Construction And Screening Of Chrebp-mirna Eukaryotic Expression Vector And Its Transfection

Objective: To construct four plasmids which can inhibit expression of ChREBP of rat adipocyte, observe effects of glucose on rat adipocyte multiplication and differentiation by lipofectamine 2000 transfecting and the regulation role of ChREBP on its lipogenesis.Methods: 1. Four short DNA targetsequence were designed according to gene-silencing technologies principle, chcmically synthesized , and reconstructed to four plasmids: pcDNA6.2-GW/EmGFP-ChREBPâ… ,pcDNA6.2-GW/EmGFP-ChREBPâ…¡,pcDNA6.2-GW/EmGFP-ChREBPâ…¢,pcDNA6.2-GW/EmGFP-ChREBPâ…£. 2. Four recombinant plasmids and control plasmid were transfected to primary rat adipocyte,respectively. 3. Effective transfection recombinant plasmids can be obeseved by fluorescence-microscope. 4. The rate of increase of primary rat adipocyte proliferation were detected by MTT.5. Effects of primary rat adipocyte differentiation by oil red O.Results: 1. Four plasmids were constracted successfully. 2. Four recombinant plasmids and control plasmid were transfected to primary rat adipocyte and was used Blasticidin to gain the cell clones that express stably. Recombinant plasmids pcDNA6.2-GW/EmGFP-ChREBPâ…¢'s fluorescent images shows batter than others .Tissue that were not digested were good in transfection unexpected. 3. Effects of glucose on rat preadipocytes differentiation by applications,that were transfected by pcDNA6.2-GW/EmGFP-ChREBPâ…¢48 h, 20mmol/L glucose(P<0.05) was higher significantly than 0mmol/L,5mmol/L and 10mmol/L glucose, 25mmol/L,15mmol/L was no significant(P>0.05). Effects of glucose on rat proliferation applications RNAi,that were transfected by pcDNA6.2-GW/EmGFP-ChREBPâ…¢48 h,15mmol/L glucose was more enhancement significantly than 10mmol/L,15mmol/L and 20mmol/L glucose(P<0.05), 5mmol/L,25mmol/L glucose was no significant(P>0.05).Conclusion: The recombinant plasmids was inefficiency express ChREBP specific RNA,but pcDNA6.2-GW/EmGFP-ChREBPâ…¢can more efficiency. Effects of glucose on rattus norvegicus preadipocytes differentiation and proliferation by applications RNAi,that were transfected by pcDNA6.2-GW/EmGFP-ChREBPâ…¢48 h,has few changes.