高血压家族史与高盐对人脐动脉平滑肌细胞转化生长因子β1/Smads及钙离子调控相关基因表达的析因分析

Interactions of family history of hypertension and high salt on the gene expression of transforming growth factor β1/Smads,ion pumps and TRPC in human umbilical artery smooth muscle cells

目的研究高血压家族史与高盐对人脐动脉平滑肌细胞(hUASMC)转化生长因子β1(TGF-β1)/Smads及钙离子调控相关基因表达的效应分析。方法取高血压阴性家族史(FH-)和阳性家族史(FH+)胎儿脐带各7例,按照析因设计采用组织块贴壁法培养hUASMC:培养基Na+终浓度为139mmol/L(FH-正常盐组和FH+正常盐组)和164mmol/L(FH-高盐组和FH+高盐组),用平滑肌细胞特异性α肌动蛋白进行免疫细胞化学方法鉴定;实时逆转录聚合酶链反应检测4组hUASMC TGF-β1、Smad2、Smad3、Smad7、细胞膜钠泵-α1亚型(α1-subunit)、α2-subunit、α3-subunit、细胞膜钙泵亚型1(PMCA1)、PMCA4、瞬时受体电位通道C亚族(TRPC)TRPC1、TRPC3、TRPC6mRNA表达;采用析因分析高血压家族史与高盐在TGF-β1/Smads及钙离子调控相关基因表达中的效应。结果高血压家族史和高盐对TGF-β1/Smads、α3-subunit、PMCA4、TRPC1、TRPC6mRNA表达无交互效应(均P>0.05),主效应分析显示高血压家族史增加TGF-β1、α3-subunit、TRPC6的表达(均P<0.05),减少Smad7的表达(均P<0.05),而高盐增加Smad3、TRPC1的表达(均P<0.05),减少PMCA4、Smad7的表达(均P<0.05)。高血压家族史和高盐对α1-subunit、α2-subunit、PMCA1、TRPC3mRNA表达有交互效应(均P<0.05)。高血压家族史为阳性时,高盐组较正常盐组TRPC3表达增加(P<0.05),α1-subunit、α2-subunit、PMCA1表达减少(P<0.05);高血压家族史为阴性时,高盐组较正常盐组TRPC3表达增加(P<0.05),对α1-subunit、α2-subunit、PMCA1表达无影响(均P>0.05);此外,在高盐水平下,高血压家族史阳性组较阴性组α1-subunit、TRPC3表达增加(P<0.05),PMCA1表达减少(P<0.05),在正常盐水平下,高血压家族史阳性组较阴性组α1-subunit、α2-subunit、PMCA1表达增加(P<0.05),对TRPC3表达无影响(均P>0.05)。结论家族史影响α3-subunit、TRPC6基因表达,而高盐影响PMCA4、TRPC1基因表达,高盐和家族史共同参与调节TGF-β1/Smads、α1-subunit、α2-subunit、PMCA1、TRPC3基因表达。

Objective To evaluate the effect of high-salt intake and family history of hypertension on the gene expression of transforming growth factor beta-1（TGF-β1）/Smads and calcium-regulation proteins in human umbilical artery smooth muscle cells（hUASMC）. Methods A lot of fourteen normal fetus were derived into positive hypertension family history group（FH＋,n=7）and negative hypertension family history group（FH-,n=7）,and their umbilical cords were collected. HUASMC isolated by tissue explants adherent method were cultured with normal medium（139mmol/L sodium chloride）or high salt medium（164mmol/L sodium chloride）. Smooth muscle cell-specificα-actin was observed through the immunocytochemical method. The real-time reverse transcription polymerase chain reaction was employed to detect the gene expressions of TGF-β1,Smad2,Smad3 and Smad7,Na＋-K＋-ATPaseα1-subunit,α2-subunit andα3-subunit,plasma membrane Ca2＋-ATPase isoform（PMCA）1and PMCA4,as well as transient receptor potential canonical channel（TRPC）1,TRPC3 and TRPC6. Factor analysis method was used to evaluate the interactions of family history of hypertension and high salt on above gene expressions. Results There was no interaction between hypertension family history and high-salt intake on the mRNA expressions of TGF-β1/Smads,α3-subunit,PMCA4,TRPC1 and TRPC6（P〈0.05）,main effects analysis showed that family history of hypertension could promote the expressions of TGF-β1,α3-subunit and TRPC6（P〈0.05）,and inhibit Smad7（P〈0.05）,while high salt intake could promote Smad3 and TRPC1（P〈0.05）,and inhibit PMCA4 and Smad7. There was interaction between hypertension family history and high salt intake on the expressions ofα1-subunit,α2-subunit,PMCA1 and TRPC3（P〈0.05）. When the family history is positive,compared with the control group,the high salt group had increased expression of TRPC3（P〈0.05）,and decreased expressions ofα1-subunit,α2-subunit and PMCA1（P〈0.05）. When the family history is negative,high salt intake could increase the expression of TRPC3（P〈0.05）,but had no effect onα1-subunit,α2-subunit and PMCA1（P〉0.05）. In FH＋group,the expressions ofα1-subunit and TRPC3increased（P〈0.05）,with PMCA1decreased（P〈0.05）in high salt condition,while in normal salt condition,the expressions ofα1-subunit,α2-subunit and PMCA1increased（P〈0.05）,with no significant change in TRPC3expression（P〉0.05）. Conclusions Family history of hypertension may affect the mRNA expressions ofα3-subunit and TRPC6,while high salt intake may affect the mRNA expressions of PMCA4 and TRPC1. Hypertension family history and high salt intake may co-regulate the mRNA expressions of TGF-β1/Smads,α1-subunit,α2-subunit,PMCA1 and TRPC3in hUASMC.