缝隙连接在同型半胱氨酸介导的自发性高血压大鼠血管平滑肌细胞增殖中的作用

目的:探讨缝隙连接(GJ)是否参与同型半胱氨酸(Hcy)介导的自发性高血压(SHR)大鼠血管平滑肌细胞(VSMCs)增殖及可能的分子机制。方法:原代培养SHR VSMCs,细胞分四组:①对照组,②Hcy组,③缝隙连接阻断剂(18α-GA)组和④Hcy+18α-GA组。MTT法及流式细胞仪检测细胞的增殖活性,免疫荧光技术观察细胞中Cx43、Cx40蛋白表达及定位,Western blot法检测细胞中Cx43、Cx40蛋白表达,染料示踪分子传递法(划痕标记染料传输法)检测细胞的缝隙连接功能。结果:①与对照组相比,Hcy组MTT法测得A值及细胞周期测得S值增高(P0.05),18α-GA组降低(P0.05);与Hcy组比,TGFβ-1+18α-GA组A值及S值均降低(P0.05)。②免疫荧光技术检测细胞中Cx43、Cx40蛋白表达呈阳性,两者共定位于胞浆。③与对照组相比,Hcy组Cx43、Cx40蛋白表达增强(P0.05),18α-GA组Cx43、Cx40表达均减弱(P0.05);与Hcy组比,Hcy+18α-GA组Cx43、Cx40表达均减弱(P0.05)。④与对照组相比,Hcy组缝隙连接功能明显增强(P0.05),18α-GA组缝隙连接功能明显减弱(P0.05),与Hcy组比,Hcy+18α-GA组缝隙连接功能显著降低(P0.05)。结论:Hcy通过上调SHR VSMCs Cx43和Cx40的蛋白表达,引起缝隙连接通讯功能的增强,促进了SHR VSMCs增殖。

Roles of gap junctions in proliferation mediated by Hcy in the spontaneous hypertensive rat vascular smooth muscle cells

Objective: To investigate whether homocysteine(Hcy)participates the proliferation of the spontaneously hypertensive rat(SHR)vascular smooth musde cell(VSMCs) and the molecular mechanism.Methods: The rat’s arota were removed.The primary SHR VSMCs were isolated and cultured in vitro,then the SHR VSMCs were divided into four groups: ①control group,②Hcy group,③18α-glycyrrhetinic acid(GA) group,④Hcy+18α-GA group.We detected proliferation of the SHR VSMCs by MTT and flow cytometry.The expression and co-localization of the connexin(Cx)43 and Cx40 proteins in the SHR VSMCs were deteced by immunofluorescence.The expression of the Cx43 and Cx40 proteins in SHR VSMCs were detected by Western blot.The molecular dye transfer method(scrape dye transfer method) was applied to detect the gap junction function in the SHR VSMCs.Results: ①The Cx43 and Cx40 proteins expression in the SHR VSMCs were positive,confocal microscopy supported the co-localization of Cx43 and Cx40 in the cytoplasm.②The S value deteced by cell cycle and A value detected by MTT in the Hcy group were increased obviously compared with those in the control group(P<0.05),decreased in 18α-GA group(P<0.05).Compared with the Hcy group,the S and A value in the Hcy+18α-GA group were significantly decreased,respectively(P<0.05).③The expression of Cx43 and Cx40 proteins in Hcy group were increased compared with the control group(P<0.05),decreased in 18α-GA group(P<0.05).Compared with the Hcy group,the expression of Cx43 and Cx40 proteins in the Hcy +18α-GA group were significantly decreased,respectively(P<0.05).④The function of gap junction detected by scrape dye transfer method in the Hcy group were enhanced compared with the control group(P<0.05),weakened in the 18α-GA group(P<0.05).Compared with the Hcy group,the function of gap junction in the Hcy +18α-GA group was significantly weakened(P<0.05).Conclusion: Hcy can enhance the function of gap junctional to stimulate the proliferation of SHR VSMCs through the expression of Cx43 and Cx40 proteins promoted.