&differentially&expressed
genes&in&the&4&month-old&group&(P&&&.05&in&four&microarray
slides).&C.&The&overlapping&region&indicates&the&82&differentially
expressed&genes&in&both&the&3&month&old&group&and&the
4&month&old&group&that&were&of&interest.
BMC&Genomics&&
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Table&2:&Group&I&of&differentially&expressed&genes:&Cell&metabolism
GenBank&Access&No.
(Clone&No.&on&Microarray)
Gene&name&Annotation*&Induction&Fold&change
3&month&&&4&month
NM_001647&(rpfat_18926)&Apolipprotein&D&(ApoD)&Lipid&metabolism&1.76&1.67
L06820(rpfat_18262)&Apolipprotein&R&(ApoR)&Lipid&metabolism&2.58&1.80
XM_(rpig_3250)&Canis&familiaris&similar&to&phosphatidic&acid
phosphatase&type&2A&isoform&1,&transcript&variant&2
Lipid&metabolism&1.27&1.14
NM_006022&(rpigfat_10082)&transforming&growth&factor&beta-stimulated
protein
modulates&the&frequency,&rate
or&extent&of&DNA-dependent
transcription
1.25&1.13
NM_007158&(rpfat_18317)&NRAS-related&gene&(D1S155E)&1.25&1.54
NM_014828&(rpfat_16693)&KIAA0737&gene&product&(KIAA&1.43
NM_003069(rpfat_18518)&SWI/SNF&related,&matrix&associated,&actin
dependent&regulator&of&chromatin,&subfamily&a,
member&1&(SMARCA1)
1.23&1.64
NM_000985&(rpfat_19876)&ribosomal&protein&L17&(RPL17),&mRNA&protein&translation&1.47&1.09
LOC484530(rpfat_15686)&ribosomal&protein&S10&protein&translation&1.33&1.15
快试试吧，可以对自己使用挽尊卡咯~◆◆
M64620&(rpfat_4179)&cathepsin&B&lysosomal&cysteine&proteinase&1.14&1.13
DQ673096(rpfat_19771)&Eukaryotic&translation&elongation&factor&1&alpha
(EEF1A)
protein&translation&1.70&1.29
X81197(rpfat_13772)&archain&1&endoplasmic&reticulum&to&Golgi
transport
2.15&1.62
AF027652&(rpfat_19670)&L-3-hydroxyacyl-CoA&dehydrogenase&precursor
(HAD)&mRNA,
Lipid&metabolism&-1.30&1.11
AY487830&(rpfat_17395)&Sus&scrofa&stearoyl-CoA&desaturase&(SCD)&gene,
exons&1&through&6&and&complete&cds
Lipid&metabolism&-1.89&1.03
DQ629164&(rpfat_4193)&ribosomal&protein&L10a&protein&translation&-1.01&1.03
NP_&(rpfat_18915)&ribosomal&protein&L32&Protein&translation&-1.79&-1.01
*according&to&description&of&the&Gene&Ontology&Biological&Process&Category
*All&the&genes&in&the&table&were&differentially&expressed&according&to&the&student&t&test&in&the&eight&microarray&slides&(P&&&.05)
Table&3:&Group&II&of&differentially&expressed&genes:&Signal&transduction*
GenBank&Access&No.
