猪前脂肪细胞诱导分化前后SSH文库的构建及差异表达基因的筛选鉴定
|
摘要
前脂肪细胞是一类具有增殖和向脂肪细胞分化能力的特异化了的前体细胞,对其增殖和分化机理的深入研究有助于了解细胞脂肪沉积的调控机理,为提高畜禽品质乃至人类的肥胖及Ⅱ型糖尿病的治疗均有较大的指导意义。本论文的研究内容包括：
1、以2日龄仔猪皮下脂肪组织为材料,采用胶原酶消化法分离培养出细胞成分均一、增殖旺盛的原代前脂肪细胞,经生长动力学研究发现所培养细胞接种后的潜伏期约为40 h,倍增时间约为61.9h;经核型分析认为所培养细胞为稳定的二倍体细胞系。通过组织块培养法发现极易漂浮的脂肪组织通过悬浮培养也能获得原代培养细胞,用吸管吹打悬浮的组织块能加速组织块中的细胞游离出来。
2、用M1、M2和M3三种不同诱导分化培养液(以DMEM/F12+100 IU/mL双抗+50 nmol/L In+100 nmol/L DEX+O.25 mmol/L IBMX为基础,Ml补加10%FBS,M2补加10%FBS和100nmol/L RSG,M3补加100nmol/L RSG)对前脂肪细胞进行诱导分化,通过分化过程中细胞形态学观察发现M2在诱导分化后的第2天大多细胞内均有小脂滴出现,而M1和M3在诱导分化后第3天部分细胞开始出现小脂滴,在脂肪积聚速度和脂滴汇聚快慢上以M2最快,M3次之,最慢的是M1；通过分化过程中细胞内脂肪含量测定发现采用M2诱导分化的细胞细胞内脂肪含量最多、增速最快,其次是M3,M1最少,与形态学观察结果一致。通过上述试验结果可知分化培养液M2对前脂肪细胞的诱导分化效果最好。
3、在M1、M2和M3三种不同诱导分化培养液对前脂肪细胞进行诱导分化过程中,于11个不同分化时间点分别提取三种细胞的总RNA,借助实时荧光定量PCR.检测了PPARα、PPARγ、C/EBPα、FASN、GPAT、FABP4、ACOX1、GAPDH和ENFP2等基因的表达变化趋势。通过比较M1和M2中各基因的表达变化发现罗格列酮对PPARγ、C/EBPα、FASN、GPAT、FABP4、ACOX1和GAPDH等基因的表达有极显著的上调作用(P<0.01),且能促进各基因协同表达达到表达高峰,而对PPARα和ENPP2两基因的表达有极显著的抑制作用(P<0.01);通过比较M2和M3中各基因的表达变化发现血清对PPARα、C/EBPα、FASN、GPAT、ACOX1、GAPDH和ENPP2等基因的表达有极显著的抑制作用(P<0.01),而对PPARγ和FABP4两基因的表达有极显著的促进作用(P<0.01)；通过比较M1、M2和M3中各基因表达量变化趋势的相关系数发现PPARγ在前脂肪细胞的分化调控中发挥作用较早,继而作用于C/EBPα,再对下游相关生脂基因进行调控和激活；通过比较参与脂肪酸生物合成的FASN和参与脂肪酸β氧化的ACOX1两基因在三种培养液中表达量的变化趋势发现细胞内脂肪酸的生物合成和脂肪酸β氧化均在进行,以维持细胞内环境的稳定,细胞内聚脂快慢取决于脂肪酸生物合成和脂肪酸β氧化两个不同生物进程的力量对比。
4、采用聚脂最快的分化培养液M2对前脂肪细胞进行诱导分化,于分化前和分化后分别提取细胞总RNA,构建前脂肪细胞诱导分化前、后抑制消减杂交文库。以分化前细胞所获cDNA为Driver,以分化后细胞所获cDNA为Tester构建的文库的消减效率为210(记为SSH-1),反向文库的消减效率为25(记为SSH-2)。其中SSH-1文库第二次PCR产物纯化后和载体PMD18-T连接,经蓝白斑筛选到3840个克隆；SSH-2筛选到2880个克隆。随机从SSH-1文库选取了960个克隆,从SSH-2文库选取了672个克隆经过PCR'扩增鉴定,SSH-1文库得到797个条带单一的阳性克隆,SSH-2文库得到536个条带单一的阳性克隆,有效阳性率分别为83.0%和79.8%。从SSH-1文库的797个阳性克隆中选取了400个插入片断在150bp-700bp的单克隆,从SSH-2文库的536个阳性克隆中选取了150个插入片断在150bp-700bp的单克隆进行斑点杂交,SSH-1文库筛选出221个呈明显差异表达的阳性克隆,SSH-2文库筛选出73个呈明显差异表达的阳性克隆,斑点杂交后的阳性率分别为55.3%和48.7%。对所有通过斑点杂交筛选的阳性克隆进行序列测定,SSH-1文库共获得183个EST,SSH-2文库共获得63个EST。
5、对测序所获246个EST序列(其中SSH-1共183个,SSH-2共63个)经载体、接头去除,单机BLAST比对去除重复EST序列,再通过NCBI的mRNA参考序列数据库(refseq_rna)、非冗余核酸数据库(nr/nt)和猪的EST数据库进行序列比对,SSH-1文库共获得130个已知基因,10个已知EST;SSH-2文库共获得15个已知基因,8个已知EST和1个全新EST,并将195条无重复EST向dbEST数据库进行了提交。
6、利用已知基因对应的人参考mRNA序列的Genebank登陆号,通过Panther和DAVID两个在线基因功能注释系统对SSH-1文库的130个已知基因和SSH-2文库的15个已知基因进行了基因功能注释。通过Panther的基因功能注释,SSH-1文库的130个已知基因中参与信号转导/细胞间通信的基因有22个,占16.9%；参与细胞结构/运动的基因有10个,占7.7%：参与细胞凋亡的有3个(1个凋亡基因,2个抑制凋亡基因),占2.3%;参与免疫防御的基因有16个,占12.3%；参与物质转运的基因有19个,占14.6%,其中参与脂类和脂肪酸转运的基因有7个(LPL, NPC2, ABCD3, APOE, SCARB1,OSBPL9和FABP4),占所有基因的5.4%,占参与物质转运基因的36.8%；参与转录、翻译与表达调控的基因有31个,占23.8%,其中参与脂肪酸生物合成的基因有2个(FASN和LTA4H),参与脂类、脂肪酸和类固醇代谢调控的基因1个(ADRP);参与物质代谢的基因有42个,占32.3%,其中参与脂肪酸脱饱和的基因有1个(CYB5R3),参与脂肪酸β氧化的基因有3个(DECR1,ACOX1和ECHS1),参与脂肪酸代谢的基因有6个(CRAT, SCD, AACS, ADIPOR1, CYP4F3和ANXA8),参与脂类代谢的基因有5个(LTA4H, DGAT2, ADIPOR1,OSBPL9和PIGH),参与磷脂代谢的基因有2个(MTMR3和PIGH),参与类固醇代谢的基因有3个(CYP4F3,OSBPL9和PPAP2B),参与胆固醇代谢的基因有1个(ECHS1);参与细胞增殖和分化的基因有10个,占7.7%,其中参与细胞周期控制的有3个(YWHAQ, BTG1和CDK8),参与细胞增殖和分化的有7个(PTMA, IL8, PCNA, BTG1,KHDRBS1,PA2G4和PGHS-2);此外,Panther上未分类的基因有22个,占16.9%,参与其它生物学进程的基因有18个,占13.8%。同时,部分基因具有多种功能,参与多种生物学进程。在SSH-2文库所分离出的15个已知基因中,参与信号转导/细胞间通信的基因有7个,占46.7%；参与细胞结构/运动、翻译与表达调控及Panther上未分类的基因均有2个,各自占13.3%；参与细胞凋亡和免疫防御的均有1个,各自占6.7%；参与物质转运的基因有3个,占20%；参与其它生物学进程的基因有5个,占33.3%。同时,部分基因具有多种功能,参与多种生物学进程。
7、利用DAVID的在线功能相关基因聚类分群系统对功能相关基因进行了聚类分组,SSH-1文库130个基因共得到21个功能相关聚类群,SSH-2文库15个基因共得到3个功能相关聚类群。
8、对Genebank上还未公布猪的mRNA序列的91基因(SSH-1共82个,SSH-2共9个),利用相应EST和猪的dbEST数据库通过末端延伸法共拼接出包括全部CDS序列的69个基因的mRNA序列(SSH-1共64个,SSH-2共5个)。
Preadipocytes are a type of special precursor cells with ability of generation and cell differentiation towards adipocytes whose deeply research of mechanism of generation and cell differentiation is helpful to comprehend regulation mechanism of fatty accumulation in cells, has major guiding significance to improve quality of livestock and poultry even to cure obesity in human and Type 2 Diabetes Mellitus. The content of this thesis includes:
1. Use subcutaneous fatty tissue got from 2-day old piglet as experimental materials, isolate and culture preadipocytes with uniform cell composition and vigorous generation by collagenase digestion method, with results that eclipse period of cultured cells after innoculation about 40 h and doubling time of that about 61.9 h trough research of Growth Kinetics, that karyotype analysis also establishes the presumption that those cultured cells are stable diploud cell line, that primary cells can be cultured with suspension culture from easily floated fatty tissue through tissue pieces culture method, and that beating upon suspended tissue pieces with pipette is able to accelerate eduction of cells from tissue pieces.
2. Carry out the induced differentiation on preadipocytes with M1, M2, M3 (M1 is the medium into which 10% FBS is added, M2 is the medium into which 10% FBS and 100 nmol/L RSG are added, M3 is the medium into which 100 nmol/L RSG is added, based on DMEM/F12+100 IU/mL anti-body+50 nmol/L In+100 nmol/L DEX+0.25 mmol/L IBMX) these three different culture media for induced differentiation, and the results show that small fat droplets appear in most of cells in M2 on 2nd day after induced differentiation through morphological observation in progress of differentiation, while some cells have the similar phenomenon on 3rd day in M1 and M3, velocity of fatty accumulation and convergence of fat droplets is the fastest in M2, faster in M3 whose velocity is superior to M1, and that assaying of fat in cells shows that fatty quantity and increasing velocity are respectively the most and the fastest in M2 as well as the least and the most slowly in M1, and that in M3 stays medium, which agrees with the morphological observation. Then conclusion that Medium2 has the best effect on induced differentiation of preadipocytes is established.
3. In the progress of induced differentiation of preadipocytes in M1, M2 and M3, respectively extract total RNA from 3 types of cells at 11 different time pionts, and detect tendency of gene expression changes of genes such as PPARα, PPARγ, C/EBPα, FASN, GPAT, FABP4, ACOX1, GAPDH and ENPP2 using real-time PCR. Reach the conclusion that RSG can very significantly up-regulate expression of genes PPARγ, C/EBPα, FASN, GPAT, FABP4, ACOX1 and GAPDH (P<0.01) by comparing expressing changes of each gene in Ml and M2, and promote coordinate expression of each gene to reach expression peak, while has very significantly inhibition on expression of PPARa and ENPP2(P<0.01); In addition, serum has been confirmed to have very significant prohibition on expression of genes PPARa, C/EBPa, FASN, GPAT, ACOX1, GAPDH and ENPP2 (P<0.01) by comparing expressing changes of each gene in M2 and M3, while can very significantly promote that of PPARy and FABP4 (P<0.01); PPARγis found to have a earilier effect in regulation of preadipocytes differentiation, then influence C/EBPα, regulate and activate genes relative to lipide bearing at downstream; by comparing tendencies of expression quantities changes of FASN taking part in fatty acid biosynthetic and ACOX1 joining fatty acidβ-oxidation in three media, we can find that the two kinds of performance of fatty acid biosynthetic and fatty acidβ-oxidation are simultaneous to maintain the stability of cellar internal environment, the velocity of fat accumulation depends on competition of two types of powers namely fatty acid biosynthetic and fatty acidβ-oxidation through comparisons of coefficient correlation of expressing tendencies of changes of each gene in M1,M2 and M3.
4. Induct preadipocytes differentiation using M2 whose fat accumulation velocity is the fastest, and respectively extract total RNA both before and after differentiation to build two SSH libraries ahead of and after preadipocytes differentiation. Creating SSH libraries with condition that the cDNA obtained before differentiation are used as Driver, while that after differentiation as Tester, we got the results pruning efficiency of the library is 210 (recorded as SSH-1), and that of backward library is 25 (recorded as SSH-2).3840 clones from SSH-1 and 2880 clones from SSH-2 are obtained after purification of the PCR product of SSH-1 library at the second time, connection to PMD18-T and blue-white spot election. Then select by random 960 clones from SSH-1 library and 672 clones from SSH-2 library to run identification by PCR,797 and 567 single strap of positive clones are got in SSH-1 library and in SSH-2 library, respectively with the effective positive rates of 83.0% and 79.8%. Select 400 monoclones with inserted fractions with length from 150bp to 700bp from 797 positive clones in SSH-1 library, and 150 monoclones with inserted fractions with length from 150bp to 700bp from 536 positive clones in SSH-2 library to carry out dot blot hybridi-zation. Results show that 221 significantly differential expressed positive clones and 73 significantly differential expressed positive clones are screened from SSH-1 and SSH-2, with effective positive rates of 55.3% and 48.7%, at last,183 ESTs and 63 ESTs are got in SSH-1 and SSH-2 after sequencing of all positive clones selected by dot blot hybridization.
5. Remove duplicated EST sequences on 246 ESTs (183 ESTs in SSH-1 and 63 ESTs in SSH-2)through the removal of vector and connecter and alignment of BLAST on single machine, then compare the sequences with the help of reference sequence database (refseq_rna) on NCBI, non-redundant nucleic acid database (refseq_rna) and EST database of swine, at last, addition of 130 known genes,10 known ESTs and 15 known genes,8 known ESTs,1 brand-new EST into SSH-1 and SSH-2 parallels submission to dbEST of 195 ESTs without duplication.
6. Note the function of 130 known genes of library SSH-1 and 15 known genes of library SSH-2 using the ID number of corresponding human reference mRNA sequences to those known genes and Panther as well as DAVID these two on-line systems for functional annotation on genes. The results show that 22 genes accounting 16.9% of 130 known genes of library SSH-1 join signal transduction/cell-cell communication,10 genes accounting 7.7% of 130 known genes of library SSH-1 join cellular structure/movement,3 genes (1 gene on Apoptosis,2 inhibiting genes on Apoptosis) accounting 2.3% of 130 known genes of library SSH-1 join Cell Apoptosis,16 genes accounting 12.3% of 130 known genes of library SSH-1 join immune defense,19 genes accounting 14.6% of 130 known genes of library SSH-1 join Material Transport, among which,7 genes (LPL, NPC2, ABCD3, APOE, SCARB1, OSBPL9 and FABP4) accounting 5.4%,36.8% of all genes, genes on transportation join lipide and fatty acid Transport,31 genes accounting 23.8% of 130 known genes of library SSH-1 join transcription, translation and regulation of expression, among which,2 genes (FASN and LTA4H) join fatty acid biosynthetic and 1 gene (ADRP) join regulation of lipide, fatty acid and steroid metabolism,42 genes accounting 32.3% of 130 known genes of library SSH-1 join substance metabolism, among which,1 gene (CYB5R3) join fatty acid desaturation,3 genes (DECR1, ACOX1 and ECHS1) join fatty acidβ-oxidation,6 genes (CRAT, SCD, AACS, ADIPOR1, CYP4F3 and ANXA8) join fatty acid metabolism,5 genes (LTA4H, DGAT2, ADIPOR1, OSBPL9 and PIGH) join lipide metabolism,2 genes (MTMR3 and PIGH) join lipometabolic metabolism,3 genes (CYP4F3, OSBPL9 and PPAP2B) join steroid metabolism,1 gene (ECHS1) join cholesterol metabolism,10 genes accounting 7.7% of 130 known genes of library SSH-1 join generation and differentiation of cells, among which,3 genes (YWHAQ, BTG1 and CDK8) join cell cycle control,7 genes (PTMA, IL8, PCNA, BTG1, KHDRBS1, PA2G4 and PGHS-2) join generation and differentiation. Additionally,22 genes accounting 16.9% of 130 known genes of library SSH-1 have not been classified yet on Panther,18 genes accounting 13.8% of 130 known genes of library SSH-1 join other biological proceedings, simultaneously, there are also some genes in possession of multi-function and joining many biological proceedings. In those 15 isolated known genes from SSH-2,7 genes accounting 46.7% join signal transduction/cell-cell communication,2 genes accounting 13.3% join cellular structure/movement,2 genes accounting 13.3% join translation and regulation of expression,2 genes accounting 13.3% haven't been classified on Panther,1 gene 6.7% join Cell Apoptosis,1 gene accounting 6.7% join immune defense,3 genes accounting 20% join Material Transport,5 genes accounting 33.3% join other biological proceedings, simultaneously, there are also some genes in possession of multi-function and joining many biological proceedings.
