AIM:To isolate and culture cardiac microvascular endothelial cells primarily,and identify it by immunofluorescence, and investigate the effect of high-glucose and Angiotensinâ…¡on 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 Collagenaseâ… , 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 Angiotensinâ…¡respectively 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 Angiotensinâ…¡and valsartan;Experimental gr -oup1:Medium with Angiotensinâ…¡(100nmol/L);Experimental group Medium with Angiotensinâ…¡(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 thatâ…§factor 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 Angiotensinâ…¡inhibited the expression of NRG-1 mRNA and NRG-1 protein of cardiac microvascular endothelial cells,and insulin and valsartan could antagonized that respectively.
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