(Clone&No.&on&Microarray)
Gene&name&Annotation&Induction&Fold&change&3
month&&&4&month
X05942&(rpfat_17661)&cAMP&dependent&protein
kinase&type&I&regulatory
(PRKAR1A)
causes&the&dissociation&of&the&inactive
holoenzyme
2.0&2.0
Z33879&(rpfat_17754)&mRNA&encoding&G-beta&like
protein&(RACK1)
a&physiological&mediator&of&agonist-induced
Ca2+&release
1.29&1.05
NM_&(rpfat_17793)&wingless-type&MMTV
integration&site&family,&member
3A&(WNT3A)
leads&to&an&increase&in&intracellular&calcium&and
activation&of&protein&kinase&C&(PKC)
1.27&1.09
U57092&(rpfat_19360)&RAB30&member&of&the&RAS&oncogene&family&2.05&2.41
U05291&(rpfat_10974)&fibromodulin&participate&in&the&assembly&of&the&extracellular
matrix&as&it&interacts&with&type&I&and&type&II
collagen&fibrils
1.28&1.04
M18981&(rpfat_8258)&S100&calcium&binding&protein
A6
helps&stimulation&Ca2+-dependent&insulin
release,&prolactin&secretion&and&exocytosis
5.96&1.61
U01160&(rpfat_8561)&transmembrane&4&superfamily
protein&(SAS)
growth-related&cellular&processes&1.09&1.21
AF268463&(rpfat_15672)&voltage-dependent&anion
channel&3&(VDAC3)
Calcium&signaling&pathway&-1.26&-1.05
NM_003248&(rpigfat_10263)&thrombospondin&4&(THBS4)&forms&a&pentamer&and&can&bind&to&heparin&and
calcium
-1.04&1.11
BC024040&(rpfat_18349)&Homo&sapiens&CXXC&finger&5,
mRNA
up-regulation&of&I-kappaB&kinase/NF-kappaB
cascade
-1.41&1.04
*according&to&description&of&the&Gene&Ontology&Biological&Process&Category
*All&the&genes&in&the&table&were&differentially&expressed&according&to&the&student&t&test&in&the&eight&microarray&slides&(P&&&.05)
BMC&Genomics&&
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Both&apoD&and&apoR&were&up-regulated&by&the&administration
of&clenbuterol.&ApoD&is&a&component&of&high&density
lipoproteins&[21].&ApoR&is&a&23-kDa&protein&found&on
very&low-density&lipoproteins&(VLDL),&on&chylomicrons,
and&in&the&d&&&1.21&g/ml&fraction&of&pig&plasma&[22].&ApoR
was&also&found&to&be&up-regulated&by&proteomic&analysis.
Although&the&physiologic&functions&of&apoD&and&apoR&in
快试试吧，可以对自己使用挽尊卡咯~◆◆
adipose&tissue&are&unknown,&they&may&respond&to&clenbuterol
stimulation&to&alter&lipid&metabolism.
Type&2&PAPs&appear&to&metabolize&a&wide&range&of&lipid
mediators&derived&from&both&glycero-&and&sphingolipids
[23,24].&SCD&is&the&enzyme&responsible&for&conversion&of
saturated&fatty&acids&into&monounsaturated&fatty&acids
Table&4:&Group&III&of&differentially&expressed&genes:&Expressed&Sequence&Tags&(ESTs)
GenBank&Access&No.&Clone&No.&on&Microarray&Induction&Fold&change
3&month&4&month
ES605520&rpfat_&1.06
ES605522&rpig_&1.42
ES605503&rpfat_&1.27
ES605504&rpig_&1.10
ES605505&rpfat_&1.21
ES605524&rpfat_&1.05
ES605516&rpfat_&1.25
ES605525&rpfat_&1.13
ES605526&rpfat_&1.12
ES605506&rpfat_&1.07
ES605507&rpfat_&1.17
ES605508&rpigfat_&1.21
ES605509&rpigfat_&1.21
ES605510&rpig_&1.09
ES605511&rpfat_&1.08
ES605528&rpfat_&1.08
ES605530&rpfat_&1.18
ES605532&rpigfat_&1.26
ES605533&rpig_&1.11
ES605535&rpfat_&1.14
ES605536&rpfat_&1.58
ES605537&rpfat_&1.14
ES605538&rpfat_&1.24
ES605539&rpfat_&1.37
ES605540&rpfat_&1.13
ES605513&rpfat_&1.22
ES605541&rpfat_&1.38
ES605543&rpigfat_&1.08
ES605514&rpfat_&1.57
ES605544&rpfat_&1.33
ES605518&rpig_&1.43
ES605515&rpfat_&1.14
ES605519&rpfat_&1.16
ES652311&rpfat_&1.21
ES652312&rpfat_&1.46
ES652313&rpfat_&1.22
ES605523&rpigfat_&1.04
ES605512&rpfat_1.00
ES605527&rpfat_1&1.03
ES605529&rpfat_1&1.02
ES605534&rpig_&1.10
ES605531&rpfat_1&1.01
ES605517&rpfat_1&1.08
ES605542&rpfat_1&1.13
ES605521&rpfat_1&-1.06
*All&the&genes&in&the&table&were&differentially&expressed&according&to&the&student&t&test&in&the&eight&microarray&slides&(P&&&.05)
BMC&Genomics&&
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(MUFA)&in&mammalian&adipocytes&[25].&HAD&is&a&mitochondrial
protein&that&catalyzes&the&oxidation&of&a&wide
variety&of&fatty&acids,&alcohols,&and&steroids&[26,27].