7. Gather and divide into groups using on-line system of DAVID of clustering and swarming on function-relative genes, and respectively get 21 groups and 3 groups from 130 genes of SSH Library land 15 genes of SSH Library 2.
8.Splice mRNA sequences of 69 genes (64 mRNA sequences in SSH-1 and 5 mRNA sequences in SSH-2) including the whole CDS sequences using terminus extension method with corresponding ESTs ans dbEST databank of swine on 91 genes (82 mRNA sequences in SSH-1 and 9 mRNA sequences in SSH-2) of swine mRNA sequences without announcement in Genebank.
1、以2日龄仔猪皮下脂肪组织为材料,采用胶原酶消化法分离培养出细胞成分均一、增殖旺盛的原代前脂肪细胞,经生长动力学研究发现所培养细胞接种后的潜伏期约为40 h,倍增时间约为61.9h;经核型分析认为所培养细胞为稳定的二倍体细胞系。通过组织块培养法发现极易漂浮的脂肪组织通过悬浮培养也能获得原代培养细胞,用吸管吹打悬浮的组织块能加速组织块中的细胞游离出来。
2、用M1、M2和M3三种不同诱导分化培养液(以DMEM/F12+100 IU/mL双抗+50 nmol/L In+100 nmol/L DEX+O.25 mmol/L IBMX为基础,Ml补加10%FBS,M2补加10%FBS和100nmol/L RSG,M3补加100nmol/L RSG)对前脂肪细胞进行诱导分化,通过分化过程中细胞形态学观察发现M2在诱导分化后的第2天大多细胞内均有小脂滴出现,而M1和M3在诱导分化后第3天部分细胞开始出现小脂滴,在脂肪积聚速度和脂滴汇聚快慢上以M2最快,M3次之,最慢的是M1；通过分化过程中细胞内脂肪含量测定发现采用M2诱导分化的细胞细胞内脂肪含量最多、增速最快,其次是M3,M1最少,与形态学观察结果一致。通过上述试验结果可知分化培养液M2对前脂肪细胞的诱导分化效果最好。
3、在M1、M2和M3三种不同诱导分化培养液对前脂肪细胞进行诱导分化过程中,于11个不同分化时间点分别提取三种细胞的总RNA,借助实时荧光定量PCR.检测了PPARα、PPARγ、C/EBPα、FASN、GPAT、FABP4、ACOX1、GAPDH和ENFP2等基因的表达变化趋势。通过比较M1和M2中各基因的表达变化发现罗格列酮对PPARγ、C/EBPα、FASN、GPAT、FABP4、ACOX1和GAPDH等基因的表达有极显著的上调作用(P<0.01),且能促进各基因协同表达达到表达高峰,而对PPARα和ENPP2两基因的表达有极显著的抑制作用(P<0.01);通过比较M2和M3中各基因的表达变化发现血清对PPARα、C/EBPα、FASN、GPAT、ACOX1、GAPDH和ENPP2等基因的表达有极显著的抑制作用(P<0.01),而对PPARγ和FABP4两基因的表达有极显著的促进作用(P<0.01)；通过比较M1、M2和M3中各基因表达量变化趋势的相关系数发现PPARγ在前脂肪细胞的分化调控中发挥作用较早,继而作用于C/EBPα,再对下游相关生脂基因进行调控和激活；通过比较参与脂肪酸生物合成的FASN和参与脂肪酸β氧化的ACOX1两基因在三种培养液中表达量的变化趋势发现细胞内脂肪酸的生物合成和脂肪酸β氧化均在进行,以维持细胞内环境的稳定,细胞内聚脂快慢取决于脂肪酸生物合成和脂肪酸β氧化两个不同生物进程的力量对比。
4、采用聚脂最快的分化培养液M2对前脂肪细胞进行诱导分化,于分化前和分化后分别提取细胞总RNA,构建前脂肪细胞诱导分化前、后抑制消减杂交文库。以分化前细胞所获cDNA为Driver,以分化后细胞所获cDNA为Tester构建的文库的消减效率为210(记为SSH-1),反向文库的消减效率为25(记为SSH-2)。其中SSH-1文库第二次PCR产物纯化后和载体PMD18-T连接,经蓝白斑筛选到3840个克隆；SSH-2筛选到2880个克隆。随机从SSH-1文库选取了960个克隆,从SSH-2文库选取了672个克隆经过PCR'扩增鉴定,SSH-1文库得到797个条带单一的阳性克隆,SSH-2文库得到536个条带单一的阳性克隆,有效阳性率分别为83.0%和79.8%。从SSH-1文库的797个阳性克隆中选取了400个插入片断在150bp-700bp的单克隆,从SSH-2文库的536个阳性克隆中选取了150个插入片断在150bp-700bp的单克隆进行斑点杂交,SSH-1文库筛选出221个呈明显差异表达的阳性克隆,SSH-2文库筛选出73个呈明显差异表达的阳性克隆,斑点杂交后的阳性率分别为55.3%和48.7%。对所有通过斑点杂交筛选的阳性克隆进行序列测定,SSH-1文库共获得183个EST,SSH-2文库共获得63个EST。
5、对测序所获246个EST序列(其中SSH-1共183个,SSH-2共63个)经载体、接头去除,单机BLAST比对去除重复EST序列,再通过NCBI的mRNA参考序列数据库(refseq_rna)、非冗余核酸数据库(nr/nt)和猪的EST数据库进行序列比对,SSH-1文库共获得130个已知基因,10个已知EST;SSH-2文库共获得15个已知基因,8个已知EST和1个全新EST,并将195条无重复EST向dbEST数据库进行了提交。
6、利用已知基因对应的人参考mRNA序列的Genebank登陆号,通过Panther和DAVID两个在线基因功能注释系统对SSH-1文库的130个已知基因和SSH-2文库的15个已知基因进行了基因功能注释。通过Panther的基因功能注释,SSH-1文库的130个已知基因中参与信号转导/细胞间通信的基因有22个,占16.9%；参与细胞结构/运动的基因有10个,占7.7%：参与细胞凋亡的有3个(1个凋亡基因,2个抑制凋亡基因),占2.3%;参与免疫防御的基因有16个,占12.3%；参与物质转运的基因有19个,占14.6%,其中参与脂类和脂肪酸转运的基因有7个(LPL, NPC2, ABCD3, APOE, SCARB1,OSBPL9和FABP4),占所有基因的5.4%,占参与物质转运基因的36.8%；参与转录、翻译与表达调控的基因有31个,占23.8%,其中参与脂肪酸生物合成的基因有2个(FASN和LTA4H),参与脂类、脂肪酸和类固醇代谢调控的基因1个(ADRP);参与物质代谢的基因有42个,占32.3%,其中参与脂肪酸脱饱和的基因有1个(CYB5R3),参与脂肪酸β氧化的基因有3个(DECR1,ACOX1和ECHS1),参与脂肪酸代谢的基因有6个(CRAT, SCD, AACS, ADIPOR1, CYP4F3和ANXA8),参与脂类代谢的基因有5个(LTA4H, DGAT2, ADIPOR1,OSBPL9和PIGH),参与磷脂代谢的基因有2个(MTMR3和PIGH),参与类固醇代谢的基因有3个(CYP4F3,OSBPL9和PPAP2B),参与胆固醇代谢的基因有1个(ECHS1);参与细胞增殖和分化的基因有10个,占7.7%,其中参与细胞周期控制的有3个(YWHAQ, BTG1和CDK8),参与细胞增殖和分化的有7个(PTMA, IL8, PCNA, BTG1,KHDRBS1,PA2G4和PGHS-2);此外,Panther上未分类的基因有22个,占16.9%,参与其它生物学进程的基因有18个,占13.8%。同时,部分基因具有多种功能,参与多种生物学进程。在SSH-2文库所分离出的15个已知基因中,参与信号转导/细胞间通信的基因有7个,占46.7%；参与细胞结构/运动、翻译与表达调控及Panther上未分类的基因均有2个,各自占13.3%；参与细胞凋亡和免疫防御的均有1个,各自占6.7%；参与物质转运的基因有3个,占20%；参与其它生物学进程的基因有5个,占33.3%。同时,部分基因具有多种功能,参与多种生物学进程。
7、利用DAVID的在线功能相关基因聚类分群系统对功能相关基因进行了聚类分组,SSH-1文库130个基因共得到21个功能相关聚类群,SSH-2文库15个基因共得到3个功能相关聚类群。
8、对Genebank上还未公布猪的mRNA序列的91基因(SSH-1共82个,SSH-2共9个),利用相应EST和猪的dbEST数据库通过末端延伸法共拼接出包括全部CDS序列的69个基因的mRNA序列(SSH-1共64个,SSH-2共5个)。
Preadipocytes are a type of special precursor cells with ability of generation and cell differentiation towards adipocytes whose deeply research of mechanism of generation and cell differentiation is helpful to comprehend regulation mechanism of fatty accumulation in cells, has major guiding significance to improve quality of livestock and poultry even to cure obesity in human and Type 2 Diabetes Mellitus. The content of this thesis includes:
1. Use subcutaneous fatty tissue got from 2-day old piglet as experimental materials, isolate and culture preadipocytes with uniform cell composition and vigorous generation by collagenase digestion method, with results that eclipse period of cultured cells after innoculation about 40 h and doubling time of that about 61.9 h trough research of Growth Kinetics, that karyotype analysis also establishes the presumption that those cultured cells are stable diploud cell line, that primary cells can be cultured with suspension culture from easily floated fatty tissue through tissue pieces culture method, and that beating upon suspended tissue pieces with pipette is able to accelerate eduction of cells from tissue pieces.
2. Carry out the induced differentiation on preadipocytes with M1, M2, M3 (M1 is the medium into which 10% FBS is added, M2 is the medium into which 10% FBS and 100 nmol/L RSG are added, M3 is the medium into which 100 nmol/L RSG is added, based on DMEM/F12+100 IU/mL anti-body+50 nmol/L In+100 nmol/L DEX+0.25 mmol/L IBMX) these three different culture media for induced differentiation, and the results show that small fat droplets appear in most of cells in M2 on 2nd day after induced differentiation through morphological observation in progress of differentiation, while some cells have the similar phenomenon on 3rd day in M1 and M3, velocity of fatty accumulation and convergence of fat droplets is the fastest in M2, faster in M3 whose velocity is superior to M1, and that assaying of fat in cells shows that fatty quantity and increasing velocity are respectively the most and the fastest in M2 as well as the least and the most slowly in M1, and that in M3 stays medium, which agrees with the morphological observation. Then conclusion that Medium2 has the best effect on induced differentiation of preadipocytes is established.
3. In the progress of induced differentiation of preadipocytes in M1, M2 and M3, respectively extract total RNA from 3 types of cells at 11 different time pionts, and detect tendency of gene expression changes of genes such as PPARα, PPARγ, C/EBPα, FASN, GPAT, FABP4, ACOX1, GAPDH and ENPP2 using real-time PCR. Reach the conclusion that RSG can very significantly up-regulate expression of genes PPARγ, C/EBPα, FASN, GPAT, FABP4, ACOX1 and GAPDH (P<0.01) by comparing expressing changes of each gene in Ml and M2, and promote coordinate expression of each gene to reach expression peak, while has very significantly inhibition on expression of PPARa and ENPP2(P<0.01); In addition, serum has been confirmed to have very significant prohibition on expression of genes PPARa, C/EBPa, FASN, GPAT, ACOX1, GAPDH and ENPP2 (P<0.01) by comparing expressing changes of each gene in M2 and M3, while can very significantly promote that of PPARy and FABP4 (P<0.01); PPARγis found to have a earilier effect in regulation of preadipocytes differentiation, then influence C/EBPα, regulate and activate genes relative to lipide bearing at downstream; by comparing tendencies of expression quantities changes of FASN taking part in fatty acid biosynthetic and ACOX1 joining fatty acidβ-oxidation in three media, we can find that the two kinds of performance of fatty acid biosynthetic and fatty acidβ-oxidation are simultaneous to maintain the stability of cellar internal environment, the velocity of fat accumulation depends on competition of two types of powers namely fatty acid biosynthetic and fatty acidβ-oxidation through comparisons of coefficient correlation of expressing tendencies of changes of each gene in M1,M2 and M3.
4. Induct preadipocytes differentiation using M2 whose fat accumulation velocity is the fastest, and respectively extract total RNA both before and after differentiation to build two SSH libraries ahead of and after preadipocytes differentiation. Creating SSH libraries with condition that the cDNA obtained before differentiation are used as Driver, while that after differentiation as Tester, we got the results pruning efficiency of the library is 210 (recorded as SSH-1), and that of backward library is 25 (recorded as SSH-2).3840 clones from SSH-1 and 2880 clones from SSH-2 are obtained after purification of the PCR product of SSH-1 library at the second time, connection to PMD18-T and blue-white spot election. Then select by random 960 clones from SSH-1 library and 672 clones from SSH-2 library to run identification by PCR,797 and 567 single strap of positive clones are got in SSH-1 library and in SSH-2 library, respectively with the effective positive rates of 83.0% and 79.8%. Select 400 monoclones with inserted fractions with length from 150bp to 700bp from 797 positive clones in SSH-1 library, and 150 monoclones with inserted fractions with length from 150bp to 700bp from 536 positive clones in SSH-2 library to carry out dot blot hybridi-zation. Results show that 221 significantly differential expressed positive clones and 73 significantly differential expressed positive clones are screened from SSH-1 and SSH-2, with effective positive rates of 55.3% and 48.7%, at last,183 ESTs and 63 ESTs are got in SSH-1 and SSH-2 after sequencing of all positive clones selected by dot blot hybridization.
5. Remove duplicated EST sequences on 246 ESTs (183 ESTs in SSH-1 and 63 ESTs in SSH-2)through the removal of vector and connecter and alignment of BLAST on single machine, then compare the sequences with the help of reference sequence database (refseq_rna) on NCBI, non-redundant nucleic acid database (refseq_rna) and EST database of swine, at last, addition of 130 known genes,10 known ESTs and 15 known genes,8 known ESTs,1 brand-new EST into SSH-1 and SSH-2 parallels submission to dbEST of 195 ESTs without duplication.
6. Note the function of 130 known genes of library SSH-1 and 15 known genes of library SSH-2 using the ID number of corresponding human reference mRNA sequences to those known genes and Panther as well as DAVID these two on-line systems for functional annotation on genes. The results show that 22 genes accounting 16.9% of 130 known genes of library SSH-1 join signal transduction/cell-cell communication,10 genes accounting 7.7% of 130 known genes of library SSH-1 join cellular structure/movement,3 genes (1 gene on Apoptosis,2 inhibiting genes on Apoptosis) accounting 2.3% of 130 known genes of library SSH-1 join Cell Apoptosis,16 genes accounting 12.3% of 130 known genes of library SSH-1 join immune defense,19 genes accounting 14.6% of 130 known genes of library SSH-1 join Material Transport, among which,7 genes (LPL, NPC2, ABCD3, APOE, SCARB1, OSBPL9 and FABP4) accounting 5.4%,36.8% of all genes, genes on transportation join lipide and fatty acid Transport,31 genes accounting 23.8% of 130 known genes of library SSH-1 join transcription, translation and regulation of expression, among which,2 genes (FASN and LTA4H) join fatty acid biosynthetic and 1 gene (ADRP) join regulation of lipide, fatty acid and steroid metabolism,42 genes accounting 32.3% of 130 known genes of library SSH-1 join substance metabolism, among which,1 gene (CYB5R3) join fatty acid desaturation,3 genes (DECR1, ACOX1 and ECHS1) join fatty acidβ-oxidation,6 genes (CRAT, SCD, AACS, ADIPOR1, CYP4F3 and ANXA8) join fatty acid metabolism,5 genes (LTA4H, DGAT2, ADIPOR1, OSBPL9 and PIGH) join lipide metabolism,2 genes (MTMR3 and PIGH) join lipometabolic metabolism,3 genes (CYP4F3, OSBPL9 and PPAP2B) join steroid metabolism,1 gene (ECHS1) join cholesterol metabolism,10 genes accounting 7.7% of 130 known genes of library SSH-1 join generation and differentiation of cells, among which,3 genes (YWHAQ, BTG1 and CDK8) join cell cycle control,7 genes (PTMA, IL8, PCNA, BTG1, KHDRBS1, PA2G4 and PGHS-2) join generation and differentiation. Additionally,22 genes accounting 16.9% of 130 known genes of library SSH-1 have not been classified yet on Panther,18 genes accounting 13.8% of 130 known genes of library SSH-1 join other biological proceedings, simultaneously, there are also some genes in possession of multi-function and joining many biological proceedings. In those 15 isolated known genes from SSH-2,7 genes accounting 46.7% join signal transduction/cell-cell communication,2 genes accounting 13.3% join cellular structure/movement,2 genes accounting 13.3% join translation and regulation of expression,2 genes accounting 13.3% haven't been classified on Panther,1 gene 6.7% join Cell Apoptosis,1 gene accounting 6.7% join immune defense,3 genes accounting 20% join Material Transport,5 genes accounting 33.3% join other biological proceedings, simultaneously, there are also some genes in possession of multi-function and joining many biological proceedings.
