【作者】 刘海峰；

【导师】 李学伟；

【作者基本信息】 四川农业大学 ， 动物遗传育种与繁殖， 2010， 博士

【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)。更多还原

【Abstract】 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.更多还原