The Study On The Effect And Intervention Of High Glucose On Vascular Smooth Muscle Cells Apoptosis And Proliferation

The study aims to demonstrate whether there is difference in the expression of anti-apoptosis gene bcl-2 between VSMCs cultured in normal glucose and high glucose and the relationship with apoptosis and proliferation. We also investigate the effect of natural plant ingredient resveratrol and shRNA plasmid vector on bcl-2 expression and VSMCs apoptosis and proliferation to provide new path to prevention and cure of diabetic vasculopathy.Part I The Study on the Effect of High Glucose on Apoptosis and Proliferation of VSMCsObjective: Comparing the VSMCs cultured in high glucose medium with normal glucose and mannitol control medium, we investigate whether there is difference in the expression of anti-apoptosis gene bcl-2 and VSMCs apoptosis and proliferation. Methods: In vitro culture of the rabbit aorta VSMCs is performed by the means of tissue sticking. The VSMCs are devided into three groups, normal glucose group (5.5mmol/L glucose), mannitol control group (5.5mmol/L glucose plus 19.5 mmol/l mannitol) and high glucose group (25mmol/L glucose). Real time quantitive-PCR and Western blotting were performed to analyze the mRNA and protein expression changes of bcl-2 in VSMCs. We use flow cytometry to analyze the apoptosis and MTT to analyze the proliferation of VSMCs. Results: Sufficient rabbit VSMCs were obtained by culture in vitro. Compared with VSMCs cultured in normal glucose and mannitol control medium, VSMCs in high glucose medium significantly increased bcl-2 expression, the cell apoptosis was decreased and the cell proliferation was enhanced. However, there was no significant difference between the former two groups. Conclusion: We conclude that high glucose stimulates bcl-2 expression, leading to less apoptosis and more proliferation of VSMCs, and results in the pathogenesis of diabetic vasculopathy.Part II The Study on the Effect of Resveratrol on Apoptosis and Proliferation of VSMCs in High Glucose MediumObjective: To determine the effect of resveratrol on bcl-2 expression and apoptosis and proliferation of VSMCs in high glucose medium. Methods: There are totally 4 groups: high glucose control group, 50μmol/L Res group, 100μmol/L Res group and 150μmol/L Res group. Real time quantitive-PCR and Western blotting were performed to analyze the expression changes of bcl-2 of VSMCs. We use flow cytometry to analyze apoptosis and MTT to analyze proliferation of VSMCs. Results: Compared to high glucose control group, the mRNA and protein expression level of bcl-2 was significantly inhibited in all three Res groups (P<0.05). And the cell proliferation was enhanced and the cell apoptosis was decreased. Among them ,the 150μmol/L Res group showed the strongest inhibiting effect. Conclusion: Res could inhibit the expression of bcl-2 of VSMCs, then enhance cell apoptosis and decrease proliferation. The effect is concentration dependent.Part III The Study on the Effect of ShRNA on bcl-2 Expression of VSMCs in High Glucose MediumObjective: To construct shRNA plasmid vector targeting rabbit bcl-2 gene and determine the effect of shRNA on bcl-2 expression and VSMCs' apoptosis and proliferation. Methods: Two shRNA plasmids vector targeting bcl-2 gene were constructed. There were totally 5 groups: high glucose control group, lipofectamine group, pHK-shRNA control group and two pbcl-2-shRNA experimental groups. Real time quantitive-PCR and Western blotting were performed to analyze the expression changes of bcl-2 in VSMCs. Results: bcl-2-shRNA plasmids were successfully constructed and confirmed by sequencing. Compared to high glucose control group, Lipofectamine group and pHK-shRNA control group, the mRNA and protein expression level of bcl-2 were significantly inhibited in both two bcl-2-shRNA experimental groups (P<0.05). pbcl-21 -shRNA showed the stronger inhibiting effect. Conclusion: The expression of bcl-2 of VSMCs is inhibited after pbcl-2-shRNA transfection, and pbcl-21 -shRNA is more effective and could be applied for further studies.