7. Gather and divide into groups using on-line system of DAVID of clustering and swarming on function-relative genes, and respectively get 21 groups and 3 groups from 130 genes of SSH Library land 15 genes of SSH Library 2.
8.Splice mRNA sequences of 69 genes (64 mRNA sequences in SSH-1 and 5 mRNA sequences in SSH-2) including the whole CDS sequences using terminus extension method with corresponding ESTs ans dbEST databank of swine on 91 genes (82 mRNA sequences in SSH-1 and 9 mRNA sequences in SSH-2) of swine mRNA sequences without announcement in Genebank.
引文
[1]Harrison R G. Observations on the living developing nerve fiber[J]. Proc Soc Exp Biol Med,1907, 4:140-143.
[2]Carrel A. On the Permanent life of tissues out side the Organism[J]. J Exp Med,1912,15:516-528.
[3]Earle W R, Schilling EL, Stark T H, et al. The mouse Fibroblast cultures and changes seen in the living cells[J]. NATL Cancer Inst,1943,4:165-212.
[4]Gey G O, Coffman W D, Kubicek M T, et al. Tissue culture studies of the proliferative capacity of cervical carcinoma and normal epithelium[J]. Cancer Res,1952,12:364-365.
[5]Ailhaud G, Amri E, Bardon S, et al. Growth and differentiation of regional adipose tissue molecular and hormonal mechanisms[J]. Int J Obesity,1991,15:87-90.
[6]Steven R Smith, Barbara Gawronska-Kozak, Lenka Janderova, et al. Agouti Expression in Human Adipose Tissue:Functional Consequences and Increased Expression in Type 2 Diabetes. Diabetes, 2003,52:2914-2922.
[7]Smas C M, Chen L, Sul H S, et al. St ructural characterization and alternate splicing of the gene encoding the preadipocyte EGF-like protein pref-1 [J]. Biochemistry,1994,33:9257-9265.
[8]Umek R M, Friedman A D, Mcknight S L, et al. CCAAT-enhancerbinding protein:a component of a differentiation switch [J]. Science,1991,251:288-292.
[9]James M Ntambi and Young-Cheul Kim. Adipocyte Differentiation and Gene Expression[J]. Journal of Nutrition,2000,130:3122S-3126S.
[10]Morrison R F and Farmer S R. Hormonal signaling and transcriptional control of adipocyte differentiation [J]. American Society for Nutritional Sciences,2000,130 (12):3116S-3121S.
[11]夏成,王哲,朱淑玲,等.犊牛前脂肪细胞的培养及其增殖与分化模型的建立[J].中国兽医科技,2004,34(5)：26-30.
[12]Satol Nakanishi N and Mit sumoto M. Culture condition supporting adipocyte conversion of st romal-vascular cells from bovine intramuscular adipocyte tissues [J]. Journal of Veterinary Medical Science,1997,58:1073-1078.
[13]Marshall S, Garvey WT and Geller M. Primary culture of isolated adipocytes:A new model to study insulin receptor regulation and insulin action [J]. Biol Chem,1984,259(10):6376-6384.
[14]王竹晨,刘建中,李燕,等.人前脂肪细胞的原代培养[J].山医科大学学报,2001,22(6)：443-446.
[15]Satol Nakanishi N and Mit sumoto M. Culture condition supporting adipocyte conversion of st romal-vascular cells from bovine intramuscular adipocyte tissues [J]. Journal of Veterinary Medical Science,1997,58:1073-1078.
[16]Sib S R, Mohua M, Samir B, et al. A new cell secreting insulin [J]. Endocrinology,2003,4 (144): 1585-1593.
[17]Hausman G J, D R Campion, J P McNamara, et al. Adiposetissue development in the fetal pig after decapition[J]. J Anim Sci,1981,53:1634-1644.
[18]Hausman G J, Novakofski J E, Martin R J, et al. The development of adipocytes in primary stromal-vascular culture of fetal pig adipose tissue [J]. Cell Tissue Res,1984,236:459-464.
[19]Sarawak A, Swanson V and Hun C Y. Insulin and hydrocortisone, but not triiodothyronine, are required for the differentiation of pig preadipocytes in primary cultures [J]. J Anim Sci,1997,75: 105-111.
[20]Brandebourg T D, Hu C Y. Isomer-specific regulation of differentiating pig preadipocytes by conjugated linoleic acids [J]. J Anim Sci,2005,83:2096-2105.
[21]Ramsay T. Porcine preadipocyte proliferation and differentiation:A role for leptin [J]. J Anim Sci, 2005,83:2066-2074.
[22]屈长青,张国华,陈粉粉,等.猪前体脂肪细胞的原代培养[J].农业生物技术学报,2005,13(5)：649-653.
[23]张罕星,朱晓彤,束刚,等.猪脂肪前体细胞分化过程中聚脂相关基因的表达模式[J].动物学报,2007,53(1)：143-150.
[24]Pilgrim C H. DNA synthesis and differentiation in developing white adipose tissue [J]. Dev Biol,1971,26:69-76.
[25]Patrick C W, Chauvin PB, Robb G L, et al. Tissue enmneered adipose frontiers in tissue engineering[J]. Science,1998,369-382.
[26]Tang Q Q, Tamara C, Otto M, et al. Commit of C3H10 T1/2 pluripotent stem cells to the adipocyte lineage [J]. PNAS,2004,101(26):9607-9611.
[27]Tamara C, Otto M, Lane D, et al. Adipose development:from stem cell to adipocyte [J]. Critical Reviews in Biochemistry and Molecular Biology,2005,40(4):229-242.
[28]Dani C, Smith A G, Dessolin S, et al. Difirentiation of embryonic stem cell into adipocyte in vitro [J]. Cell Sci,1997,110:1279-1285.
[29]Hugo E R, Brandebourg T D, Comstock C E, et al. Anovel human adipocyte cell line that produces prolactin [J]. Endocrinology,2005,147(1):306-313.
[30]Nathan E, Wolins, Benjamin K. Quaynor, OP9 mouse stromal cells rapidly differentiate into adipocytes:Characterization of a useful new model of adipogenesis [J]. J Lipid Res,2005,11: 1194-1198.
[31]Dorothy B Hausman, Hea Jin Park and Gary J Hausman. Isolation and Culture of Preadipocytes from Rodent White Adipose Tissue. Adipose Tissue Protocols,2008:201-219.
[32]Rodriguez A M, Elabd C, Delteil F, et al. Adipocyte differentiation of multipotent cells established from human adipose tissue[J]. Biochem Biophys Res Commun,2004,315 (2):255-263.
[33]Ntambi J M.Adipocyte differentiation and gene expression [J]. J Nutr,2000,13(12):3122s-3126s.
[34]Zhang H, Noohr J, Jensen CH, et al. Insulin-like growth factor-1/insulin bypasses P-ref-1/FAL-mediated inhibition of adipocyte differentiation[J]. Biochem,2003, 278(23):20906-20914.
[35]倪毓辉,郭锡熔,陈荣华,等.脂肪细胞分化的表型特征、分子标志和调控[J].国外医学儿科学分册,2004,31(6)：306-308.
[36]Hug C, Lodish H F. Diabetes, obesity, and Acrp30/adiponectin [J]. Biotechniques,2002, 33(3):654-658.
[37]Otto T C and Lane M D. Adipose development:from stem cell to adipocyte[J]. Critical Reviews in Biochemistry and Molecular Biology,2005,40(4):229-242.
[38]Farmer S R. Regulation of PPARgamma activity during adipogenesis[J]. International Journal of Obesity,2005,29(Suppl 1):13-16.
[39]Lekstrom-Himes J A. The role of C/EBP (epsilon) in the terminal stages of granulocyte differentiation[J]. Stem Cells,2001,19(2):125-133.
[40]Gregoire F M. Adipocyte differentiation:from fibroblast to endocrine cell[J]. Experimental Biology and Medicine (Maywood),2001,226:997-1002.
[41]Richards M P, Poch S M, Coon C N, et al. Feed restriction significantly alters lipogenic gene expression in broiler breeder chickens[J]. Journal of Nutrition,2003,133:707-715.
[42]Matsubara Y, Sato K, Ishii H, et al. Changes in mRNA expression of regulatory factors involved in adipocyte differentiation during fatty acid induced adipogenesis in chicken[J]. Comparative Biochemistry and Physiology Part A:Molecular & Integrative Physiology,2005,141(1):108-115,
[43]Stahl A. Current review of fatty acid transport proteins (SLC27)[J]. Pflugers Archiv European Journal of Physiology,2004,447:722-727.
[44]Ferre P. The Biology of Peroxisome Proliferator-Activated Receptors:Relationship with Lipid Metabolism and Insulin Sensitivity [J]. Diabetes,2004,53(suppl 1):s43-s50.
[45]Tsuchida A, Yamauchi T, Kadowaki T, et al. Nuclear Receptors as Targets for Drug Development: Molecular Mechanisms for Regulation of Obesity and Insulin Resistance by Peroxisome Proliferator-Activated Receptor y, CREB-Binding Protein, and Adiponectin[J]. Pharmacol Sci, 2005,97 (2):164-170.
[46]Evan D Rosen, Pasha Sarraf, Amy E Troy, et al. PPARγ is required for the differentiation of adipose tissue in vivo and in vitro [J]. Mol Cell,1999,4:611-617.
[47]Liping Qiao, Paul S MacLean, Jerome Schaack, et al. C/EBP alpha regulates human adiponectin gene transcription through an intronic enhancer[J]. Diabetes,2005,54(6):1744-1754.
[48]Zhang Jiang-Wen, Dwight J Klemm, Charles Vinson, et al. Role of CREB in Transcriptional Regulation of CCAAT/Enhancer-binding Protein P Gene during Adipogenesis [J]. J Biol Chem, 2004,279(6):4471-4478.
[49]Youngjin Choi, Young-Cheul Kim, Yong-Bong Han, et al. The trans-10, cis-12 Isomer of Conjugated Linoleic Acid Downregulates Stearoyl-CoA Desaturase 1 Gene Expression in 3T3-L1 Adipocytes[J]. Journal of Nutrition,2000,130:1920-1924.
[50]Yeh W C, Cao Z, Classon M, et al. Cascade regulation of terminal adipocyte differentiation by three members of the C/EBP family of leucine zipper proteins[J]. Genes & Dev,1995,9:168-181.
[51]Tanaka T, Yoshida N, Kishimoto T, et al. Defective adipocyte differentiation in mice lacking the C/EBP beta and/or C/EBP delta gene[J]. EMBO J,1997,16(24):7432-7443.
[52]Jae Bum Kim, Harold M Wright, Margaret Wright, et al. ADD1/SREBP1 activates PPARy through the production of endogenous ligand [J]. Proc Natl Acad Sci USA,1998,95(8):4333-4337.
[53]Lluis Fajas, Kristina Schoonjans, Laurent Gelman, et al. Regulation of Peroxisome Proliferator-Activated Receptor y Expression by Adipocyte Differentiation and Determination Factor 1/Sterol Regulatory Element Binding Protein 1:Implications for Adipocyte Differentiation and Metabolism [J]. Mol Cell Biol,1999,19(8):5495-5503.
[54]Sato K, Nakanishi N and Mitsumoto M. Culture conditions supporting adipose conversion of stromal-vascular cells from bovine intramuscular adipose tissues [J]. Journal of Veterinary Medical Science,1996,58:1073-1078.
[55]Mur C, Arribas M, Benito M, et al. Essential role of insulin-like growth factor I receptor in insulin-induced fetal brown adipocyte differentiation[J]. Endocrinology,2003,144:581-593.
[56]Lin J, Arnold H B, Della-Fera M A, et al. Myostatin knockout in mice increases myogenesis and decreases adipogenesis[J]. Biochemical and Biophysical Research Communications,2002,291: 701-706.
[57]Van Hall G, Steensberg A, Sacchetti M, et al. Interleukin-6 stimulates lipolysis and fat oxidation in humans[J]. Journal of Clinical Endocrinology & Metabolism,2003,88:3005-3010.
[58]Tang Q Q, Otto T C, Lane MD, et al. CCAAT/enhancer-binding protein β is required for mitotic clonal expansion during adipogenesis[J]. Natl Acad Sci USA,2003,100(3):850-855.
[59]Smas C M, Chen I, Zhao L, et al. Transcriptional Repression of pref-1 by Glucocorticoids Promotes 3T3-L1 Adipocyte Differentiation [J]. J Bio l Chem,1999,274(6):12632-12641.
[60]Tang Q Q, Jiang M S, Lane M D. Repressive effect of SP1 on the COBRA alpha gene promoter: role in adipocyte differentiation [J]. Mol Cell Biod,1999,19(7):4855-4865.
[61]Aubert J, Saint-Marc P, Belmonte N, et al. Prostacyclin IP receptor up-regulates the early expression of C/EBP beta and C/EBP delta in preadipose cells[J].Mol Cell Endocrinol, 2000,160(1-2):149-156.
[62]Zhang H H, Halbleib M, Ahmad F, et al. Tumor necrosis factor-alpha stimulates lipolysis in differentiated human adipocytes through activation of extracellular signal-related kinase and elevation of intracellular cAMP[J]. Diabetes,2002,51:2929-2935.
[63]Smas C M, Chen L, Sul H S, et al. Structural characterization and alternate splicing of the gene encoding the preadipocyte EGF-like protein Pref-1 [J]. Biochemistry,1994,33:9257-9265.
[64]Smas C M, Chen L, Sul H S. Cleavage of membrane-associated pref-1 generates a soluble inhibitor of adipocyte differentiation[J]. Mol Cell Biol,1997,17:977-988.
[65]Man-Shiow Jiang and M Daniel Lane. Sequential repression and activation of the CCAAT enhancer-binding protein-a (C/EBPα) gene during adipogenesis[J]. PNAS,2000,97(23): 12519-12523.
[66]Francisco Felipe, M Luisa Bonet, Joan Ribot, et al. Modulation of Resistin Expression by Retinoic Acid and Vitamin A Status [J]. Diabetes,2004,53:882-889.
[67]Brandebourg T D and Hu C Y. Regulation of differentiating pig preadipocytes by retinoic acid [J]. Anim. Sci,2005,83:98-107.
[68]Dani C, Smith A G, Dessolin S, et al. Differentiation of embryonic stem cells into adipocytes in vitro[J]. J Cell Sci,1997,110(11):1279-1285.
[69]Stephen F Konieczny and Charles P Emerson.5-azacytidine induction of stable mesodermal stem cell lineages from 10T1/2 cells:Evidence for regulatory genes controlling determination[J]. Cell, 1984,38(3):791-800.
[70]Chapman A B, Knight D M, Dieckmann B S, et al. Analysis of gene expression during differentiation of adipogenic cells in culture and hormonal control of the developmental program[J]. J Biol Chem,1984,259(24):15548-15555.
[71]RubinC S, Hirsch A, Fung C, et al. Development of hormone receptors and hormonal responsiveness in vitro Insulin receptors and insulin sensitivity in the preadipocyte and adipocyte forms of 3T3-L1 cells[J]. J Biol Chem,1978,253(20):7570-7578.
[72]Wang P, Renes J, Bouwman F, et al. Absence of an adipogenic effect of rosiglitazone on mature 3T3-L1 adipocytes:increase of lipid catabolism and reduction of adipokine expression[J]. Diabetologia,2007,50:654-665.
[73]Naima Moustaid, Francoise Lasnier, Bernard Hainque, et al. Analysis of gene expression during adipogenesis in 3T3-F442A preadipocytes:Insulin and dexamethasone control[J]. J Cell Biochem, 1990,42:243-254.
[74]卢慧玲,王宏伟,Katherine Cianflone,等.诱导SW872前脂肪细胞分化的最优方法探讨[J]. 基础医学与临床,2006,26(3)：15-318.
