| 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. |