The&identification&of&these&molecules&is&not&sufficient&to
draw&out&the&clenbuterol&physiological&pathway.&However,
with&the&study&of&the&ESTs&in&Table&4,&some&new&functional
genes&related&to&lipid&metabolism&will&be&identified.
The&mechanism&of&clenbuterol&reducing&fat&accumulation
will&be&revealed&on&the&molecular&level&based&on&these&differentially
expressed&genes.
Other&cellular&metabolism&related&genes&in&group&I
Of&the&other&cellular&metabolism&related&genes,&EEF1A
(eukaryotic&translation&elongation&factor&1&alpha)&is
responsible&for&the&enzymatic&delivery&of&aminoacyl
tRNAs&to&the&ribosome.&EEF1A&up-regulation&indicates
that&translation&activity&in&adipose&cells&is&enhanced&by
clenbuterol&stimulation.&Aarchain&1&may&be&involved&in
快试试吧，可以对自己使用挽尊卡咯~◆◆
vesicle&structure&or&trafficking&[28]&and&its&up&regulation
suggests&that&transport&of&cargos&from&the&endoplasmic
reticulum&(ER)&to&the&Golgi&was&enhanced.&Cathepsin&B&is
a&lysosomal&cysteine&proteinase&composed&of&a&dimer&with
disulfide-linked&heavy&and&light&chains.&D1S155E&(NRASrelated
gene),&SMARCA1&(SWI/SNF&related,&matrix&associated,
actin&dependent&regulator&of&chromatin,&subfamily
a,&member&1)&and&KIAA0737&(KIAA0737&gene&product)
modulate&the&frequency,&rate&or&extent&of&DNA-dependent
transcription.
Together,&these&findings&indicate&that&some&metabolic
processes,&such&as&DNA&transcription,&protein&translation
Table&5:&Group&IV&of&differentially&expressed&genes:&Various&categories*
GenBank&Access&No.
(Clone&No.&on&Microarray)
Gene&name&Annotation&Change&test/control&3
month&&&4&month
AF246221&(rpfat_12517)&transmembrane&protein&BRI&developmental&processes&1.27&1.13
AF178980&(rpigfat_9892)&D-prohibitin&mRNA&developmental&processes&1.54&1.24
NM_000089&(rpfat_12612)&collagen,&type&I,&alpha&2&(COL1A2)&developmental&processes&2.48&1.53
Z74616&(rpfat_16033)&mRNA&encoding&Pro-alpha-2&chain&of&type&I
procollagen&(COL1A2)
cell&structure&and&mobility&4.20&2.52
AB033007&(rpfat_15395)&mRNA&for&KIAA1181&protein&cellular&localization&1.29&1.08
X06700&(rpfat_18309)&mRNA&3'&region&for&pro-alpha1(III)
collagen(COL3A1)
cell&structure&and&mobility&3.16&4
Z74615&(rpfat_8523)&mRNA&for&prepro-alpha1(I)&collagen
(COL1A1)
cell&structure&and&mobility&2.86&2.86
X14420&(rpfat_19990)&mRNA&for&pro-alpha-1&type&3&collagen
(COL3A1)
cell&structure&and&mobility&3.29&3.33
BC093076&(rpigfat_10250)&Homo&sapiens&peptidylprolyl&isomerase&A
(cyclophilin&A)
protein&folding&and&stabilization&1.28&1.05
NM_006136&(rpfat_18939)&capping&protein&(actin&filament)&muscle&Z-line,
alpha
cell&mobility&2.02&1.61
NM_005915&(rpfat_15643)&minichromosome&maintenance&deficient&(mis5,
S.&pombe)&6
cell&cycle&1.75&1.62
*according&to&description&of&the&Gene&Ontology&Biological&Process&Category
*All&the&genes&in&the&table&were&differentially&expressed&according&to&the&student&t&test&in&the&eight&microarray&slides&(P&&&.05).