[75]Gharbi-Chihi J, Grimaldi P, Torresani J, et al. Triiodothyronine and adipose conversion of OB17 preadipocytes:binding to high affinity sites and effects on fatty acid synthetizing and esterifying enzymes[J]. J Recept Res,1981,2(2):153-173.
[76]Wabitsch M, Heinze E, Hauner H, et al. Biological effects of human growth hormone in rat adipocyte precursor cells and newly differentiated adipocytes in primary culture[J]. Metabolism, 1996,45(1):34-42.
[77]李舜,周杰,郑智勇,等.共轭亚油酸对大鼠前体脂肪细胞增殖与分化的影响[J].中国畜牧杂志,2007,43(11)：19-21.
[78]Litthauer D and Serrero G. The primary culture of mouse adipocyte precursor cells in defined medium[J]. Biochem Physiology,1992,101(1):59-64.
[79]Yves Reyne, Jean Nougues and Jean-Paul Dulor. Differentiation of rabbit adipocyte precursor cells in a serum-free medium In Vitro Cell[J]. Dev Biol,1989,25(8):747-752.
[80]Suryawan A, Swanson L V and Hu C Y. Insulin and hydrocortisone, but not triiodothyronine, are required for the differentiation of pig preadipocytes in primary culture[J]. J Anim Sci,1997,75(1): 105-111.
[81]Entenmann G and Hauner H. Relationship between replication and differentiation in cultured human adipocyte precursor cells[J]. Am JPhysiol Cell Physiol,1996,270(39):C1011-C1016.
[82]Heid C A, Stevens J, Livak K J, et al.Real time quantitative PCR[J].Genome Research,1996, 6(10):986-994.
[83]Zhu SH F. The Detected Technology of Real-time Fluorogenic PCR [M]. Beijing:China Measure Press,2003.
[84]Walker N J. Real time and quantitative PCR:applications to mechanism based toxicology [J]. Biochem Mol Toxicol,2001,15 (3):121-127.
[85]Woo T H, Patel B K, Cinco M, et al. Identification of Leptospira biflexa by real-time homogeneous detection of rapid cycle PCR product [J]. Journal of Microbiological Methods,1999,35:23-30.
[86]Giulietti A, Overbergh L, Valckx D, et al. An overviewof real-time quantitative PCR:application to quantify cytokine gene expression [J]. Methods,2001,25 (4):386-401.
[87]Nami Okumura, Naoko-Kishi Nishizawa, Yosuke Umehara, et al. A dioxygenase gene (Ids2) expressed under iron deficiency conditions in the roots of Hordeum vulgare[J]. Plant Molecular Biology,1994,4(25):705-719.
[88]George P, Douglas T, Yang F, et al. Combining SSH and cDNA microarrays for rapid ide ntification of diferentially expressed genes[J]. Nucleic Acids Research,1999,127(6):1517-1523.
[89]Sturtevant J. Applications of differential display reverse transcription PCR to molecular pathogenesis and medical mycology [J].Clinical Microbial Rev,2000,13:408-427.
[90]SteinauM, Unger E, Jones J and Rajeevan M. Differential display PCR of peripheral blood for biomarker discovery in chronic fatigue syndrome [J]. J Mol Med,2004,82:750-755.
[91]Ripamonte P, Merighe G K, Meirelles F V. Development and optimization of a fluorescent differential display PCR system for studying bovine embryo development in vitro [J]. GenetMol Res,2005,4(4):726-733.
[92]Hong ping Jiang, Dong-chun Kang, Deborah Alexandre, et al. Arapidsubtration hybridization approach for identifying and cloning diferentially expressed genes[J]. Proc Natl Acad Sci,2000, 97(23):12684-12689.
[93]Wang T W, Lu L, Wang D, et al. Isolation and characterization of senescence-induced cDNAs encoding deoxy-hypusine synthase and eukaryotic inittranslation iation factor 5A from tomato[J]. J Biol Chem,2001,276:17541-17549.
[94]Hubank M and Schatz D G. Identifying diferences in mRNA expression by representational dinference analysis of cDNA[J]. Nucleic Acids Res,1994,22 (25):5640-5648.
[95]Hubank M, Bryntesson F, Schatz D G. Cloning of apop tosis-related genes by representational difference analysis of cDNA [J]. Methods Mol Biol,2004,282:255-273.
[96]Bowler L D. Representational difference analysis of cDNA [J]. Methods Mol Med,2004,94:49-66.
[97]Sung B, Jung K J, Chung H Y. cDNA representational difference analysis used in the identification of genes related to the aging process in rat kidney [J]. Mech Ageing Dev,2005,126(8):882-891.
[98]Yang J L, LI G, Zhang F L, et al. Identification of variations of gene expression of visceral adipose and renal tissue in type 2 diabetic rats using cDNA representational difference analysis [J]. Chin Med J,2003,116(4):529-533.
[99]Kang Dong-chul, Raphael LaFance, Zao-zhong Su, et al. Reciprocal subtraction differential RNA display:An efficient and rapid procedure for isolating differentially expressed gene sequences [J]. Proc Natl Acad Sci USA,1998,95:13788-13793.
[100]Velculescu V E, Zhang L and Vogelstein B. Serial analysis of gene expression [J]. Science, 1995,270:484-487.
[101]Richards M, Tan S P. and Tan J H. The transcriptome profile of human embryonic stem cells as defined by SAGE [J]. Stem Cells,2004,22(1):51-64.
[102]Quere R, Manchon L and Lejeune M. Mining SAGE data allows largescale, sensitive screening of antisense transcript expression [J]. Nucleic Acids Res,2004,32(20):e163.
[103]Keime C, Damiola F, Mouchiroud D, et al. Identitag,a relational database for SAGE tag identification and interspecies comparison of SAGE libraries [J]. BMC Bioinformatics,2004, 5:143.
[104]Boheler K R and Tarasov K V. SAGE analysis to identify embryonic stem cell predominant transcripts [J]. Methods Mol Biol,2006,329:195-221.
[105]Richards M, Tan S P, Bongso A, et al.Reverse serial analysis of gene expression (SAGE)characterization of orphan SAGE tags from human embryonic stem cells identifies the presence of novel transcripts and antisense transcription of key pluripotency genes [J]. Stem Cells, 2006,24(5):1162-1173.
[106]Sjostedt A. A microarray analysis of the murine macrophage response to infection with Francisella tularensis LVS [J]. J Med Microbiol,2006,55:1023-1033.
[107]Suzuki T, Tazoe H and Isemura M. DNA microarray analysis of changes in gene expression induced by 1,25-dihydroxyvitamin D3 in human promyelocytic leukemia HL-60 cells [J]. Biomed Res,2006,27(3):99-109.
[108]Andersson H and Hartmanova B. A microarray analysis of the murine macrophage response to infection with Francisella tularensis LVS [J]. J Med Microbiol,2006,55(Pt8):1023-1033.
[109]Peeters J K and Vander Spek P J.Growing applications and advancements in microarray technology and analysis tools [J]. Cell Biochem Biophys,2005,43(1):149-166.
[110]MaurerM, Molidor R, Sturn A, et al. MARS:Microarray analysis, retrieval, and storage system [J]. BMC Bioinformatics,2005,6:101.
[111]Qin L, Rueda L, Ali A,et al. Spot detection and image segmentation in DNA microarray data [J]. Appl Bioinformatics,2005,4(1):1-11.
[112]Diatchenko L, Chris Lau Y F, Campbeu A P, et al. Suppression subtractive hybridization:A method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA,1996,93(12):6025-6030.
[113]Diatchenko L, Lukyanov S, Lau Y F, et al. Suppression subtractive hybridization:a versatile method for identifying differentially expressed genes[J]. Methods Enzymol,1999,303:349-380.
[114]何鹏,江渝.抑制性消减杂交技术及其应用研究进展[J].国外医学临床生物化学与检验学分册,2004,25(4)：653-853.
[115]ShirinM, Sushmita S, Vivek K, et al. Suppression subtractive hybridization coupled with microarray analysis to examine differential expression of genes in virus infected cells Biol [J]. Proced Online,2004,6(1):94-104.
[115]Yao J, Coussens P M, Saama P, et al. Generation of expressed sequence tags from a normalized porcine skeletal muscle cDNA library[J]. Anim Biotechnol,2002,13(2):211-222.
[116]Zhao S H, Nettleton D, Liu W, et al. Complementary DNA macroarray analyses of differential gene expression in porcine fetal and postnatal muscle[J]. J Anim Sci,2003, (9):2179-2188.
[117]Diatchenko L, Chris Lau Y F, Campbeu A P, et al. Suppression subtractive hybridization:A method for generating differentially regulated or tissue-specific cDNA probes and libraries[J]. Proc Natl Acad Sci USA,1996,93(12):6025-6030.
[118]Kuang J and Ashorn C L. At least two kinase phosphoralate the MPM-2 epitope during Xenopusooc-yte maturation [J]. J Cell Biol,1993,123:859-868.
[119]Carmeci C, Thompson D A, Kuang W W, et al. Moesin expression is associated with the estrogen receptor-negative breast cancer phenotype [J]. Surgery,1998,124(2):211-217.
[120]Jin H, Cheng X, Diatchenko L, et al. Differential screening of a subtracted cDNA library:a method to search for genes preferentially expressed in multiple tissues [J]. Biotechniques,1997, 23(6):1084-1086.
[121]Harris A J, Shaddock J G, Manjanatha M G, et al. Identification of differentially expressed genes in aflatoxin B1-treated cultured primary rat hepatocytes and Fischer 344 rats [J]. Carcinogenesis, 1998,19(8):1451-1458.
[122]Ligon A H, Pershouse M A, Jasser S, et al. Differentially expressed gene products in glioblastoma cells suppressed for tumorigenicity [J]. J Neurovirol,1998,4(2):217-226.
[123]VonStein O D, Thies W G, Hofmann M, et al.A high throughput screening for rarely transcribed differentially expressed genes[J]. Nucleic Acids Res,1997,25(13):2598-2602.
[124]Hebrok M, Kim S K, Melton D A, et al. Screening for novel pancreatic genes expressed during embryogenesis [J]. Diabetes,1999,48(8):1550-1556.
[125]Miller F R, Barnbabas N, Liu X, et al. Differential display, subtractive hybridization and application of methodology to search for point mutations to identify genetic defects responsible for progression in MCF10AT model of human breast disease [J]. Electrophoresis,1999, 20(2):256-260.
[126]Murphy M, Godson C, Cannon S, et al. Suppression subtractive hybridization identifies high glucose levels as a stimulus for expression of connective tissue growth factor and other genes in human mesangial cells [J]. J Biol Chem,1999,274(9):5830-5834.
[127]Matsuyama T,Yasumura N, Funakoshi M, et al. Maize genes specifically expressed in the outermost cells of root cap [J]. Plant Cell Physiol,1999,40(5):469-476.
[128]Heine H, Delude R L, Monks B G, et al. Bacterial lipopolysaccharide induces expression of the stress response genes hop and H411 [J]. J Biol Chem,1999,274(30):21049-21055.
[129]Tchernitsa O I, Zuber J, Ser C, et al.Gene expression profiling of fibroblasts resistant toward oncogene 2 mediated transformation reveals preferential transcription of negative growth regulators[J]. Oncogene,1999,18(39):5448-5454.
[130]Wang X, Li X, Yaish-Ohad S, et al. Molecular cloning and expression of the rat monocyte chemotactic protein-3 gene:a possible role in stroke [J]. Brain Res Mol Brain Res,1999, 71(2):304-312.
[131]Yang G P, Ross D T, Kuang W W, et al. Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes [J]. Nucleic Acids Res,1999,27(6):1517-1523.
[132]Kostic C and Shaw P H. Isolation and characterization of sixteen novel p53 response genes [J]. Oncogene,2000,19 (35):3978-3987.
[133]Ye Z and Connor J R. Identification of iron responsive genes by screening cDNA libraries from suppression subtractive hybridization with antisense probes from three iron conditions[J]. Nucleic Acids Res,2000,28(8):1802-1807.
[134]Stier S, Totzke G, Grunewald E, et al. Identification of syntenin and other TNF-inducible genes in human umbilical arterial endothelial cells by suppression subtractive hybridization [J]. FEBS Lett, 2000,467(223):299-304.
[135]Zylka M J, Reppert S M. Discovery of a putative heme-binding protein family (SOUL/HBP) by two-tissue suppression subtractive hybridization and database searches [J]. Brain Res Mol Brain Res,1999,74(122):175-181.
[136]Bleicher F, Couble M L, Buchaille R, et al. New genes involved in odontoblast differentiation [J]. Adv Dent Res,2001,15:30-33.
[137]Liu Z W, Zhao M J, Li Z P, et al. Identification of Up-regulated Genes in rat regenerating liver tissue by suppression subtrative hybridization [J]. Shengwu Huaxue yu Shengwu Wuli Xuebao (Shanghai),2001,33(2):191-197.
[138]Rimner A,Wischhusen J, Naumann U, et al. Identification by suppression subtractive hybridization of p21 as a radio-in-ducible gene in human glioma cells [J]. Anticancer Res,2001, 21(5):3505-3508.
[139]Luo M J and Lai M D. Identification of differentially expressed genes in normal mucosa, adenoma and adenocarcinoma of colon by SSH [J]. World J Gastroenterol,2001,7(5):726-731.
[140]Bahn S C, Bae M S, Park Y B, et al. Molecular cloning and characterization of a novel low temperature-induced gene from barley (Hordeum vulgare L) [J]. Biochim Biophys Acta,2001, 1522(2):134-137.
[141]Lin S, Chugh S, Pan X, et al. Identification of up-regulated Ras-like GTPase, Raplb by suppression subtractive hybridization [J]. Kidney Int,2001,60(6):2129-2141.
[142]Stassar M J, Devitt G, Brosius M, et al. Identification of human renal cell carcinoma associated genes by suppression subtractive hybridization [J]. J Cancer,2001,85(9):1372-1382.
[143]Gao B, Allen R, Maier T, et al. Identification of putative parasitism genes expressed in the esophageal gland cells of the soybean cyst nematode Heterodera glycines [J]. Mol Plant Microbe Interact,2001,14(10):1247-1254.
[144]Niak C H, Su K W, Ko R C, et al. Identification of some heat-in-duced genes of Trichinella spiralis [J]. Parasitology,2001,123(3):293-300.
[145]Hinderhofer K and Zentgraf U. Identification of a transcription factor specifically expressed at the onset of leaf senescence [J]. Planta,2001,213(3):469-473.
[146]Larose M, Bouchard C, Chagnon Y C. A new gene related to human obesity identified by suppression subtractive hybridization [J]. Int J Obes Relat Metab Disord,2001,25(6):770-776.
[147]Du Z, Cong H, Yao Z, et al. Identification of putative downstream genes of Oct24 by suppression-subtractive hybridization [J]. Biochem Biophys Res Commun,2001,282(3):701-706.
[148]Atalay A, Crook T, Ozturk M, et al. Identification of genes induced by BRCA 1 in breast cancer cells [J]. Biochem Biophys Res Commun,2002,299(5):839-846.
[149]Li E M, Chen W Y, Chen A Y, et al. Identification of overexpressed genes induced by nitric oxid in human esophageal carcinoma cell line[J]. Aizheng,2002,21(6):610-614.
[150]Fellenberg J, Dechant M J, Ewerbeck V, et al. Identification of drug-regulated genes in osteosarcoma cells [J]. Int J Cancer,2003,105(5):636-643.
[151]Liu L Z, Qian G S, Zhou X D, et al. Expression of a new lung cancer drug resistance-related gene in lung cancer tissues and lung cancer cell strains [J]. Aizheng,2003,22(2):171-174.
[152]Rebrikov D V, Bodanova E A, Bulina M E, et al. A new planarian extrachromosomal virus-like element revealed by subtraction hybridization [J]. Mol Biol (Mosk),2002,36(6):1002-1011.
[153]李文海,邓学梅,李宁,等.SSH法结合定量PCR技术研究双肌臀猪肌肉组织的差异表达基因[J].生物化学与生物物理进展,2005,32(4)：353-358.
[154]毕延震,熊远著,徐德全,等.猪杂种一代与亲本肌肉组织间正反向消减cDNA文库的构建[J].畜牧兽医学报,2005,36(5)：417-421.
[155]黄涛,熊远著,徐德全,等.大梅与梅山猪背最长肌正反消减文库的构建及序列分析[J].农业生物技术学报,2006,14(4)：456-461.
[156]Rentzsch R, Orengo C A. Protein function prediction-the power of multiplicity[J]. Trends Biotechnol,2009,27(4):210-219.
