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

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

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,0.5mg/L)干预组、高浓度CsA(5mg/L)干预组以及苯肾上腺素(PE)刺激组(5mg/L CsA+10μmol/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 IαmRNA以及蛋白的表达水平。结果0.5mg/L CsA组PKG IαmRNA以及蛋白的表达水平与对照组相比差异无统计学意义,而5mg/L CsA组PKG IαmRNA及蛋白的表达明显高于对照组,5mg/L CsA+10μmol/L PE组中PKG IαmRNA的表达比5mg/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。

Objective： To dicuss the regulation of expression of cGMP-dependent protein kinase In （PKG I ） by calcineurin （CaN） in vascular smooth tousle cells （VSMCs） proliferation. Methods： Cul- tured 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 ＋ 10μmoL/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 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 μmoL/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 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 expression in cultured VSMCs proliferation. Once CaN was inhibited by its special inhibitor CsA, the expression of PKG Ⅰ increased obviously. And the expression reduced significantly when CaN was actived by PE again.