Table&6:&Real-time&PCR&validation&of&microarray&positive&results
Gene*
(Clone&No.&on&Microarray)
Hog&3/hog&4&Sow&3/sow&4
Change&(test/control)&P&value&Change&(test/control)&P&value
ApoD&(rpfat_1&0.013&7.49&0.002
PRKAR1A(rpfat_1&0.014&8.18&0.046
COL1A1&(rpfat_&0.&0.00001
COL1A2&(rpfat_1&0.&0.0078
COL1A2&(rpfat_1&0.&0.0002
COL3A1(rpfat_18309)&Not&detected&Not&detected&Not&detected&Not&detected
COL3A1&(rpfat_19990)&Not&detected&Not&detected&Not&detected&Not&detected
*&The&full&names&of&each&gene&are&listed&in&Tables&2-5.
Five&of&the&seven&genes&were&confirmed&by&real-time&PCR.
BMC&Genomics&&
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FPrigouteroem&3ic&study&of&adipose&tissue&of&pigs&with/without&the&administration&of&clenbuterol
Proteomic&study&of&adipose&tissue&of&pigs&with/without&the&administration&of&clenbuterol.&A.&Two-dimensional&gel
快试试吧，可以对自己使用挽尊卡咯~◆◆
analysis&of&total&proteins&from&adipose&tissue&treated&with/without&clenbuterol.&(13%&SDS-PAGE,&silver&stain)&600&±&50&protein
spots&were&detected.&B.&Two&differentially&expressed&protein&spots&(Amplified&from&the&frame&in&A).
A􀋊
Test&pig&Control&pig
Apo&R
Unknown
protein
Test&pig
pH3&10
Control&pig
pH3&10
SDS-PAGE
B􀋊
Table&7:&Real-time&PCR&validation&of&microarray&negative&results
Gene*&(Clone&No.
on&Microarray)
Hog&1/hog&2&Sow&1/sow&2&Hog&3/hog&4&Sow&3/sow&4
Change
(test/control)
P&value&Change
(test/control)
P&value&Change
(test/control)
P&value&Change
(test/control)
P&value
SCD&(rpfat_1&0.&0.&0.&0.9943
HSL&(rpfat_1&0.&0.&0.&0.5294
PMP22a
(rpfat_18575)
1.15&0.&0.&0.&0.1175
PHPT1b
(rpfat_15312)
0.75&0.&0.&0.&0.4869
*&The&full&names&of&each&gene&are&listed&in&Tables&2-5.
a&:&PMP22:&peripheral&myelin&protein&22
b&:PHPT1:&Phosphohistidine&phosphatase&1
Three&genes&(HSL,&PMP22&and&PHPT1)&were&confirmed&by&real-time&PCR.
BMC&Genomics&&
Page&9&of&12
(page&number&not&for&citation&purposes)
and&protein&translocation,&were&enhanced&in&the&adipose
cells&by&clenbuterol&stimulation.
cAMP&signaling&pathway&related&genes&in&group&II
The&beta-androgenic&receptor&(β-AR)&signal&pathway&has
been&well&described&previously&[11].&Clenbuterol&as&a
beta-agonist&works&by&binding&with&β-AR&which&then
transmits&signals&into&the&cell&along&the&G&protein-mediated
cAMP&signal&pathway&[29].&However,&the&molecular
level&mechanisms&by&which&clenbuterol&affects&adipose
accumulation&are&still&unknown.&To&examine&the&expression
level&of&genes&related&to&cAMP&or&G-protein,&a&total&of
11&genes&were&found&directly&related&to&cAMP&or&G-protein
in&the&microarray&gene&list.&Only&two&of&the&eleven&were
found&to&be&up-regulated,&the&cAMP&dependent&protein
kinase&type&I&regulatory&gene&(PRKAR1A)&and&RAB30
(Table&3).&Up-regulation&of&PRKAR1A&was&confirmed&by
the&real-time&PCR.&No&reports&have&been&found&concerning
functions&of&PRKAR1A&and&RBA30&in&adipose&cells.