[157]Drysdale R A, Crosby M A. FlyBase Consortium, FlyBase genes and gene models[J]. Nucleic Acids Res,2005,33(Database issue):D390-395.
[158]Mewes H W, Frishman D, Mayer KF, et al. MIPS:analysis and annotation of proteins from whole genomes in 2005[J]. Nucleic Acids Res,2006,34(Database issue):D169-172.
[159]Brown D, Sjolander K. Functional classification using phylogenomic inference[J]. PLoS Comput Biol,2006,2(6):e77.
[160]Krishnamurthy N, Brown DP, Kirshner D, et al. PhyloFacts:an online structural phylogenomic encyclopedia for protein functional and structural classification[J]. Genome Biol,2006,7(9):R83.
[161]Thomas P D, Mi H, Lewis S. Ontology annotation:mapping genomic regions to biological function[J]. Curr Opin Chem Biol,2007,11(1):4-11.
[162]Thomas P D, Kejariwal A, Guo N, et al. Applications for protein sequence-function evolution data:mRNA/protein expression analysis and coding SNP scoring tools[J]. Nucleic Acids Res,2006, 34(Web Server issue):W645-650.
[163]Huang da W, Sherman B T, Tan Q, et al. The DAVID Gene Functional Classification Tool:a novel biological module-centric algorithm to functionally analyze large gene lists[J]. Genome Biol, 2007,8(9):R183.
[164]Mi H, Lazareva-Ulitsky, B Loo, et al. The PANTHER database of protein families, subfamilies, functions and pathways[J]. Nucleic Acids Res,2005,33, D284-D288.
[165]Thomas P D, Campbell M J, Kejariwal A, et al. PANTHER:a library of protein families and subfamilies indexed by function[J]. Genome Res,2003,13,2129-2141.
[166]Venter J C, Adams M D, Myers E W, et al. The sequence of the human genome[J]. Science,2001, 291(5507):1304-1351.
[167]Mi H, Vandergriff J, Campbell M, et al. Assessment of genome-wide protein function classification for Drosophila melanogaster[J]. Genome Res,2003,13(9):2118-2128.
[168]Sonnhammer E L, Eddy S R, Durbin R. Pfam:a comprehensive database of protein domain families based on seed alignments[J]. Proteins,1997,28(3):405-420.
[169]Schultz J, Milpetz F, Bork P, et al. SMART, a simple modular architecture research tool: identification of signaling domains[J]. Proc Natl Acad Sci USA,1998,95(11):5857-5864.
[170]Thomas P D, Kejariwal A, Campbell M J, et al. PANTHER:a browsable database of gene products organized by biological function, using curated protein family and subfamily classification[J]. Nucleic Acids Res,2003,31(7):334-341.
[171]Mi H, Guo N, Kejariwal A, et al. PANTHER version 6:protein sequence and function evolution data with expanded representation of biological pathways[J]. Nucleic Acids Res,2007,35: D247-252.
[172]Dennis G J, Sherman B T, Hosack D A, et al. DAVID:Database for Annotation, Visualization, and Integrated Discovery[J]. Genome Biol,2003,4(5):P3.
[173]Huang da W, Sherman B T, Tan Q, et al. DAVID Bioinformatics Resources:expanded annotation database and novel algorithms to better extract biology from large gene lists[J]. Nucleic Acids Res, 2007,35(Web Server issue):W169-175.
[174]Stuart K Kim. Functional genomics:The worm scores a knockout[J]. Current Biology,2001, 11(3):R85-R87.
[175]Ying Cai, Ying Gao, Qi Sheng, et al. Characterization and potential function of a novel testis-specific nucleoporin BS-63[J]. Mol Reprod Dev,2002,61(1):126-134.
[176]Schaefer B C. Revolutions in Rapid Amplification of cDNA Ends:New Strategies for Polymerase Chain Reaction Cloning of Full-Length cDNA Ends[J]. Analytical Biochemistry,1995,227(2): 255-273.
[177]Sandro Banfi, Alessandro Guffanti and Giuseppe Borsani. How to get the best of dbEST[J]. Trends in Genetics,1998,14(2):80-81.
[178]Gill R W, Sanseau P. Rapid in silico cloning of genes using expressed sequence tags (ESTs)[J]. Biotechnol Annu Rev,2000,5:25-44.
[179]The FANTOM Consortium. Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs [J]. Nature,2002,420 (6915):563-573.
[180]Boguski M S, Tolstoshev C M, and Bassett D E. Gene discovery in dbEST[J]. Science,1994, 265(5181):1993-1994.
[181]张德礼,丁培国,凌伦奖,等.人类新基因C17orf32的电子克隆和编码区序列RT-PCR验证[J].生物化学与生物物理进展,2002,29(4)：543-549.
[182]刘庆坡,冯英,董辉.水稻线粒体丝氨酸羟甲基转移酶基因的电子克隆[J].生物信息学,2005,3(1)：5-9.
[183]黄骥,王建飞,张红生,等.水稻葡萄糖-6-磷酸脱氢酶cDNA的电子克隆[J].遗传学报,2002,29(11)：1012-1016.
[1]A ilhand G, Amn E, Bardon S, et al. Growth and differentiation of regional adipose tissue molecular and hormonal mechanisms[J]. International Journal of Obesity,1991,15:87-90.
[2]Steven R Smith, Barbara Gawronska-Kozak, Lenka Janderova,et al. Agouti Expression in Human Adipose Tissue:Functional Consequences and Increased Expression in Type 2 Diabetes [J]. Diabetes, Dec 2003,52:2914-2922.
[3]Patricia A. Zuk, Min Zhu, Peter Ashjian, et al. Human Adipose Tissue Is a Source of Multipotent Stem Cells[J]. Molecular Biology of the Cell,2002,12(13):4279-4295.
[4]屈长青,张国华,陈粉粉,等.猪前体脂肪细胞的原代培养[J].农业生物技术学报,2005,13(5)：649-653.
[5]王竹晨,刘建中,李燕,等.人前脂肪细胞的原代培养[J].中山医科大学学报,2001,22：443-447.
[6]夏成,王哲,朱淑玲,等.犊牛前脂肪细胞的培养及其增殖与分化模型的建立[J].中国兽医科技,2004,34：26-30.
[7]陈世义,马刚,于海滨等.人工真皮成纤维细胞的分离培养与鉴定[J].吉林大学学报(医学版),2002,28(4)：437-439
[8]Van R L, Bayliss C E and Roncari D A. Cytological and enzymological characterization of human adult adipocyte precursors in culture. Clin Invest,1976,58:699-704
[9]朱晓海,何清濂,林子豪,等.人前脂肪细胞培养及增殖与分化模型的建立[J].中华整形烧伤外科杂志,1999,15(3)：199-204
[10]McGarrity G J, J Sarama, and V Vanaman. Cell culture techniques. ASMNews,1985,51:170-183.
[11]Ryan J. Understanding and managing cell culture conlamination. New York:Cornig Inc,1994, 180-195.
[1]Ailhaud G, Amri E, Bardon S, et al. Growth and differentiation of regional adipose tissue molecular and hormonal mechanisms[J]. Int J Obesity,1991,15:87-90.
[2]Steven R Smith, Barbara Gawronska-Kozak, Lenka Janderova, et al. Agouti Expression in Human Adipose Tissue:Functional Consequences and Increased Expression in Type 2 Diabetes[J]. Diabetes,2003,52:2914-2922.
[3]M Wabitschl, R E Brenner, I Melzner, et al. Characterization of a human preadipocyte cell strain with high capacity for adipose differentiation[J]. Int J Obesity,2001,25:8-15.
[4]Patricia A Zuk, Min Zhu, Peter Ashjian, et al. Human Adipose Tissue Is a Source of Multipotent Stem Cells[J]. Mol Biol Cell,2002,13:4279-4295.
[5]Karsten Hemmrich, Dennis von Heimburg,Kathrin Cierpka, et al. Optimization of the differentiation of human preadipocytes in vitro[J]. Differentiation,2005,73:28-35.
[6]J.Mark Brown and Michael K Mcintosh. Conjugated Linoleic Acid in Humans:Regulation of Adiposity and Insulin Sensitivity [J]. J Nutr,2003,133(10):3041-3046.
[7]卢慧玲,王宏伟,Katherine Cianflone,等.促酰化蛋白(ASP)诱导3T3-L1前脂肪细胞分化[J].中国生物化学与分子生物学报,2004,20(3)：383-386.
[8]Barak Y, Nelson MC, Ong ES, et al. PPAR gamma is required for placental, cardiac, and adipose tissue development[J]. Mol Cell,1999,4:585-595.
[9]Rosen E D, Walkey C J, Puigserver P, et al. Transcriptional regulation of adipogenesis[J]. Gene DEV,2000,14:1293-1307.
[10]Linhart HG, Ishimura-Oka K, DeMayo F, et al. C/EBPalpha is required for differentiation of white, but not brown, adipose tissue[J]. PNAS,2001,98:12532-12537.
[11]S Kersten, J Seydoux, J M Peters, et al. Peroxisome proliferator-activated receptor a mediates the adaptive response to fasing[J]. Journal of Clinical Investigation,1999,103(11):1489-1498.
[12]Chieh J Chou, Martin Haluzik, Charmaine Gregory, et al. WY14643, a peroxisome proliferator-activated receptor a (PPARa) agonist, improves hepatic and muscle steatosis and reverses insulin resistance in lipoatrophic A-ZIP/F-1 mice[J]. Journal of Biological Chemistry, 2002,277(27):24484-24489.
[13]S Mandard, M M"uller, and S Kersten. Peroxisome proliferator-activated receptor a target genes[J]. Cellular and Molecular Life Sciences,2004,61(4):393-416.
[14]Hana Koutnikova, Terrie-Anne Cock, Mitsuhiro Watanabe, et al. Compensation by the muscle limits the metabolic consequences of lipodystrophy in PPARy hypomorphic mice[J]. Proceedings of the national academy of sciences of the united states of America,2003,100(24), supplement 2:14457-14462.
[15]Rinke Stienstra, Caroline Duval, Michael Muller, et al. PPARs, Obesity, and Inflammation[J]. PPAR Research, Volume 2007, Article ID 95974,10 pages.
[16]P Wang, J Renes, F Bouwman, et al. Absence of an adipogenic effect of rosiglitazone on mature 3T3-L1 adipocytes:increase of lipid catabolism and reduction of adipokine expression[J]. Diabetologia,2007,50:654-665.
[17]David S Kumpl and Frank W Booth. Sustained rise in triacylglycerol synthesis and increased epididymal fat mass when rats cease voluntary wheel running [J]. J Physiol Biochem,2005,565 (3): 911-925.
[18]B LEDERER and H G HERS. A kinetic study of glycerophosphate acyltransferase of rat adipocytes in relation to its control by noradrenaline[J]. Biochem J,1985,226:269-274.
[19]Thomas E. BROAD and Richard G. HAM.Growth and adipose differentiation of sheep preadipocyte fibroblasts in serum-free medium[J]. European Journal of Biochemistry,1983, 135:33-39.
[20]Wilfried Schmidt, Gisela Poll-Jordan, and Georg Loffler. Adipose Conversion of 3T3-L1 Cells in a Serum-free Culture System Depends on Epidermal Growth Factor, Insulin-like Growth Factor I, Corticosterone, and Cyclic AMP[J]. The Journal of Biological Chemistry,1990,265(26): 15489-15495.
[21]C. Boone, F Gre'goire, and C Remade. Culture of porcine stromal-vascular cells in serum-free medium:Differential action of various hormonal agents on adipose conversion[J]. Journal of Animal Science,2000,78:885-895.
[22]Haiyan Huang, M Daniel Lane and Qi-Qun Tang. Effect of serum on the down-regulation of CHOP-10 during differentiation of 3T3-L1 preadipocytes[J]. Biochemical and Biophysical Research Communications,2005,338:1185-1188.
[23]朱惠娟,邓洁英,龚凤英,等.无血清原代培养人前脂肪细胞并诱导分化[J].基础医学与临床,2006,26(7)：770-773.
[24]J L Ramirez-Zacarias, F Castro-Mufiozledo and W Kuri-Harcuch. Quantitation of adipose conversion and triglycerides by staining intracyctoplasmic lipids with Oil red O[J]. Histochemistry, 1992,97:493-497.
[25]Eli Eisenberg and Erez Y Levanon. Human housekeeping genes are compact[J]. Trends Genet, 2003,19(7):362-365.
[26]Erkens T, Van Poucke M, Vandesompele J, et al. Development of a new set of reference genes for normalization of real-time RT-PCR data of porcine backfat and longissimus dorsi muscle, and evaluation with PPARGC1A[J]. BMC Biotechnol,2006,6:41.
[27]Ann-Britt Nygard, Claus B Jorgensen, Susanna Cirera, et al. Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR[J]. BMC Mol Biol,2007,8:67.
[28]郭玉姣,唐国庆,李学伟,等.猪脂肪组织中IGF2和IGFBP3基因表达的发育性变化及其品种差异[J].遗传,2008,30(5)：602-606.
[29]张罕星,朱晓彤,束刚,等.猪前脂肪细胞分化过程中聚脂相关基因的表达模式[J].动物学报,2007,53(1)：143-150.
[30]张学进,罗清礼,何为民,等.甲状腺相关眼病及正常人眼眶成纤维细胞与前脂肪细胞培养及分化诱导[J].眼科研究,2007,25(2)：81-85.
[31]Kumpl D S and Booth F W. Sustained rise in triacylglycerol synthesis and increased epididymal fat mass when rats cease voluntary wheel running [J]. Journal of Physiology and Biochemistry,2005, 565 (3):911-925.
[32]Farmer S R. Regulation of PPARα activity during adipogenesis[J]. International Journal of Obesity, 2005,29:13-16.
[33]Gilles Ferry, Edwige Tellier, Anne Try, et al. Autotaxin is released from adipocytes, catalyzes lysophosphatidic acid synthesis, and activates preadipocyte proliferation, Up-regulated expression with adipocyte differentiation and obesity[J]. Journal of Biological Chemistry,2003,278(20): 18162-18169.
[1]Diatchenko L, Lau Y F, Campbell A P, et al. Suppression subtractive hybridization:A method for generating differentially regulated or tissue specific cDNA probes and libraries[J]. Proc Natl Acad Sci USA,1996,93(12):6025-6030.
[2]Gurskaya N G, Diatchenko L, Chenchik A, et al. Equalizing cDNA subtraction based on selective suppression of polymerase chain reaction:cloning of Jurkat cell transcripts induced by phytohemaglutinin and phorbol 12-myristate 13-acetate[J]. Anal Biochem,1996,240(1):90-97.
[3]Xu D Q, Xiong Y Z, Ling X F, et al. Identification of a differential gene HUMMLC2B between F1 hybrids Landrace×Yorkshire and their female parents Yorkshire[J]. Gene,2005,352:118-126.
[4]Ponsuksili S,Tesfaye D, El-Halawany, et al. Stage-spccific expressed sequence tags obtained during preimplantation bovine development by differential display RT-PCR and suppression subteaction hybridization[J]. Prenatal Diagnosis,2002,229(12):1135-1142.
[5]Paul D Thomas, Michael J Campbell, Anish Kejariwal, et al. PANTHER:a library of protein families and subfamilies indexed by function[J]. Genome Res,2003,13:2129-2141.
[6]Huaiyu Mi, Nan Guo, Anish Kejariwal, et al. PANTHER version 6:protein sequence and function evolution data with expanded representation of biological pathways[J]. Nucl. Acids Res,2007,35: D247-D252.
[7]Dennis G Jr, Sherman BT, Hosack DA, et al. DAVID:Database for Annotation, Visualization, and Integrated Discovery[J]. Genome Biol,2003,4(5):P3.
[8]Huang D W, Sherman B T, Lempicki R A. Systematic and integrative analysis of large gene lists using DAVID Bioinformatics Resources[J]. Nature Protoc,2009,4(1):44-57.
[9]Davoli R, Zambonelli P, Bigi D, et al. Analysis of expressed sequence tags of porcine skeletal muscle[J]. Gene,1999,233:181-188.
[10]Vilson M E, Sonstegard T S, SmithT P, et al. Differential gene expression during elongation in the preimplantation pig embryo[J]. Gene,2000,26 (1):9-14.
[11]Lee S H, Zhao S H, Recknor J C, et al. Transcriptional profiling using a novel cDNA array identifies differential gene expression during porcine embryo elongation[J]. Mol Reprod Dev,2005, 71(2):129-139.
[12]Stain O D, Thies W G, Hofmann M. A high throughput screening for rarely transcribed differentially expressed genes[J]. Nucleic Acids Res,1991,25:2598-2602.
