Construction Eukaryotic Expression Plasmid Of Insig2 And Its Influence To Related Genes Of Fat Metabolism

Objective:To construct the transfection eukaryotic expression plasmid pcDNA3.1(+)-insig2, which were transfected into 3T3-L1 cells, then observe the influence of cell cycle and apoptosis,genes express in the insig-SREBPS-SCAP-downstream signal passageway and adipocyte differentiation and synthesis.Method: Insig2 gene of the mouse was amplified from 3T3-L1 pre-adipocytes by RT-PCR,then cloned into the eukaryotic expression vectors pcDNA3.1(+) After being confirmed by PCR ,double restriction enzyme digestion analysis and DNA sequencing, pcDNA3.1(+)-insig2 were transfected into 3T3-L1 cells by lipofectamine 2000. The expression of insig2 in the 3T3-L1 cells were detected by RT-PCR and immunohistochemistry,then to observe the cell cycle and apoptosis by flow cytometry after being transfected into 3T3-L1 cells, and to detect the adipocyte differentiation by Oil Red "O" strain and genes express in the insig-SREBPS-SCAP-downstream genes signal passageway during cell differentiation by RT-PCR.Result: The eukaryotic expression plasmid of pcDNA3.1(+)-insig2 were constructed.RT-PCR and immunohistochemistry showed pcDNA3.1(+)-insig2 was transfected in 3T3-L1 preadipocytes successfully.we establish insig2 stable express cell line. The cell cycle apoptosis and growth curve of 3T3-L1 cells were not influenced after transfected with pcDNA3.1 (+)-insig2 (P>0.05). During the process of cell differentiation ,we observed the adipocyte differentiation degree in pcDNA3.1(+)-insig2 group was decreased than in pcDNA3.1(+) group and 3T3-L1 cells group by Oil Red "O" strain, but not different between pcDNA3.1(+) group and 3T3-L1 cells group (P>0.05) ; RT-PCR show the insig2,insig1,SREBPS,FAS,AP2 mRNA expressions were up-regulation during cell differentiation in the three groups ,but except SCAP (P>0.05) . However, in the pcDNA3.1(+)-insig2 group the insig2 mRNA expression was up-regulation faster and the insig1,SREBPS,FAS,AP2 mRNA expressions were up-regulation slower than in pcDNA3.1(+) group and 3T3-L1 cells group (P<0.05) . Conclusion: The eukaryotic expression plasmid of pcDNA3.1(+) -insig2 is successfully constructed, then transfected to the 3T3-L1 and the cell line which expressed insig2 was obtained. The cell cycle , apoptosis were not influenced after transfecting insig2, but insig2 gene may have a depressant effect on adipocyte differentiation and expression of related genes.