Other&signal&transduction&related&genes&in&group&II
Another&seven&signal&transduction&genes&were&found&to&be
differentially&expressed&(Table&3).&Five&genes&(RACK1,
WNT3A,&VDAC3,&S100A6,&THBS4)&encoding&signal&transduction
molecules&are&calcium&related.&This&indicates&that
clenbuterol&may&have&an&impact&on&calcium&signaling
pathways&in&cells.&Both&RACK1&and&WNT3A&activate&protein
kinase&C,&while&protein&kinase&A&has&previously&been
reported&as&the&target&of&clenbuterol.&All&this&data&suggests
that&clenbuterol's&effects&on&adipose&cell&may&be&more
complex&than&previously&suggested.
Fibromodulin&participates&in&the&assembly&of&the&extracellular
matrix&as&it&interacts&with&type&I&and&type&II&collagen
fibrils&[30].&Several&collagen&genes&were&also&found&to&be
up-regulated&(Table&5).&This&indicates&that&clenbuterol
may&have&an&effect&on&cell&collagen&synthesis&which&may
快试试吧，可以对自己使用挽尊卡咯~◆◆
alter&the&cell&matrix&composition.&It&is&difficult&to&determine
whether&this&has&any&relationship&to&trigalloyl&glycerol
accumulation.
ESTs&sequence&in&group&IV
45&differentially&expressed&ESTs&are&listed&in&Table&4.&Specific
conclusions&can&not&be&derived&from&these&ESTs
results&at&this&time.&Some&key&genes&for&lipid&metabolism,
which&are&unknown&at&this&time,&may&be&found&from&this
list&in&the&future.
Clenbuterol&stimulates&genes&up-regulated
Only&three&genes&were&found&to&be&down-regulated&in&the
82&differentially&expressed&genes.&To&further&investigate
whether&some&genes&were&down-regulated&by&the&administration
of&clenbuterol,&PHPT1&(phosphohistidine&phosphatase
1)&and&PMP22&(peripheral&myelin&protein&22)
were&analyzed&by&real-time&PCR&(Table&7).&These&two
genes&were&down-regulated&in&the&3&month-old&group&(p
=&0.03),&but&they&were&not&statistically&differentially
expressed&in&the&4&month-old&group&(p&=&0.09)&by&the
microarray&analysis.&The&PCR&results&showed&that&the
mRNA&expression&of&these&two&genes&were&not&changed
significantly&(P&&&0.05)&which&confirms&the&unusual
microarray&result&that&very&few&genes&were&suppressed.
This&data&suggests&that&clenbuterol&stimulates&gene&up-regulation
in&adipose&cells.
Conclusion
Pig&fat&accumulation&was&reduced&dramatically&with&clenbuterol
treatment.&Histological&sections&and&global&evaluation
of&gene&expression&after&administration&of
clenbuterol&in&pigs&identified&profound&changes&in&adipose
cells.&With&clenbuterol&stimulation,&adipose&cell&volumes
decreased&and&their&gene&expression&profile
changed,&which&indicate&some&metabolism&processes&have
been&also&altered.&Although&the&biological&functions&of&the
differentially&expressed&genes&are&not&completely&known,
higher&expressions&of&these&molecules&in&adipose&tissue
might&contribute&to&the&reduction&of&fat&accumulation.
Among&these&genes,&five&lipid&metabolism&related&genes
were&of&special&interest&for&further&study,&including&apoD
and&apoR.&The&apoR&expression&was&increased&at&both&the
RNA&and&protein&levels.&The&apoR&may&be&one&of&the&critical
molecules&through&which&clenbuterol&reduces&fat
accumulation.
Methods
Animal&sampling&and&clenbuterol&treatment
Eight&Chinese&miniature&pigs&were&used&in&the&experiments.
Four&hogs&and&four&sows,&all&4&weeks&old,&were
housed&in&the&Nutrition&and&Metabolism&Laboratory&at
the&China&Agriculture&University.&They&were&raised&under
exactly&the&same&conditions&and&were&fed&the&same&diets
until&8&weeks&(average&body&weight&17&kg).&They&were&randomly
divided&into&4&groups&with&each&group&having&two
pigs&with&the&same&gender&and&the&same&parents.&For&the
following&4&weeks,&one&pig&in&each&group&was&fed&25&mg/
kg&clenbuterol&twice&daily&in&their&diets&as&the&test&pig,
while&the&other&was&fed&the&same&diet&without&clenbuterol
as&the&control.&Then&one&group&of&hogs&and&one&group&of
sows&were&slaughtered&for&analysis.&These&two&groups&are
referred&to&as&the&3&month-old&pigs.&The&other&two&groups
were&fed&with/without&50&mg/kg&clenbuterol&twice&daily&in
快试试吧，可以对自己使用挽尊卡咯~◆◆
their&diets&for&another&4&weeks&and&slaughtered&for&analysis.