[13]陈数珍,马文丽,郑文岭.宫颈癌相关基因的筛选及生物信息学分析[J].南方医科大学学报,2008,28(4)：585-588.
[14]薛净,李瑜羌,聂玉强,等.苦参碱对人肝癌细胞株HepG2基因表达谱的影响[J].胃肠病学和肝病学杂志,2007,16(4)：341-343.
[15]廖之君,马文丽,梁爽,等.荧光交换标记法基因表达芯片的数据挖掘[J].福建医科大学学报,2008,42(3)：236-239.
[1]Cohen B A, Mitra R D, Hughes J D, et al. A computational analysis of whole-genome expression data revaels chromosomal domains of gene expression[J]. Nat genet,2000,26(2):183-186.
[2]Bussemaker H J, Li H, Siggia E D. Regulatory element detection using correlation with expression[J]. Nat genet,2001,27(2):167-171.
[2]Carrel A. On the Permanent life of tissues out side the Organism[J]. J Exp Med,1912,15:516-528.
[3]Earle W R, Schilling EL, Stark T H, et al. The mouse Fibroblast cultures and changes seen in the living cells[J]. NATL Cancer Inst,1943,4:165-212.
[4]Gey G O, Coffman W D, Kubicek M T, et al. Tissue culture studies of the proliferative capacity of cervical carcinoma and normal epithelium[J]. Cancer Res,1952,12:364-365.
[5]Ailhaud G, Amri E, Bardon S, et al. Growth and differentiation of regional adipose tissue molecular and hormonal mechanisms[J]. Int J Obesity,1991,15:87-90.
[6]Steven R Smith, Barbara Gawronska-Kozak, Lenka Janderova, et al. Agouti Expression in Human Adipose Tissue:Functional Consequences and Increased Expression in Type 2 Diabetes. Diabetes, 2003,52:2914-2922.
[7]Smas C M, Chen L, Sul H S, et al. St ructural characterization and alternate splicing of the gene encoding the preadipocyte EGF-like protein pref-1 [J]. Biochemistry,1994,33:9257-9265.
[8]Umek R M, Friedman A D, Mcknight S L, et al. CCAAT-enhancerbinding protein:a component of a differentiation switch [J]. Science,1991,251:288-292.
[9]James M Ntambi and Young-Cheul Kim. Adipocyte Differentiation and Gene Expression[J]. Journal of Nutrition,2000,130:3122S-3126S.
[10]Morrison R F and Farmer S R. Hormonal signaling and transcriptional control of adipocyte differentiation [J]. American Society for Nutritional Sciences,2000,130 (12):3116S-3121S.
[11]夏成,王哲,朱淑玲,等.犊牛前脂肪细胞的培养及其增殖与分化模型的建立[J].中国兽医科技,2004,34(5)：26-30.
[12]Satol Nakanishi N and Mit sumoto M. Culture condition supporting adipocyte conversion of st romal-vascular cells from bovine intramuscular adipocyte tissues [J]. Journal of Veterinary Medical Science,1997,58:1073-1078.
[13]Marshall S, Garvey WT and Geller M. Primary culture of isolated adipocytes:A new model to study insulin receptor regulation and insulin action [J]. Biol Chem,1984,259(10):6376-6384.
[14]王竹晨,刘建中,李燕,等.人前脂肪细胞的原代培养[J].山医科大学学报,2001,22(6)：443-446.
[15]Satol Nakanishi N and Mit sumoto M. Culture condition supporting adipocyte conversion of st romal-vascular cells from bovine intramuscular adipocyte tissues [J]. Journal of Veterinary Medical Science,1997,58:1073-1078.
[16]Sib S R, Mohua M, Samir B, et al. A new cell secreting insulin [J]. Endocrinology,2003,4 (144): 1585-1593.
[17]Hausman G J, D R Campion, J P McNamara, et al. Adiposetissue development in the fetal pig after decapition[J]. J Anim Sci,1981,53:1634-1644.
[18]Hausman G J, Novakofski J E, Martin R J, et al. The development of adipocytes in primary stromal-vascular culture of fetal pig adipose tissue [J]. Cell Tissue Res,1984,236:459-464.
[19]Sarawak A, Swanson V and Hun C Y. Insulin and hydrocortisone, but not triiodothyronine, are required for the differentiation of pig preadipocytes in primary cultures [J]. J Anim Sci,1997,75: 105-111.
[20]Brandebourg T D, Hu C Y. Isomer-specific regulation of differentiating pig preadipocytes by conjugated linoleic acids [J]. J Anim Sci,2005,83:2096-2105.
[21]Ramsay T. Porcine preadipocyte proliferation and differentiation:A role for leptin [J]. J Anim Sci, 2005,83:2066-2074.
[22]屈长青,张国华,陈粉粉,等.猪前体脂肪细胞的原代培养[J].农业生物技术学报,2005,13(5)：649-653.
[23]张罕星,朱晓彤,束刚,等.猪脂肪前体细胞分化过程中聚脂相关基因的表达模式[J].动物学报,2007,53(1)：143-150.
[24]Pilgrim C H. DNA synthesis and differentiation in developing white adipose tissue [J]. Dev Biol,1971,26:69-76.
[25]Patrick C W, Chauvin PB, Robb G L, et al. Tissue enmneered adipose frontiers in tissue engineering[J]. Science,1998,369-382.
[26]Tang Q Q, Tamara C, Otto M, et al. Commit of C3H10 T1/2 pluripotent stem cells to the adipocyte lineage [J]. PNAS,2004,101(26):9607-9611.
[27]Tamara C, Otto M, Lane D, et al. Adipose development:from stem cell to adipocyte [J]. Critical Reviews in Biochemistry and Molecular Biology,2005,40(4):229-242.
[28]Dani C, Smith A G, Dessolin S, et al. Difirentiation of embryonic stem cell into adipocyte in vitro [J]. Cell Sci,1997,110:1279-1285.
[29]Hugo E R, Brandebourg T D, Comstock C E, et al. Anovel human adipocyte cell line that produces prolactin [J]. Endocrinology,2005,147(1):306-313.
[30]Nathan E, Wolins, Benjamin K. Quaynor, OP9 mouse stromal cells rapidly differentiate into adipocytes:Characterization of a useful new model of adipogenesis [J]. J Lipid Res,2005,11: 1194-1198.
[31]Dorothy B Hausman, Hea Jin Park and Gary J Hausman. Isolation and Culture of Preadipocytes from Rodent White Adipose Tissue. Adipose Tissue Protocols,2008:201-219.
[32]Rodriguez A M, Elabd C, Delteil F, et al. Adipocyte differentiation of multipotent cells established from human adipose tissue[J]. Biochem Biophys Res Commun,2004,315 (2):255-263.
[33]Ntambi J M.Adipocyte differentiation and gene expression [J]. J Nutr,2000,13(12):3122s-3126s.
[34]Zhang H, Noohr J, Jensen CH, et al. Insulin-like growth factor-1/insulin bypasses P-ref-1/FAL-mediated inhibition of adipocyte differentiation[J]. Biochem,2003, 278(23):20906-20914.
[35]倪毓辉,郭锡熔,陈荣华,等.脂肪细胞分化的表型特征、分子标志和调控[J].国外医学儿科学分册,2004,31(6)：306-308.
[36]Hug C, Lodish H F. Diabetes, obesity, and Acrp30/adiponectin [J]. Biotechniques,2002, 33(3):654-658.
[37]Otto T C and Lane M D. Adipose development:from stem cell to adipocyte[J]. Critical Reviews in Biochemistry and Molecular Biology,2005,40(4):229-242.
[38]Farmer S R. Regulation of PPARgamma activity during adipogenesis[J]. International Journal of Obesity,2005,29(Suppl 1):13-16.
[39]Lekstrom-Himes J A. The role of C/EBP (epsilon) in the terminal stages of granulocyte differentiation[J]. Stem Cells,2001,19(2):125-133.
[40]Gregoire F M. Adipocyte differentiation:from fibroblast to endocrine cell[J]. Experimental Biology and Medicine (Maywood),2001,226:997-1002.
[41]Richards M P, Poch S M, Coon C N, et al. Feed restriction significantly alters lipogenic gene expression in broiler breeder chickens[J]. Journal of Nutrition,2003,133:707-715.
[42]Matsubara Y, Sato K, Ishii H, et al. Changes in mRNA expression of regulatory factors involved in adipocyte differentiation during fatty acid induced adipogenesis in chicken[J]. Comparative Biochemistry and Physiology Part A:Molecular & Integrative Physiology,2005,141(1):108-115,
[43]Stahl A. Current review of fatty acid transport proteins (SLC27)[J]. Pflugers Archiv European Journal of Physiology,2004,447:722-727.
[44]Ferre P. The Biology of Peroxisome Proliferator-Activated Receptors:Relationship with Lipid Metabolism and Insulin Sensitivity [J]. Diabetes,2004,53(suppl 1):s43-s50.
[45]Tsuchida A, Yamauchi T, Kadowaki T, et al. Nuclear Receptors as Targets for Drug Development: Molecular Mechanisms for Regulation of Obesity and Insulin Resistance by Peroxisome Proliferator-Activated Receptor y, CREB-Binding Protein, and Adiponectin[J]. Pharmacol Sci, 2005,97 (2):164-170.
[46]Evan D Rosen, Pasha Sarraf, Amy E Troy, et al. PPARγ is required for the differentiation of adipose tissue in vivo and in vitro [J]. Mol Cell,1999,4:611-617.
[47]Liping Qiao, Paul S MacLean, Jerome Schaack, et al. C/EBP alpha regulates human adiponectin gene transcription through an intronic enhancer[J]. Diabetes,2005,54(6):1744-1754.
[48]Zhang Jiang-Wen, Dwight J Klemm, Charles Vinson, et al. Role of CREB in Transcriptional Regulation of CCAAT/Enhancer-binding Protein P Gene during Adipogenesis [J]. J Biol Chem, 2004,279(6):4471-4478.
[49]Youngjin Choi, Young-Cheul Kim, Yong-Bong Han, et al. The trans-10, cis-12 Isomer of Conjugated Linoleic Acid Downregulates Stearoyl-CoA Desaturase 1 Gene Expression in 3T3-L1 Adipocytes[J]. Journal of Nutrition,2000,130:1920-1924.
[50]Yeh W C, Cao Z, Classon M, et al. Cascade regulation of terminal adipocyte differentiation by three members of the C/EBP family of leucine zipper proteins[J]. Genes & Dev,1995,9:168-181.
[51]Tanaka T, Yoshida N, Kishimoto T, et al. Defective adipocyte differentiation in mice lacking the C/EBP beta and/or C/EBP delta gene[J]. EMBO J,1997,16(24):7432-7443.
[52]Jae Bum Kim, Harold M Wright, Margaret Wright, et al. ADD1/SREBP1 activates PPARy through the production of endogenous ligand [J]. Proc Natl Acad Sci USA,1998,95(8):4333-4337.
[53]Lluis Fajas, Kristina Schoonjans, Laurent Gelman, et al. Regulation of Peroxisome Proliferator-Activated Receptor y Expression by Adipocyte Differentiation and Determination Factor 1/Sterol Regulatory Element Binding Protein 1:Implications for Adipocyte Differentiation and Metabolism [J]. Mol Cell Biol,1999,19(8):5495-5503.
[54]Sato K, Nakanishi N and Mitsumoto M. Culture conditions supporting adipose conversion of stromal-vascular cells from bovine intramuscular adipose tissues [J]. Journal of Veterinary Medical Science,1996,58:1073-1078.
[55]Mur C, Arribas M, Benito M, et al. Essential role of insulin-like growth factor I receptor in insulin-induced fetal brown adipocyte differentiation[J]. Endocrinology,2003,144:581-593.
[56]Lin J, Arnold H B, Della-Fera M A, et al. Myostatin knockout in mice increases myogenesis and decreases adipogenesis[J]. Biochemical and Biophysical Research Communications,2002,291: 701-706.
[57]Van Hall G, Steensberg A, Sacchetti M, et al. Interleukin-6 stimulates lipolysis and fat oxidation in humans[J]. Journal of Clinical Endocrinology & Metabolism,2003,88:3005-3010.
[58]Tang Q Q, Otto T C, Lane MD, et al. CCAAT/enhancer-binding protein β is required for mitotic clonal expansion during adipogenesis[J]. Natl Acad Sci USA,2003,100(3):850-855.
[59]Smas C M, Chen I, Zhao L, et al. Transcriptional Repression of pref-1 by Glucocorticoids Promotes 3T3-L1 Adipocyte Differentiation [J]. J Bio l Chem,1999,274(6):12632-12641.
[60]Tang Q Q, Jiang M S, Lane M D. Repressive effect of SP1 on the COBRA alpha gene promoter: role in adipocyte differentiation [J]. Mol Cell Biod,1999,19(7):4855-4865.
[61]Aubert J, Saint-Marc P, Belmonte N, et al. Prostacyclin IP receptor up-regulates the early expression of C/EBP beta and C/EBP delta in preadipose cells[J].Mol Cell Endocrinol, 2000,160(1-2):149-156.
[62]Zhang H H, Halbleib M, Ahmad F, et al. Tumor necrosis factor-alpha stimulates lipolysis in differentiated human adipocytes through activation of extracellular signal-related kinase and elevation of intracellular cAMP[J]. Diabetes,2002,51:2929-2935.
[63]Smas C M, Chen L, Sul H S, et al. Structural characterization and alternate splicing of the gene encoding the preadipocyte EGF-like protein Pref-1 [J]. Biochemistry,1994,33:9257-9265.
[64]Smas C M, Chen L, Sul H S. Cleavage of membrane-associated pref-1 generates a soluble inhibitor of adipocyte differentiation[J]. Mol Cell Biol,1997,17:977-988.
[65]Man-Shiow Jiang and M Daniel Lane. Sequential repression and activation of the CCAAT enhancer-binding protein-a (C/EBPα) gene during adipogenesis[J]. PNAS,2000,97(23): 12519-12523.
[66]Francisco Felipe, M Luisa Bonet, Joan Ribot, et al. Modulation of Resistin Expression by Retinoic Acid and Vitamin A Status [J]. Diabetes,2004,53:882-889.
[67]Brandebourg T D and Hu C Y. Regulation of differentiating pig preadipocytes by retinoic acid [J]. Anim. Sci,2005,83:98-107.
[68]Dani C, Smith A G, Dessolin S, et al. Differentiation of embryonic stem cells into adipocytes in vitro[J]. J Cell Sci,1997,110(11):1279-1285.
[69]Stephen F Konieczny and Charles P Emerson.5-azacytidine induction of stable mesodermal stem cell lineages from 10T1/2 cells:Evidence for regulatory genes controlling determination[J]. Cell, 1984,38(3):791-800.
[70]Chapman A B, Knight D M, Dieckmann B S, et al. Analysis of gene expression during differentiation of adipogenic cells in culture and hormonal control of the developmental program[J]. J Biol Chem,1984,259(24):15548-15555.
[71]RubinC S, Hirsch A, Fung C, et al. Development of hormone receptors and hormonal responsiveness in vitro Insulin receptors and insulin sensitivity in the preadipocyte and adipocyte forms of 3T3-L1 cells[J]. J Biol Chem,1978,253(20):7570-7578.
[72]Wang P, Renes J, Bouwman F, et al. Absence of an adipogenic effect of rosiglitazone on mature 3T3-L1 adipocytes:increase of lipid catabolism and reduction of adipokine expression[J]. Diabetologia,2007,50:654-665.
[73]Naima Moustaid, Francoise Lasnier, Bernard Hainque, et al. Analysis of gene expression during adipogenesis in 3T3-F442A preadipocytes:Insulin and dexamethasone control[J]. J Cell Biochem, 1990,42:243-254.
[74]卢慧玲,王宏伟,Katherine Cianflone,等.诱导SW872前脂肪细胞分化的最优方法探讨[J]. 基础医学与临床,2006,26(3)：15-318.
[75]Gharbi-Chihi J, Grimaldi P, Torresani J, et al. Triiodothyronine and adipose conversion of OB17 preadipocytes:binding to high affinity sites and effects on fatty acid synthetizing and esterifying enzymes[J]. J Recept Res,1981,2(2):153-173.
[76]Wabitsch M, Heinze E, Hauner H, et al. Biological effects of human growth hormone in rat adipocyte precursor cells and newly differentiated adipocytes in primary culture[J]. Metabolism, 1996,45(1):34-42.
[77]李舜,周杰,郑智勇,等.共轭亚油酸对大鼠前体脂肪细胞增殖与分化的影响[J].中国畜牧杂志,2007,43(11)：19-21.
[78]Litthauer D and Serrero G. The primary culture of mouse adipocyte precursor cells in defined medium[J]. Biochem Physiology,1992,101(1):59-64.
