| Part One:Text One:Objective:Down regulate the expression of ROCK Ⅰ and ROCK Ⅱ genes in hu--man aortic vascular smooth muscle cells.Methods:First,siRNA technique was used to reduce the ROCK Ⅰ and ROCK Ⅱgenes of HA-VSMCs respectively.After 24h,the transfection effect was observed under the inverted microscope.Cells were divided into 6 groups after transfection of 48h:blank control group,ROCK ⅠsiRNA group,ROCK ⅡsiRNA group,TGF-β1 group and ROCK ⅠsiRNA+TGF-β1 groups,ROCK ⅡsiRNA+TGF-β1 groups.The blank control group,the ROCK ⅠsiRNA group and the ROCK ⅡsiRNA group were cultured in the complete medium.The groups of TGF-β1 ROCK ⅠsiRNA+TGF-β1 and ROCK ⅡsiRNA+TGF-β1 were cultured in the complete medium containing 5ng/ml TGF-β1.Results:Compared with the blank control group,the expression of ROCKprotein in TGF-β1 groups increased significantly(P<0.05),but there was no obvious change in the expression of ROCK Ⅱ protein(P>0.05).Compared with the blank control group,the corresponding target protein of the ROCK ⅠsiRNA group and ROCK ⅡsiRNA group decreased significantly(all P<0.05).Compared with ROCK IsiRNA+TGF-β1 group and ROCK ⅡsiRNA+TGF-β1 group,the corresponding target protein ROCK Ⅰ and ROCK Ⅱ expression decreased significantly in HA-VSMCs(P<0.0 5).Conclusion:The ROCK ⅠsiRNA and ROCK ⅡsiRNA transfection models of HA-VSMCs were successfully constructed.TGF-β1 can induce the expression of ROCK Ⅰ protein in HA-VSMCs,which can be inhibited by ROCK ⅠsiRNA transfection.Text Two:Objective:To investigate the effect of ROCK Ⅰ/Ⅱ gene on HA-VSMCs phenotype transformation induced by TGF-β1.Methods:The HA-VSMCs cultured in vitro was divided into 5 groups:blank control group,TGF-β1 group,ROCK Ⅰ siRNA +TGF-β1 group,ROCK Ⅱ siRNA+TGF-β1 group and Y-27632+TGF-β1 group(Y-27632 as specific inhibitor of ROCK Ⅰ).After different pretreatments,after 24h,Western blot and fluorescence quantitative polymerase chain reaction(RT-PCR)were used to detect the protein expression and mRNA content of the contractile marker protein α-smooth muscle actin(α-SMA),the smooth muscle 22 α(SM22-α),the synthetic marker protein osteopontin(OPN),respectively.Results:Compared with the TGF-β1 group,the protein expression and mRNA content of the ROCK Ⅰ siRNA+TGF-β1 groups increased significantly(P<0.05),while the protein expression and mRNA content of OPN decreased significantly(P<0.05).Compared with TGF-β1 group,there was no significant difference in protein expression and mRNA content between ROCK ⅡsiRNA+TGF-β1 groups(P>0.05).Conclusion:TGF-β1 can induce the transformation of HA-VSMCs from the contractile phenotype to the constitutive phenotype,and ROCK Ⅰ gene play an important role in this transformation,while the effect of ROCK Ⅱ gene is not obvious.Text Three:Objective:To investigate the effects of Rho-associated coiled-coil containing protein kinase Ⅰ/Ⅱ(ROCK Ⅰ/Ⅱ)on migration and proliferation in human aortic vascular smooth muscle cells(HA-VSMCs)induced by transforming growth factorβ1(TGF-β1).Methods:The HA-VSMCs cultured in vitro was divided into 5 groups:blank control group,TGF-β1 group,ROCK Ⅰ siRNA +TGF-β1 group,ROCK ⅡsiRNA+TGF-β1 group and Y-27632+TGF-β1 group.After different intervention treatment,the cell migration and proliferative energy were detected by Transwell and CCK-8 tests.Results:Compared with the blank control group,the cell migration number of TGF-β1 was significantly increased(P<0.05).Compared with the TGF-β1 group,the cell migration number of the ROCK IsiRNA+TGF-β1 group and Y-27632+TGF-β1 group decreased[(61 + 1.8)vs.(208.3 + 18.9);(63.3 + 7.4)vs.(208.3 + 18.9),all P<0.05],while ROCK IIsiRNA+ TGF-β1 group cell migration was not significant changes(P>0.05).Compared with the blank control group,the OD value of TGF-β1 group increased[1.350 + 0.057)vs.