These&two&groups&are&referred&to&as&the&4&month-old
pigs.&Approximately&1&g&biopsies&of&different&tissues,
including&the&back&fat&adipose&tissues&(at&the&fifth&lumbar
vertebra&level),&skeleton&muscle&(biceps&femoris&and&sural
muscle),&liver,&heart,&kidney,&spleen&and&lung,&were&taken
from&each&pig.&Samples&were&washed&in&sterile&water,&snap
frozen&in&liquid&nitrogen&and&stored&at&-80°C.
BMC&Genomics&&
Page&10&of&12
(page&number&not&for&citation&purposes)
Histology
Tissue&samples&were&fixed&in&4%&formaldehyde&in&PBS,
embedded&in&paraffin,&and&sectioned&(5&μm&sections).
After&deparaffinization&and&rehydration,&the&sections&were
washed&three&times&with&PBS&and&stained&with&H&E.
RNA&preparation,&RNA&labeling&and&DNA&microarray
hybridization
Total&RNA&of&the&adipose&tissue&was&extracted&with&TRIZOL
reagent&(Invitrogen,&Gaithersburg,&MD,&USA)&and
further&purified&with&an&RNeasy&mini&kit&(Qiagen,&Valencia,
CA,&USA)&according&to&the&manufacturers'&instructions.
The&porcine&cDNA&microarray&was&produced&at&the&China
Agricultural&University.&A&total&of&11520&spots&representing
3358&genes&(ESTs)&were&included&on&the&microarray
slide.&3358&genes&(ESTs)&were&cloned&from&the&porcine
adipose&cDNA&library&and&printed&in&triplicate&on&each
slide.&(More&details&on&the&specific&genes&and&probe
sequences&are&given&by&Guo&et&al.&[31]).
A&cDNA&microarray&hybridization&analysis&was&performed
with&Pronto!™&Universal&Microarray&Kits&(Corning,&MA,
USA)&according&to&the&manufacturers&instructions.&Briefly,
reverse&transcription&was&done&with&10&μg&total&RNA&as&the
template&to&synthesize&cDNA&incorporating&the&fluorescence
dyes&Cy3-dCTP&or&Cy5-dCTP.&Probes&were&purified
on&a&Qiagen&spin&column&(Qiagen,&Valencia,&CA,&USA).
Dye-labelled&cDNA&was&mixed&together&with&dry&dyelabeled
cDNA&for&hybridization&(see&Pronto&Universal
hybridization&kit&Quick&Reference&Guide).
To&avoid&dye&bias,&the&experiments&were&performed&in
duplicate&by&dye&swap&with&Cy5-dCTP&first&used&with&the
test&pig&and&Cy3-dCTP&used&with&the&control&pig&and&then
Cy3-dCTP&used&with&the&test&pig&and&Cy5-dCTP&used&with
the&control&pig.
Eight&cDNA&microarray&slides&were&hybridized&for&the
eight&pigs.
DNA&microarray&Imaging&and&data&analysis
Arrays&were&scanned&with&a&ScanArray&Express&scanner
(Parckard&Bioscience,&Kanata,&OT,&USA)&with&the&obtained
images&analyzed&with&GenePix&Pro&4.0&(Axon&Instruments,
Foster&City,&CA)&and&Acuity&4.0&(Axon&Instruments,&Foster
City,&CA).&The&resulting&microarray&data&was&then&normalized
using&the&space&and&intensity-dependent&normalization
in&the&LOWESS&program&[32].&Each&gene&was
represented&in&triplicate&on&each&slide.&The&intensity
(median)&of&each&spot&was&analyzed&using&the&student&ttest
to&identify&the&differentially&expressed&genes&(P&&
0.05).&Low&quality&spots&were&filtered&out&before&the&student
t-test&analysis.&Low&quality&spots&refer&to&stained&spots
with&bad&images&or&spots&with&intensities&(median)&lower
快试试吧，可以对自己使用挽尊卡咯~◆◆
than&200&(too&weak)&or&greater&than&60000&(saturated).