[79]Yves Reyne, Jean Nougues and Jean-Paul Dulor. Differentiation of rabbit adipocyte precursor cells in a serum-free medium In Vitro Cell[J]. Dev Biol,1989,25(8):747-752.
[80]Suryawan A, Swanson L V and Hu C Y. Insulin and hydrocortisone, but not triiodothyronine, are required for the differentiation of pig preadipocytes in primary culture[J]. J Anim Sci,1997,75(1): 105-111.
[81]Entenmann G and Hauner H. Relationship between replication and differentiation in cultured human adipocyte precursor cells[J]. Am JPhysiol Cell Physiol,1996,270(39):C1011-C1016.
[82]Heid C A, Stevens J, Livak K J, et al.Real time quantitative PCR[J].Genome Research,1996, 6(10):986-994.
[83]Zhu SH F. The Detected Technology of Real-time Fluorogenic PCR [M]. Beijing:China Measure Press,2003.
[84]Walker N J. Real time and quantitative PCR:applications to mechanism based toxicology [J]. Biochem Mol Toxicol,2001,15 (3):121-127.
[85]Woo T H, Patel B K, Cinco M, et al. Identification of Leptospira biflexa by real-time homogeneous detection of rapid cycle PCR product [J]. Journal of Microbiological Methods,1999,35:23-30.
[86]Giulietti A, Overbergh L, Valckx D, et al. An overviewof real-time quantitative PCR:application to quantify cytokine gene expression [J]. Methods,2001,25 (4):386-401.
[87]Nami Okumura, Naoko-Kishi Nishizawa, Yosuke Umehara, et al. A dioxygenase gene (Ids2) expressed under iron deficiency conditions in the roots of Hordeum vulgare[J]. Plant Molecular Biology,1994,4(25):705-719.
[88]George P, Douglas T, Yang F, et al. Combining SSH and cDNA microarrays for rapid ide ntification of diferentially expressed genes[J]. Nucleic Acids Research,1999,127(6):1517-1523.
[89]Sturtevant J. Applications of differential display reverse transcription PCR to molecular pathogenesis and medical mycology [J].Clinical Microbial Rev,2000,13:408-427.
[90]SteinauM, Unger E, Jones J and Rajeevan M. Differential display PCR of peripheral blood for biomarker discovery in chronic fatigue syndrome [J]. J Mol Med,2004,82:750-755.
[91]Ripamonte P, Merighe G K, Meirelles F V. Development and optimization of a fluorescent differential display PCR system for studying bovine embryo development in vitro [J]. GenetMol Res,2005,4(4):726-733.
[92]Hong ping Jiang, Dong-chun Kang, Deborah Alexandre, et al. Arapidsubtration hybridization approach for identifying and cloning diferentially expressed genes[J]. Proc Natl Acad Sci,2000, 97(23):12684-12689.
[93]Wang T W, Lu L, Wang D, et al. Isolation and characterization of senescence-induced cDNAs encoding deoxy-hypusine synthase and eukaryotic inittranslation iation factor 5A from tomato[J]. J Biol Chem,2001,276:17541-17549.
[94]Hubank M and Schatz D G. Identifying diferences in mRNA expression by representational dinference analysis of cDNA[J]. Nucleic Acids Res,1994,22 (25):5640-5648.
[95]Hubank M, Bryntesson F, Schatz D G. Cloning of apop tosis-related genes by representational difference analysis of cDNA [J]. Methods Mol Biol,2004,282:255-273.
[96]Bowler L D. Representational difference analysis of cDNA [J]. Methods Mol Med,2004,94:49-66.
[97]Sung B, Jung K J, Chung H Y. cDNA representational difference analysis used in the identification of genes related to the aging process in rat kidney [J]. Mech Ageing Dev,2005,126(8):882-891.
[98]Yang J L, LI G, Zhang F L, et al. Identification of variations of gene expression of visceral adipose and renal tissue in type 2 diabetic rats using cDNA representational difference analysis [J]. Chin Med J,2003,116(4):529-533.
[99]Kang Dong-chul, Raphael LaFance, Zao-zhong Su, et al. Reciprocal subtraction differential RNA display:An efficient and rapid procedure for isolating differentially expressed gene sequences [J]. Proc Natl Acad Sci USA,1998,95:13788-13793.
[100]Velculescu V E, Zhang L and Vogelstein B. Serial analysis of gene expression [J]. Science, 1995,270:484-487.
[101]Richards M, Tan S P. and Tan J H. The transcriptome profile of human embryonic stem cells as defined by SAGE [J]. Stem Cells,2004,22(1):51-64.
[102]Quere R, Manchon L and Lejeune M. Mining SAGE data allows largescale, sensitive screening of antisense transcript expression [J]. Nucleic Acids Res,2004,32(20):e163.
[103]Keime C, Damiola F, Mouchiroud D, et al. Identitag,a relational database for SAGE tag identification and interspecies comparison of SAGE libraries [J]. BMC Bioinformatics,2004, 5:143.
[104]Boheler K R and Tarasov K V. SAGE analysis to identify embryonic stem cell predominant transcripts [J]. Methods Mol Biol,2006,329:195-221.
[105]Richards M, Tan S P, Bongso A, et al.Reverse serial analysis of gene expression (SAGE)characterization of orphan SAGE tags from human embryonic stem cells identifies the presence of novel transcripts and antisense transcription of key pluripotency genes [J]. Stem Cells, 2006,24(5):1162-1173.
[106]Sjostedt A. A microarray analysis of the murine macrophage response to infection with Francisella tularensis LVS [J]. J Med Microbiol,2006,55:1023-1033.
[107]Suzuki T, Tazoe H and Isemura M. DNA microarray analysis of changes in gene expression induced by 1,25-dihydroxyvitamin D3 in human promyelocytic leukemia HL-60 cells [J]. Biomed Res,2006,27(3):99-109.
[108]Andersson H and Hartmanova B. A microarray analysis of the murine macrophage response to infection with Francisella tularensis LVS [J]. J Med Microbiol,2006,55(Pt8):1023-1033.
[109]Peeters J K and Vander Spek P J.Growing applications and advancements in microarray technology and analysis tools [J]. Cell Biochem Biophys,2005,43(1):149-166.
[110]MaurerM, Molidor R, Sturn A, et al. MARS:Microarray analysis, retrieval, and storage system [J]. BMC Bioinformatics,2005,6:101.
[111]Qin L, Rueda L, Ali A,et al. Spot detection and image segmentation in DNA microarray data [J]. Appl Bioinformatics,2005,4(1):1-11.
[112]Diatchenko L, Chris Lau Y F, Campbeu A P, et al. Suppression subtractive hybridization:A method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA,1996,93(12):6025-6030.
[113]Diatchenko L, Lukyanov S, Lau Y F, et al. Suppression subtractive hybridization:a versatile method for identifying differentially expressed genes[J]. Methods Enzymol,1999,303:349-380.
[114]何鹏,江渝.抑制性消减杂交技术及其应用研究进展[J].国外医学临床生物化学与检验学分册,2004,25(4)：653-853.
[115]ShirinM, Sushmita S, Vivek K, et al. Suppression subtractive hybridization coupled with microarray analysis to examine differential expression of genes in virus infected cells Biol [J]. Proced Online,2004,6(1):94-104.
[115]Yao J, Coussens P M, Saama P, et al. Generation of expressed sequence tags from a normalized porcine skeletal muscle cDNA library[J]. Anim Biotechnol,2002,13(2):211-222.
[116]Zhao S H, Nettleton D, Liu W, et al. Complementary DNA macroarray analyses of differential gene expression in porcine fetal and postnatal muscle[J]. J Anim Sci,2003, (9):2179-2188.
[117]Diatchenko L, Chris Lau Y F, Campbeu A P, et al. Suppression subtractive hybridization:A method for generating differentially regulated or tissue-specific cDNA probes and libraries[J]. Proc Natl Acad Sci USA,1996,93(12):6025-6030.
[118]Kuang J and Ashorn C L. At least two kinase phosphoralate the MPM-2 epitope during Xenopusooc-yte maturation [J]. J Cell Biol,1993,123:859-868.
[119]Carmeci C, Thompson D A, Kuang W W, et al. Moesin expression is associated with the estrogen receptor-negative breast cancer phenotype [J]. Surgery,1998,124(2):211-217.
[120]Jin H, Cheng X, Diatchenko L, et al. Differential screening of a subtracted cDNA library:a method to search for genes preferentially expressed in multiple tissues [J]. Biotechniques,1997, 23(6):1084-1086.
[121]Harris A J, Shaddock J G, Manjanatha M G, et al. Identification of differentially expressed genes in aflatoxin B1-treated cultured primary rat hepatocytes and Fischer 344 rats [J]. Carcinogenesis, 1998,19(8):1451-1458.
[122]Ligon A H, Pershouse M A, Jasser S, et al. Differentially expressed gene products in glioblastoma cells suppressed for tumorigenicity [J]. J Neurovirol,1998,4(2):217-226.
[123]VonStein O D, Thies W G, Hofmann M, et al.A high throughput screening for rarely transcribed differentially expressed genes[J]. Nucleic Acids Res,1997,25(13):2598-2602.
[124]Hebrok M, Kim S K, Melton D A, et al. Screening for novel pancreatic genes expressed during embryogenesis [J]. Diabetes,1999,48(8):1550-1556.
[125]Miller F R, Barnbabas N, Liu X, et al. Differential display, subtractive hybridization and application of methodology to search for point mutations to identify genetic defects responsible for progression in MCF10AT model of human breast disease [J]. Electrophoresis,1999, 20(2):256-260.
[126]Murphy M, Godson C, Cannon S, et al. Suppression subtractive hybridization identifies high glucose levels as a stimulus for expression of connective tissue growth factor and other genes in human mesangial cells [J]. J Biol Chem,1999,274(9):5830-5834.
[127]Matsuyama T,Yasumura N, Funakoshi M, et al. Maize genes specifically expressed in the outermost cells of root cap [J]. Plant Cell Physiol,1999,40(5):469-476.
[128]Heine H, Delude R L, Monks B G, et al. Bacterial lipopolysaccharide induces expression of the stress response genes hop and H411 [J]. J Biol Chem,1999,274(30):21049-21055.
[129]Tchernitsa O I, Zuber J, Ser C, et al.Gene expression profiling of fibroblasts resistant toward oncogene 2 mediated transformation reveals preferential transcription of negative growth regulators[J]. Oncogene,1999,18(39):5448-5454.
[130]Wang X, Li X, Yaish-Ohad S, et al. Molecular cloning and expression of the rat monocyte chemotactic protein-3 gene:a possible role in stroke [J]. Brain Res Mol Brain Res,1999, 71(2):304-312.
[131]Yang G P, Ross D T, Kuang W W, et al. Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes [J]. Nucleic Acids Res,1999,27(6):1517-1523.
[132]Kostic C and Shaw P H. Isolation and characterization of sixteen novel p53 response genes [J]. Oncogene,2000,19 (35):3978-3987.
[133]Ye Z and Connor J R. Identification of iron responsive genes by screening cDNA libraries from suppression subtractive hybridization with antisense probes from three iron conditions[J]. Nucleic Acids Res,2000,28(8):1802-1807.
[134]Stier S, Totzke G, Grunewald E, et al. Identification of syntenin and other TNF-inducible genes in human umbilical arterial endothelial cells by suppression subtractive hybridization [J]. FEBS Lett, 2000,467(223):299-304.
[135]Zylka M J, Reppert S M. Discovery of a putative heme-binding protein family (SOUL/HBP) by two-tissue suppression subtractive hybridization and database searches [J]. Brain Res Mol Brain Res,1999,74(122):175-181.
[136]Bleicher F, Couble M L, Buchaille R, et al. New genes involved in odontoblast differentiation [J]. Adv Dent Res,2001,15:30-33.
[137]Liu Z W, Zhao M J, Li Z P, et al. Identification of Up-regulated Genes in rat regenerating liver tissue by suppression subtrative hybridization [J]. Shengwu Huaxue yu Shengwu Wuli Xuebao (Shanghai),2001,33(2):191-197.
[138]Rimner A,Wischhusen J, Naumann U, et al. Identification by suppression subtractive hybridization of p21 as a radio-in-ducible gene in human glioma cells [J]. Anticancer Res,2001, 21(5):3505-3508.
[139]Luo M J and Lai M D. Identification of differentially expressed genes in normal mucosa, adenoma and adenocarcinoma of colon by SSH [J]. World J Gastroenterol,2001,7(5):726-731.
[140]Bahn S C, Bae M S, Park Y B, et al. Molecular cloning and characterization of a novel low temperature-induced gene from barley (Hordeum vulgare L) [J]. Biochim Biophys Acta,2001, 1522(2):134-137.
[141]Lin S, Chugh S, Pan X, et al. Identification of up-regulated Ras-like GTPase, Raplb by suppression subtractive hybridization [J]. Kidney Int,2001,60(6):2129-2141.
[142]Stassar M J, Devitt G, Brosius M, et al. Identification of human renal cell carcinoma associated genes by suppression subtractive hybridization [J]. J Cancer,2001,85(9):1372-1382.
[143]Gao B, Allen R, Maier T, et al. Identification of putative parasitism genes expressed in the esophageal gland cells of the soybean cyst nematode Heterodera glycines [J]. Mol Plant Microbe Interact,2001,14(10):1247-1254.
[144]Niak C H, Su K W, Ko R C, et al. Identification of some heat-in-duced genes of Trichinella spiralis [J]. Parasitology,2001,123(3):293-300.
[145]Hinderhofer K and Zentgraf U. Identification of a transcription factor specifically expressed at the onset of leaf senescence [J]. Planta,2001,213(3):469-473.
[146]Larose M, Bouchard C, Chagnon Y C. A new gene related to human obesity identified by suppression subtractive hybridization [J]. Int J Obes Relat Metab Disord,2001,25(6):770-776.
[147]Du Z, Cong H, Yao Z, et al. Identification of putative downstream genes of Oct24 by suppression-subtractive hybridization [J]. Biochem Biophys Res Commun,2001,282(3):701-706.
[148]Atalay A, Crook T, Ozturk M, et al. Identification of genes induced by BRCA 1 in breast cancer cells [J]. Biochem Biophys Res Commun,2002,299(5):839-846.
[149]Li E M, Chen W Y, Chen A Y, et al. Identification of overexpressed genes induced by nitric oxid in human esophageal carcinoma cell line[J]. Aizheng,2002,21(6):610-614.
[150]Fellenberg J, Dechant M J, Ewerbeck V, et al. Identification of drug-regulated genes in osteosarcoma cells [J]. Int J Cancer,2003,105(5):636-643.
[151]Liu L Z, Qian G S, Zhou X D, et al. Expression of a new lung cancer drug resistance-related gene in lung cancer tissues and lung cancer cell strains [J]. Aizheng,2003,22(2):171-174.
[152]Rebrikov D V, Bodanova E A, Bulina M E, et al. A new planarian extrachromosomal virus-like element revealed by subtraction hybridization [J]. Mol Biol (Mosk),2002,36(6):1002-1011.
[153]李文海,邓学梅,李宁,等.SSH法结合定量PCR技术研究双肌臀猪肌肉组织的差异表达基因[J].生物化学与生物物理进展,2005,32(4)：353-358.
[154]毕延震,熊远著,徐德全,等.猪杂种一代与亲本肌肉组织间正反向消减cDNA文库的构建[J].畜牧兽医学报,2005,36(5)：417-421.
[155]黄涛,熊远著,徐德全,等.大梅与梅山猪背最长肌正反消减文库的构建及序列分析[J].农业生物技术学报,2006,14(4)：456-461.
[156]Rentzsch R, Orengo C A. Protein function prediction-the power of multiplicity[J]. Trends Biotechnol,2009,27(4):210-219.
[157]Drysdale R A, Crosby M A. FlyBase Consortium, FlyBase genes and gene models[J]. Nucleic Acids Res,2005,33(Database issue):D390-395.
[158]Mewes H W, Frishman D, Mayer KF, et al. MIPS:analysis and annotation of proteins from whole genomes in 2005[J]. Nucleic Acids Res,2006,34(Database issue):D169-172.
[159]Brown D, Sjolander K. Functional classification using phylogenomic inference[J]. PLoS Comput Biol,2006,2(6):e77.
[160]Krishnamurthy N, Brown DP, Kirshner D, et al. PhyloFacts:an online structural phylogenomic encyclopedia for protein functional and structural classification[J]. Genome Biol,2006,7(9):R83.
[161]Thomas P D, Mi H, Lewis S. Ontology annotation:mapping genomic regions to biological function[J]. Curr Opin Chem Biol,2007,11(1):4-11.
