Differential Expression Of Calcium Operated Calcium Channels And Sodium Calcium Exchangers In Vascular Smooth Muscle Cells And Vascular Endothelial Cells

[Objectives]By studying the differences in the expression of TRPC,ORAI1 channel and related calcium regulatory molecule NCX in vascular smooth muscle cells and vascular endothelial cells in calcium operated calcium channels,we are tring to finding out what only inhibit the proliferation of vascular smooth muscle cells without affecting the proliferation of vascular endothelial cells.Key molecules provide a new theoretical basis for shortening the duration of dual antiplatelet therapy after PCI.[Methods]1.The differences in transcription levels of TRPC family,like as TRPC1,TRPC3,TRPC4,TRPC5 and TRPC6,ORAI1,NCX family like as NCX1 and NCX2 in vascular smooth muscle cells and vascular endothelial cells were detected by real-time fluorescent quantitative PCR.2.The differences in expression levels of the above genes in vascular smooth muscle cells and vascular endothelial cells were further confirmed by Western Blot.3.The genes with significant difference in expression in Western Blot were selected to establish high and low expression target cell lines by lentiviral transfection overexpression and siRNA interference technology,and were identified by fluorescence microscopy and fluorescence quantitative qPCR and Western Blot..4,CCK8 proliferation was assayed to detect the effects of overexpression and interference with target genes on the proliferation rate of vascular smooth muscle cells and vascular endothelial cells.[Results]1.The results of qPCR assay were:trpc1,trpc6,ncx1 and orai1 were higher in endothelial cells than smooth muscle cells,while trpc3,trpc4,trpc5 and ncx2 were lower in endothelial cells than smooth muscle cells.Analysis,P<0.01,the differences were significant and statistically significant.2.The results of Western Blot test were showed that the protein expression level of TRPC6 and ORAI1 in endothelial cells were significantly higher than that of smooth muscle cells.The results of this experiment were consistent with those of qPCR.The protein expression levels of TRPC4 and TRPC5 in endothelial cells were significantly lower than those of smooth muscle cells.The experimental results were also consistent with the qPCR results.The protein expression levels of TRPC1,TRPC3,NCX1 and NCX2 in endothelial cells and smooth muscle cells were not significantly different,P>0.05,no statistical significance.3.SMC-orail and EC-orail over-expression stable cell lines were established by lentiviral transfection.The positive rates of transfection were 90%and 80%,respectively,and the overexpressing cell line SMC-orai1 was detected by WB.The expression level of orai1 in SMC and EC cells was significantly up-regulated in EC-orai1 transcript level,P<0.05,and the difference was statistically significant.4.TRPC4 siRNA and TRPC5 siRNA were transfected into EC cells and SMC cells.The transfected EC and SMC cells were observed under fluorescence microscope.The positive rate of siRNA trpc4 and siRNA trpc5 transfection were 90%and 90%in EC cells.The positive rate of transfection of siRNA trpc4 and siRNA trpc5 were 95%and 95%in SMC cells.The protein expression levels of TRPC4 and TRPC5 in HCAEC and HCASMC cells after WB detection were significantly lower than those in the blank group and NC group,P<0.01,the difference was statistically significant.5.The results of CCK8 test were showed that the proliferation rate of SMC cells were significantly increased after overexpression of ORAI1 protein,P<0.05,the difference was statistically significant,while the proliferation rate of EC cells was not significant,P>0.05,no statistical significance.SiRNA was alone interfered with the expression of TRPC4 or TRPC5 in SMC cells,but the proliferation rate was decreased slightly,but the difference was not significant,P>0.05,which was not statistically significant,while at the same time interfering with the expression of TRPC4 and TRPC5 in SMC,the proliferation rate of SMC Significantly decreasing,the difference was significant,P<0.05,which was statistically significant.However,the proliferation rate of TRPC4 and TRPC5 in EC cells were not significantly afifected by the interference with TRPC4 and TRPC5.[Conclusions]1.TRPC4,TRPC5,TRPC6 and ORAI1 were significantly different in EC cells and SMC cells at the transcriptional level and protein expression level,while TRPC1,TRPC3,NCX1 and NCX2 were only different at the transcriptional levels.2.Overexpression of ORAI1 can promote the proliferation of SMC cells without affecting the proliferative capacity of EC cells.3.Co-inhibition of TRPC4 and TRPC5 expression can only inhibit the proliferation of SMC without affecting the proliferative capacity of EC.4.ORAI1,TRPC4,and TRPC5 are important targets for regulating the proliferation rate of vascular endothelial cells and vascular smooth muscle cells.5.To provide a new theoretical basis for shortening the time of dual antiplatelet therapy after PCI by inhibiting the two gene targets such as TRPC4 and TRPC5.