Differentially&expressed&genes&were&defined&as&genes&with
P&values&less&than&0.05&in&the&eight&microarray&slides.&The
mean&ratios&of&the&differentially&expressed&genes&(ESTs)
were&calculated&as&the&&ratio&of&the&median&&of&three&spots
to&indicate&the&trends&in&the&changes&of&the&mRNA&abundance.
The&microarray&data&from&this&research&has&been&deposited
in&the&NCBI&Gene&Expression&Omnibus&data&repository
under&accession&numbers&GSE8093&[33].
Quantitative&Real-Time&PCR
Fluorescent&real-time&PCR&was&used&to&confirm&the&transcriptional
differences&observed&in&the&microarray&results,
The&real-time&PCR&was&done&on&an&ABI&Prism&9700
Sequence&Detection&system&(Applied&Biosystems,&Foster,
CA,&USA)&using&SYBRgreen&technology&as&described&by&Li
et&al.&[34].&The&PCR&primer&sequences&used&for&the&realtime
PCR&are&shown&in&Table&8.&The&PCR&reaction&volume
was&20&μl&with&the&following&program:
50°C&2&min&(activate&the&uracil-N-glycosylase&enzyme);
95°C&10&min&(initial&denaturation&of&the&cDNA)&;
40&cycles&(1&sec&at&various&elevated&temperatures&for&data
acquisition):&95°C&20&sec,&annealing&(according&to&the
specifications&for&each&primer)&20&sec,&72°C&20&
72°C&10&min&(reformation&of&fully&duplexed&DNA);
The&dissolve&curve&analysis&used&heating&from&65&to&95°C,
with&increases&of&0.2°C&per&step&with&the&system&held&1&sec
at&each&temperature.
All&samples&were&measured&in&triplicate.&Expression&was
quantified&by&the&relative&standard&curve&method.&A&standard
graph&of&the&cycle&threshold&(CT)&values&was&obtained
from&serial&dilutions&(10-1–10-8&copies/well)&of&Glyceraldehyde-
3-phosphate&dehydrogenase&(GAPDH,&a&housekeeping
gene)&cDNA.&The&quantification&was&normalized
to&an&endogenous&RNA&control&of&the&GAPDH&or&beta2-
microglobulin&(B2M).&An&independent&sample&t-test&was
used&to&analyze&differences&in&mRNA&expression&with/
without&administration&of&clenbuterol.&Differences&were
considered&to&be&statistically&significant&at&P&&&0.05.&Reactions
for&which&the&housekeeping&gene's&CT&values&were
less&than&15&or&more&than&25&were&discarded&in&the&calculations
because&the&start&cDNA&concentration&was&not
appropriate&or&the&cDNA&quality&was&not&good&enough.
BMC&Genomics&&
Page&11&of&12
(page&number&not&for&citation&purposes)
Proteome&analysis
Protein&extraction&from&adipose&cells,&two-dimensional
electrophoresis&and&in-gel&digestion&were&conducted&as
described&by&Lee&et&al.&[35].&After&in-gel&digestion,&samples
were&dissolved&in&4&ml&0.5%&aqueous&trifluoroacetic&acid
for&mass&spectrometric&analysis&on&a&Bruker&REFLEX&III
MALDI-TOF-MS&(Bruker-Franzen,&Bremen,&Germany)&in
positive&ion&mode&at&an&accelerating&voltage&of&20&kV&with
an&a-cyano-4-hydroxy&cinnamic&acid&matrix.&The&resulting
peptide&mass&fingerprint&(PMF)&was&then&used&in&a&search
of&the&SWISS-PROT&and&NCBInr&databases&using&the&Mascot
search&engine&[20]&with&a&tolerance&of&±&0.2&D&and&one
missed&cleavage&site.
快试试吧，可以对自己使用挽尊卡咯~◆◆
Authors'&contributions
Jin&Zhang&participated&in&the&experimental&design,&animal
feeding&and&sampling,&and&the&microarray&creation&and
hybridization.&Jin&Zhang&also&completed&the&microarray
data&analysis,&real-time&PCR&experiments&and&drafted&the
manuscript.&Qiang&He&conducted&the&proteomic&research.