[162]Thomas P D, Kejariwal A, Guo N, et al. Applications for protein sequence-function evolution data:mRNA/protein expression analysis and coding SNP scoring tools[J]. Nucleic Acids Res,2006, 34(Web Server issue):W645-650.
[163]Huang da W, Sherman B T, Tan Q, et al. The DAVID Gene Functional Classification Tool:a novel biological module-centric algorithm to functionally analyze large gene lists[J]. Genome Biol, 2007,8(9):R183.
[164]Mi H, Lazareva-Ulitsky, B Loo, et al. The PANTHER database of protein families, subfamilies, functions and pathways[J]. Nucleic Acids Res,2005,33, D284-D288.
[165]Thomas P D, Campbell M J, Kejariwal A, et al. PANTHER:a library of protein families and subfamilies indexed by function[J]. Genome Res,2003,13,2129-2141.
[166]Venter J C, Adams M D, Myers E W, et al. The sequence of the human genome[J]. Science,2001, 291(5507):1304-1351.
[167]Mi H, Vandergriff J, Campbell M, et al. Assessment of genome-wide protein function classification for Drosophila melanogaster[J]. Genome Res,2003,13(9):2118-2128.
[168]Sonnhammer E L, Eddy S R, Durbin R. Pfam:a comprehensive database of protein domain families based on seed alignments[J]. Proteins,1997,28(3):405-420.
[169]Schultz J, Milpetz F, Bork P, et al. SMART, a simple modular architecture research tool: identification of signaling domains[J]. Proc Natl Acad Sci USA,1998,95(11):5857-5864.
[170]Thomas P D, Kejariwal A, Campbell M J, et al. PANTHER:a browsable database of gene products organized by biological function, using curated protein family and subfamily classification[J]. Nucleic Acids Res,2003,31(7):334-341.
[171]Mi H, Guo N, Kejariwal A, et al. PANTHER version 6:protein sequence and function evolution data with expanded representation of biological pathways[J]. Nucleic Acids Res,2007,35: D247-252.
[172]Dennis G J, Sherman B T, Hosack D A, et al. DAVID:Database for Annotation, Visualization, and Integrated Discovery[J]. Genome Biol,2003,4(5):P3.
[173]Huang da W, Sherman B T, Tan Q, et al. DAVID Bioinformatics Resources:expanded annotation database and novel algorithms to better extract biology from large gene lists[J]. Nucleic Acids Res, 2007,35(Web Server issue):W169-175.
[174]Stuart K Kim. Functional genomics:The worm scores a knockout[J]. Current Biology,2001, 11(3):R85-R87.
[175]Ying Cai, Ying Gao, Qi Sheng, et al. Characterization and potential function of a novel testis-specific nucleoporin BS-63[J]. Mol Reprod Dev,2002,61(1):126-134.
[176]Schaefer B C. Revolutions in Rapid Amplification of cDNA Ends:New Strategies for Polymerase Chain Reaction Cloning of Full-Length cDNA Ends[J]. Analytical Biochemistry,1995,227(2): 255-273.
[177]Sandro Banfi, Alessandro Guffanti and Giuseppe Borsani. How to get the best of dbEST[J]. Trends in Genetics,1998,14(2):80-81.
[178]Gill R W, Sanseau P. Rapid in silico cloning of genes using expressed sequence tags (ESTs)[J]. Biotechnol Annu Rev,2000,5:25-44.
[179]The FANTOM Consortium. Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs [J]. Nature,2002,420 (6915):563-573.
[180]Boguski M S, Tolstoshev C M, and Bassett D E. Gene discovery in dbEST[J]. Science,1994, 265(5181):1993-1994.
[181]张德礼,丁培国,凌伦奖,等.人类新基因C17orf32的电子克隆和编码区序列RT-PCR验证[J].生物化学与生物物理进展,2002,29(4)：543-549.
[182]刘庆坡,冯英,董辉.水稻线粒体丝氨酸羟甲基转移酶基因的电子克隆[J].生物信息学,2005,3(1)：5-9.
[183]黄骥,王建飞,张红生,等.水稻葡萄糖-6-磷酸脱氢酶cDNA的电子克隆[J].遗传学报,2002,29(11)：1012-1016.
[1]A ilhand G, Amn E, Bardon S, et al. Growth and differentiation of regional adipose tissue molecular and hormonal mechanisms[J]. International Journal of Obesity,1991,15:87-90.
[2]Steven R Smith, Barbara Gawronska-Kozak, Lenka Janderova,et al. Agouti Expression in Human Adipose Tissue:Functional Consequences and Increased Expression in Type 2 Diabetes [J]. Diabetes, Dec 2003,52:2914-2922.
[3]Patricia A. Zuk, Min Zhu, Peter Ashjian, et al. Human Adipose Tissue Is a Source of Multipotent Stem Cells[J]. Molecular Biology of the Cell,2002,12(13):4279-4295.
[4]屈长青,张国华,陈粉粉,等.猪前体脂肪细胞的原代培养[J].农业生物技术学报,2005,13(5)：649-653.
[5]王竹晨,刘建中,李燕,等.人前脂肪细胞的原代培养[J].中山医科大学学报,2001,22：443-447.
[6]夏成,王哲,朱淑玲,等.犊牛前脂肪细胞的培养及其增殖与分化模型的建立[J].中国兽医科技,2004,34：26-30.
[7]陈世义,马刚,于海滨等.人工真皮成纤维细胞的分离培养与鉴定[J].吉林大学学报(医学版),2002,28(4)：437-439
[8]Van R L, Bayliss C E and Roncari D A. Cytological and enzymological characterization of human adult adipocyte precursors in culture. Clin Invest,1976,58:699-704
[9]朱晓海,何清濂,林子豪,等.人前脂肪细胞培养及增殖与分化模型的建立[J].中华整形烧伤外科杂志,1999,15(3)：199-204
[10]McGarrity G J, J Sarama, and V Vanaman. Cell culture techniques. ASMNews,1985,51:170-183.
[11]Ryan J. Understanding and managing cell culture conlamination. New York:Cornig Inc,1994, 180-195.
[1]Ailhaud G, Amri E, Bardon S, et al. Growth and differentiation of regional adipose tissue molecular and hormonal mechanisms[J]. Int J Obesity,1991,15:87-90.
[2]Steven R Smith, Barbara Gawronska-Kozak, Lenka Janderova, et al. Agouti Expression in Human Adipose Tissue:Functional Consequences and Increased Expression in Type 2 Diabetes[J]. Diabetes,2003,52:2914-2922.
[3]M Wabitschl, R E Brenner, I Melzner, et al. Characterization of a human preadipocyte cell strain with high capacity for adipose differentiation[J]. Int J Obesity,2001,25:8-15.
[4]Patricia A Zuk, Min Zhu, Peter Ashjian, et al. Human Adipose Tissue Is a Source of Multipotent Stem Cells[J]. Mol Biol Cell,2002,13:4279-4295.
[5]Karsten Hemmrich, Dennis von Heimburg,Kathrin Cierpka, et al. Optimization of the differentiation of human preadipocytes in vitro[J]. Differentiation,2005,73:28-35.
[6]J.Mark Brown and Michael K Mcintosh. Conjugated Linoleic Acid in Humans:Regulation of Adiposity and Insulin Sensitivity [J]. J Nutr,2003,133(10):3041-3046.
[7]卢慧玲,王宏伟,Katherine Cianflone,等.促酰化蛋白(ASP)诱导3T3-L1前脂肪细胞分化[J].中国生物化学与分子生物学报,2004,20(3)：383-386.
[8]Barak Y, Nelson MC, Ong ES, et al. PPAR gamma is required for placental, cardiac, and adipose tissue development[J]. Mol Cell,1999,4:585-595.
[9]Rosen E D, Walkey C J, Puigserver P, et al. Transcriptional regulation of adipogenesis[J]. Gene DEV,2000,14:1293-1307.
[10]Linhart HG, Ishimura-Oka K, DeMayo F, et al. C/EBPalpha is required for differentiation of white, but not brown, adipose tissue[J]. PNAS,2001,98:12532-12537.
[11]S Kersten, J Seydoux, J M Peters, et al. Peroxisome proliferator-activated receptor a mediates the adaptive response to fasing[J]. Journal of Clinical Investigation,1999,103(11):1489-1498.
[12]Chieh J Chou, Martin Haluzik, Charmaine Gregory, et al. WY14643, a peroxisome proliferator-activated receptor a (PPARa) agonist, improves hepatic and muscle steatosis and reverses insulin resistance in lipoatrophic A-ZIP/F-1 mice[J]. Journal of Biological Chemistry, 2002,277(27):24484-24489.
[13]S Mandard, M M"uller, and S Kersten. Peroxisome proliferator-activated receptor a target genes[J]. Cellular and Molecular Life Sciences,2004,61(4):393-416.
[14]Hana Koutnikova, Terrie-Anne Cock, Mitsuhiro Watanabe, et al. Compensation by the muscle limits the metabolic consequences of lipodystrophy in PPARy hypomorphic mice[J]. Proceedings of the national academy of sciences of the united states of America,2003,100(24), supplement 2:14457-14462.
[15]Rinke Stienstra, Caroline Duval, Michael Muller, et al. PPARs, Obesity, and Inflammation[J]. PPAR Research, Volume 2007, Article ID 95974,10 pages.
[16]P Wang, J Renes, F Bouwman, et al. Absence of an adipogenic effect of rosiglitazone on mature 3T3-L1 adipocytes:increase of lipid catabolism and reduction of adipokine expression[J]. Diabetologia,2007,50:654-665.
[17]David S Kumpl and Frank W Booth. Sustained rise in triacylglycerol synthesis and increased epididymal fat mass when rats cease voluntary wheel running [J]. J Physiol Biochem,2005,565 (3): 911-925.
[18]B LEDERER and H G HERS. A kinetic study of glycerophosphate acyltransferase of rat adipocytes in relation to its control by noradrenaline[J]. Biochem J,1985,226:269-274.
[19]Thomas E. BROAD and Richard G. HAM.Growth and adipose differentiation of sheep preadipocyte fibroblasts in serum-free medium[J]. European Journal of Biochemistry,1983, 135:33-39.
[20]Wilfried Schmidt, Gisela Poll-Jordan, and Georg Loffler. Adipose Conversion of 3T3-L1 Cells in a Serum-free Culture System Depends on Epidermal Growth Factor, Insulin-like Growth Factor I, Corticosterone, and Cyclic AMP[J]. The Journal of Biological Chemistry,1990,265(26): 15489-15495.
[21]C. Boone, F Gre'goire, and C Remade. Culture of porcine stromal-vascular cells in serum-free medium:Differential action of various hormonal agents on adipose conversion[J]. Journal of Animal Science,2000,78:885-895.
[22]Haiyan Huang, M Daniel Lane and Qi-Qun Tang. Effect of serum on the down-regulation of CHOP-10 during differentiation of 3T3-L1 preadipocytes[J]. Biochemical and Biophysical Research Communications,2005,338:1185-1188.
[23]朱惠娟,邓洁英,龚凤英,等.无血清原代培养人前脂肪细胞并诱导分化[J].基础医学与临床,2006,26(7)：770-773.
[24]J L Ramirez-Zacarias, F Castro-Mufiozledo and W Kuri-Harcuch. Quantitation of adipose conversion and triglycerides by staining intracyctoplasmic lipids with Oil red O[J]. Histochemistry, 1992,97:493-497.
[25]Eli Eisenberg and Erez Y Levanon. Human housekeeping genes are compact[J]. Trends Genet, 2003,19(7):362-365.
[26]Erkens T, Van Poucke M, Vandesompele J, et al. Development of a new set of reference genes for normalization of real-time RT-PCR data of porcine backfat and longissimus dorsi muscle, and evaluation with PPARGC1A[J]. BMC Biotechnol,2006,6:41.
[27]Ann-Britt Nygard, Claus B Jorgensen, Susanna Cirera, et al. Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR[J]. BMC Mol Biol,2007,8:67.
[28]郭玉姣,唐国庆,李学伟,等.猪脂肪组织中IGF2和IGFBP3基因表达的发育性变化及其品种差异[J].遗传,2008,30(5)：602-606.
[29]张罕星,朱晓彤,束刚,等.猪前脂肪细胞分化过程中聚脂相关基因的表达模式[J].动物学报,2007,53(1)：143-150.
[30]张学进,罗清礼,何为民,等.甲状腺相关眼病及正常人眼眶成纤维细胞与前脂肪细胞培养及分化诱导[J].眼科研究,2007,25(2)：81-85.
[31]Kumpl D S and Booth F W. Sustained rise in triacylglycerol synthesis and increased epididymal fat mass when rats cease voluntary wheel running [J]. Journal of Physiology and Biochemistry,2005, 565 (3):911-925.
[32]Farmer S R. Regulation of PPARα activity during adipogenesis[J]. International Journal of Obesity, 2005,29:13-16.
[33]Gilles Ferry, Edwige Tellier, Anne Try, et al. Autotaxin is released from adipocytes, catalyzes lysophosphatidic acid synthesis, and activates preadipocyte proliferation, Up-regulated expression with adipocyte differentiation and obesity[J]. Journal of Biological Chemistry,2003,278(20): 18162-18169.
[1]Diatchenko L, Lau Y F, Campbell A P, et al. Suppression subtractive hybridization:A method for generating differentially regulated or tissue specific cDNA probes and libraries[J]. Proc Natl Acad Sci USA,1996,93(12):6025-6030.
[2]Gurskaya N G, Diatchenko L, Chenchik A, et al. Equalizing cDNA subtraction based on selective suppression of polymerase chain reaction:cloning of Jurkat cell transcripts induced by phytohemaglutinin and phorbol 12-myristate 13-acetate[J]. Anal Biochem,1996,240(1):90-97.
[3]Xu D Q, Xiong Y Z, Ling X F, et al. Identification of a differential gene HUMMLC2B between F1 hybrids Landrace×Yorkshire and their female parents Yorkshire[J]. Gene,2005,352:118-126.
[4]Ponsuksili S,Tesfaye D, El-Halawany, et al. Stage-spccific expressed sequence tags obtained during preimplantation bovine development by differential display RT-PCR and suppression subteaction hybridization[J]. Prenatal Diagnosis,2002,229(12):1135-1142.
[5]Paul D Thomas, Michael J Campbell, Anish Kejariwal, et al. PANTHER:a library of protein families and subfamilies indexed by function[J]. Genome Res,2003,13:2129-2141.
[6]Huaiyu Mi, Nan Guo, Anish Kejariwal, et al. PANTHER version 6:protein sequence and function evolution data with expanded representation of biological pathways[J]. Nucl. Acids Res,2007,35: D247-D252.
[7]Dennis G Jr, Sherman BT, Hosack DA, et al. DAVID:Database for Annotation, Visualization, and Integrated Discovery[J]. Genome Biol,2003,4(5):P3.
[8]Huang D W, Sherman B T, Lempicki R A. Systematic and integrative analysis of large gene lists using DAVID Bioinformatics Resources[J]. Nature Protoc,2009,4(1):44-57.
[9]Davoli R, Zambonelli P, Bigi D, et al. Analysis of expressed sequence tags of porcine skeletal muscle[J]. Gene,1999,233:181-188.
[10]Vilson M E, Sonstegard T S, SmithT P, et al. Differential gene expression during elongation in the preimplantation pig embryo[J]. Gene,2000,26 (1):9-14.
[11]Lee S H, Zhao S H, Recknor J C, et al. Transcriptional profiling using a novel cDNA array identifies differential gene expression during porcine embryo elongation[J]. Mol Reprod Dev,2005, 71(2):129-139.
[12]Stain O D, Thies W G, Hofmann M. A high throughput screening for rarely transcribed differentially expressed genes[J]. Nucleic Acids Res,1991,25:2598-2602.
[13]陈数珍,马文丽,郑文岭.宫颈癌相关基因的筛选及生物信息学分析[J].南方医科大学学报,2008,28(4)：585-588.
[14]薛净,李瑜羌,聂玉强,等.苦参碱对人肝癌细胞株HepG2基因表达谱的影响[J].胃肠病学和肝病学杂志,2007,16(4)：341-343.
[15]廖之君,马文丽,梁爽,等.荧光交换标记法基因表达芯片的数据挖掘[J].福建医科大学学报,2008,42(3)：236-239.
[1]Cohen B A, Mitra R D, Hughes J D, et al. A computational analysis of whole-genome expression data revaels chromosomal domains of gene expression[J]. Nat genet,2000,26(2):183-186.
[2]Bussemaker H J, Li H, Siggia E D. Regulatory element detection using correlation with expression[J]. Nat genet,2001,27(2):167-171.