Effect Of High-glucose And Angiotensin Ⅱ On The Expression Of The NRG-1mRNA And NRG-1 Protein Of Cardiac Microvascular Endothelial Cells

AIM:To isolate and culture cardiac microvascular endothelial cells primarily,and identify it by immunofluorescence, and investigate the effect of high-glucose and AngiotensinⅡon the expression of the NRG-1 mRNA and NRG-1 protein of Cardiac Microvascular Endothelial Cells, and the possible pathogenesis of Diabetic Cardiomyopathy.METHODS:(1) Isolated and cultured cardiac microvascular endothelial cells primarily,and identified it after that. In this experiment, we got heart tissues from healthy and clean Sprague-Dawley (SD) rats of four days old. Digested heart tissue with CollagenaseⅠ, adherent cultured nucleated cells after it was digested,and removed culture medium and passaged CMEC when endothelial cells had grown in primary monolayer fusion. Second-generation CMEC were identified using immunofluoresce -nce with antibody of factor VIII—related antigen and antibody of CD31—related antigen,which were important marker of CMEC.(2) Intervened CMEC with high-glucose and AngiotensinⅡrespectively after that were cultured successfully.This experiment included two parts: Test A and Test B. Test A: Microvascular Endothelial Cells were divided into three groups after isolation and identification, Control group:Medium without glucose and insulin;Experimental group1:Medium with glucose(10mmol/L);Experimental group 2:Medium with glucose(10mmol/L) and insulin(10-5U/L). Test B: Microvascular Endothelial Cells were also divided into three groups after isolation and identificatio -n, Control group:Medium without AngiotensinⅡand valsartan;Experimental gr -oup1:Medium with AngiotensinⅡ(100nmol/L);Experimental group Medium with AngiotensinⅡ(100nmol/L)and valsartan(10μmol/L).Collected CMEC after 24 hours respectively, RT-PCR detected the expression of NRG-1 mRNA ,and Westernblot detected the expression of NRG-1 protein.RESULTS: (1)Cultured cardiac microvascular endothelial cells primarily, and cell purity was about 95%,showed a typical paving pebble-like structure ;(2) Immunofluorescence, showed thatⅧfactor and CD31 were main in the cytoplasm, showed red fluorescence and green fluorescence respectively;(3) In Test A, compared with the control group and Experimental group 2 ,the expression of the NRG-1 mRNA and NRG-1 protein in Experimental group 1 decreased significantly,and the difference was significan(tP﹤0.05);Compared with the control group, the expression of the NRG-1 mRNA and NRG-1 protein in Experimental group 2 decreased,but the difference was not significant(P>0.05).(4) In Test B, compared with the control group and Experimental group 2 ,the expression of the NRG-1 mRNA and NRG-1 protein in Experimental group 1 decreased significantly,and the difference was significan(tP﹤0.05);Compared with the control group, the expression of the NRG-1 mRNA and NRG-1 protein in Experimental group 2 decreased,but the difference was not significant(P>0.05). CONCLUSION:high glucose and AngiotensinⅡinhibited the expression of NRG-1 mRNA and NRG-1 protein of cardiac microvascular endothelial cells,and insulin and valsartan could antagonized that respectively.