Qiu&Y.&Liu&helped&with&the&real-time&PCR&for&validation&of
the&microarray&data.&Wei&Guo&helped&with&the&animal
feeding,&sampling,&microarray&creation&and&hybridization.
Wei&W&Zhang&performed&the&statistical&analyses.&Xiao&X.
Hu,&Mei&X.&Deng&and&Ning&Li&designed&and&oversaw&the
research&and&assisted&in&writing&the&manuscript.&All
authors&read&and&approved&the&final&manuscript.
Additional&material
Acknowledgements
This&work&was&supported&by&the&Natural&Scientific&Foundation&of&China,&the
National&Basic&Research&Development&Program&()&and&the
National&High&Technology&Research&and&Development&Program&of&China
().
References
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Additional&file&1
Original&data&of&the&research.&The&data&include&four&tables&as&follow.&Table
1.&Clenbuterol&residue&in&porcine&blood&and&adipose.&Table&2.&Body&weight
of&pigs&treated&with/without&clenbuterol.&Table&3.&Cross-sectional&area&of
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Table&8:&Primers&used&for&the&real-time&PCR&analysis
Genes&&&clone&number&Primer&sequence&(5'&to&3')&PCR&product&size&(bp)&Annealing&Temperature
GAPDH&For.&ATGGTGAAGGTCGGAGTGA&Rev.
ATGGGTAGAATCATACTGGA
154&bp&57°C
Beta&2-microglobulin&(B2M)&For.&TGG&TCTTTCTACCTTCTGGCCC&Rev.
TGTGATGCCGGTTAGTGGTCTC
166&bp&60°C
Apolipoprotein&D&(rpfat_18926)&For.&AGATCCCAGTGAGCTTTGAG&Rev.
CGTAGTTCTCATAGTCGGTG
233&bp&58°C
PRKAR1A&(rpfat_17661)&For.&GGCGACGAGGTGCTATCAG&Rev.
ATGGCATCAAAAATATCAC
159&bp&60°C
COL1A1&(rpfat_8523)&For.&TCAAGATGTGCCACTCCGACT&Rev.
GCCTGTCTCCATGTTGCAGAA
104&bp&60°C
COL1A2&(rpfat_16033)&For.&ATATGCACCTTGGACATCGGT&Rev.
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241&bp&60°C
COL3A1&(rpfat_19990)&For.&CCTGCTGGAAAGAATGGTGAC&Rev.
ACGTTCACCGGTTTCACCTT
132&bp&60°C
COL1A2&(rpfat_17393)&For.&CCTGGCTCTAGAGGTGAACG&Rev.
AGCAGGACCAGGATTACCAG
247&bp&60°C
COL3A1&(rpfat_18309)&For.&TTTCTTTTATGGCTCCCCCTG&Rev.
GCGTGTTCGATATTCGAAGAC
101&bp&60°C
SCD&(rpfat_16685)&For.&AAGGAACTAGAAGGCTGCTC&Rev.
TGTAGAGCAGCAGCCATCAC
156&bp&58°C
PHPT1&(rpfat_15312)&For.&GAAGACACAGTTGAGGACAC&Rev.
GGACATTGTTCGGAGGATAG
110&bp&60°C
HSL&(rpfat_11096)&For.&TCCGAATGGAGTCTGCACTGT&Rev.
CTTCCACTCTGACCTCCAACG
128&bp&60°C
PMP22&(rpfat_18575)&For.&CATGAACATTTGCACCACTTG&Rev.
GTCAGCACCTAATGGTATGGA
133&bp&60°C
For.:&Forward
Rev.:&Reverse
快试试吧，可以对自己使用挽尊卡咯~◆◆
Publish&with&BioMed&Central&and&every
scientist&can&read&your&work&free&of&charge
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disseminating&the&results&of&biomedical&research&in&our&lifetime.&
Sir&Paul&Nurse,&Cancer&Research&UK
Your&research&papers&will&be:
available&free&of&charge&to&the&entire&biomedical&community
peer&reviewed&and&published&immediately&upon&acceptance
cited&in&PubMed&and&archived&on&PubMed&Central
yours&—&you&keep&the&copyright
Submit&your&manuscript&here:
BioMedcentral
BMC&Genomics&&
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32.&The&microarray&database&[
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自由译者，英汉互译，承接各种翻译任务，质优价廉。QQ&
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