(1.252 + 0.061),P<0.05],and compared with the TGF-β1 group,ROCK IsiRNA+TGF-β1 group,ROCK IIsiRNA+TGF-β1 group and Y-27632+TGF-β1 group had no significant changes(P>0.05).Conclusion:ROCK Ⅰ plays an important role in the HA-VSMCs migration induced by TGF-β1,but the role of ROCK Ⅰ and ROCK Ⅱ in HA-VSMCs proliferation induced by TGF-β1 is not obvious.Part Two:Text One:Objective:To explore the effect of co culture of human aortic smooth muscle cells and endothelial cells on the synthesis of COL Ⅰ and COL Ⅲ protein.Methods:The HA-VSMCs and HA-ECs were cultured in vitro,and the VSMC-EC co culture model was established by Transwell compartment.VSMC was in the upper chamber,EC in the lower room.After 24h,the cells were divided into EC control group,VSMC control group,VSMC-EC co culture group,EC+TGF-β1 group,VSMC+TGF-β1 group,and VSMC-EC co culture+TGF-β1 groups.After cultured 6h,12h,24h and 48 h,the contents of COL Ⅰ and COL Ⅲ in culture supernatants were detected by ELISA.Results:Compared with the EC control group and the VSMC control group,the expression of COL Ⅰ and COL Ⅲ protein in the co culture of VSMC-EC increased significantly(P<0.01).The expression of COL Ⅰ and COL Ⅲ protein in the VSMC-EC co culture +TGF-β1 group was significantly higher than that of the EC+TGF-β1 group and the VSMC+TGF-β1 group.Compared with VSMC-EC co culture group,the expression level of COL Ⅰ and COL Ⅲ increased significantly in VSMC-EC co cultured +TGF-β1 group(P<0.01).Conclusion:VSMC-EC co culture significantly increased the protein synthesis of COL Ⅰ and COL Ⅲ,while TGF-β1 significantly promoted this process.Text Two:Objective:To explore the effect of human aortic VSMC-EC co culture induced by TGF-β1 on the protein synthesis of ROCK Ⅰ and RhoA.Methods:The VSMC and EC of human aorta in vitro were divided into:VSMC control group,VSMC+TGF-β1 group,VSMC-EC co culture group,VSMC-EC co culture+TGF-β1 group.After 24 hours culture,Western blot was used to detect ROCK Ⅰ protein and RhoA protein expression.Results:Compared with VSMC control group,there was no significant difference in ROCK Ⅰ and RhoA protein expression in VSMC-EC co culture group(P>0.05).Compared with VSMC+TGF-β1 group,there was no significant difference in the expression of ROCK Ⅰ and RhoA protein in VSMC-EC co cultured+TGF-β1 group(P>0.05).Compared with group VSMC,the expression of ROCKI protein in VSMC+TGF-β1 group increased significantly(P<0.05),while the expression of RhoA protein was not significantly different(P>0.05).Compared with the VSMC-EC co culture group,the expression of ROCK Ⅰ protein in the group of VSMC-EC co cultured +TGF-β1 increased significantly(P<0.01),and the expression of RhoA protein increased significantly(P<0.05).Conclusion:The co culture of HA-VSMCs with migration ability and HA-ECs under the action of TGF-β1 can promote the expression of ROCK Ⅰ and RhoA proteins.Text Three:Objective:In order to explore the effect of co culture of human aortic smooth muscle cells which downs regulation of ROCK Ⅰ gene expression and endothelial cells on the synthesis of COL Ⅰ and COL Ⅲ protein and its mechanism.Methods:The HA-VSMCs gene was downregulated by siRNA transfection in vitro,and then the transfected HA-VSMCs and untransfected HA-ECs were divided into 5 groups:VSMC-EC co culture group,VSMC-EC co culture +TGF-β1 group,VSMC-EC co culture+ROCK IsiRNA+TGF-β1 group,VSMC-EC co culture+siRNA-C+TGF-β1 group and VSMC-EC co culture+Y-27632+TGF-β1 group.After 24h,the contents of COL Ⅰ and COL Ⅲ in different groups were detected by ELISA and the expression levels of ROCK Ⅰ and RhoA in different groups were detected by Western blot.Results:Compared with VSMC-EC co culture of +TGF-beta 1 group,VSMC-EC co culture +ROCKIsiRNA+TGF-β1 groups and VSMC-EC co culture +Y-27632+TGF-β1 group COL Ⅰ and COL Ⅲ protein expression decreased significantly(P<0.01).Compared with VSMC-EC co cultured +TGF-β1 group,there was no significant difference in the expression of COL Ⅰ and COL Ⅲ in VSMC-EC co cultured+siRNA-C+TGF-β1 group(P>0.05).Compared with VSMC-EC co culture group,the expression level of COL Ⅰ and COL Ⅲ increased significantly in VSMC-EC co cultured +TGF-(31 group(P<0.01).Compared with the VSMC-EC co culture+TGF-β1 group,there was no significant difference in the expression of ROCK Ⅰand RhoA protein in VSMC-EC co culture+ROCK IsiRNA+TGF-β1 group and VSMC-EC co culture +Y-27632+TGF-β1 group(P>0.05).Compared with VSMC-EC co cultured +TGF-(31 group,there was no significant difference in the expression of ROCK Ⅰ and RhoA protein in VSMC-EC co cultured +siRNA-C+TGF-β1 group(P>0.0 5).Compared with the VSMC-EC co culture group,the expression of ROCK Ⅰ and RhoA proteins increased significantly in VSMC-EC co cultured+TGF-β1 group(P<0.01).Conclusion:Down regulation of ROCK Ⅰ gene of HA-VSMCs inhibited the synthesis of COL Ⅰ and COL Ⅲ protein in VSMC-EC co culture induced by TGF-β1,but had no effect on the expression of ROCK Ⅰ and RhoA protein in VSMC-EC co culture induced by TGF-β1. |
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