钙调神经磷酸酶对血管平滑肌细胞增殖中蛋白激酶G表达的影响

目的:探讨钙调神经磷酸酶(CaN)对血管平滑肌细胞(VSMCs)增殖中蛋白激酶G(PKG Iα)表达的影响.方法:对大鼠VSMCs培养并行细胞鉴定,分为对照组、低浓度环孢菌素A(CsA,0.5 mg/L)干预组、高浓度CsA(5 mg/L)干预组以及苯肾上腺素(PE)刺激组(5 mg/L CsA 10 μmol/L PE).其中CsA为CaN特异性抑制剂,PE为已知的CaN激活剂,能够刺激细胞增殖.应用RT-PCR、免疫细胞化学及Western blot法定性及定量测定VSMCs增殖中PKG Iα mRNA以及蛋白的表达水平.结果:0.5 mg/L CsA组PKG Iα mRNA以及蛋白的表达水平与对照组相比差异无统计学意义,而5 mg/L CsA组PKG Iα mRNA及蛋白的表达明显高于对照组,5 mg/L CsA 10 μmol/L PE组中PKG Iα mRNA的表达比5 mg/L CsA组减少了32.2%,蛋白的表达减少了36.7%,但仍明显高于对照组.结论:CaN能够调节培养VSMCs中PKG Iα的表达,增殖细胞用CaN特异性抑制剂CsA灭活CaN后,PKG Iα的表达明显增加,给予PE再次激活CaN后,PKG Iα的表达明显减少.

Regulation of expression of cGMP-dependent protein kinase Iα by calcineurin in vascular smooth mucle cells proliferation

OBJECTIVE: To discuss the regulation of expression of cGMP-dependent protein kinase I(alpha) (PKG I(alpha)) by calcineurin (CaN) in vascular smooth muscle cells (VSMCs) proliferation. METHODS: Cultured wistar rat aortic VSMCs were used as an experimental model. CaN was inhibited by its special inhibitor cyclosporin A (CsA). Phenylephrine (PE) was given to stimulate VSMCs to proliferate. All of cultured cells were divided into four groups:control group, 0.5 mg/L CsA group, 5 mg/L CsA group and 5 mg/L CsA+10 micromol/L PE group. The mRNA and protein expressions were assayed by RT-PCR, immunocytochemistry and Western blot analysis. RESULTS: The OD ratio of PKG I(alpha) mRNA expression in 0.5 mg/L CsA group resembled that in the control group while that in 5 mg/L CsA group was significantly higher than that in the control group (P<0.01). Although the result of 5 mg/L CsA+10 micromol/L PE group was lower than that in 5 mg/L CsA group (the expression of mRNA decreased 32.2%; the production of PKG I(alpha) protein decreased 36.7%, P<0.01), but still higher than that in the control group obviously (P<0.05). This kind of complexion was also validated by immunocytochemistry and Western blot analysis. CONCLUSION: CaN is a regulator of PKG I(alpha) expression in cultured VSMCs proliferation. Once CaN was inhibited by its special inhibitor CsA, the expression of PKG I(alpha) increased obviously. And the expression reduced significantly when CaN was actived by PE again.