蛋白激酶GIa基因在蛛网膜下腔出血后对血管调控作用的研究

目的观察血性脑脊液对大鼠脑动脉平滑肌细胞(CASMC)的促增殖作用以及携带蛋白激酶GIa(PKGIa)基因的重组腺病毒载体(Ad-PKGIa)的干预作用,探讨PKGIa基因在蛛网膜下腔出血后继发慢性脑血管痉挛分子机制中可能的调控作用。方法相同密度(10~6/cm~2)接种后分为血性脑脊液组(B组)、生理盐水转染组(AN组)、血性脑脊液空载体组(KB组)和血性脑脊液转染组(AB组)。另设生理盐水对照组(N组)。采用组织块法原代培养CASMC。采用RT-PCR和Western blot检测Ad-PKGIa转染CASMC后PKGIa基因的表达。采用MTT法、~3H-TdR掺入法检测血性脑脊液对CASMC增殖的刺激作用以及Ad-PKGIa的抑制作用。结果与转染前比较,Ad-PKGIa转染后1 d,CASMC内PKGIa mRNA水平和蛋白表达明显增高(P0.05)。与N组比较,B组、KB组~3H-TdR和MTT明显增多和升高(P0.05);与B组比较,AN组和AB组~3H-TdR和MTT明显减少和降低(P0.05);与KB组比较,AB组~3H-TdR和MTT明显减少和降低(P0.05)。结论 PKGIa信号途径在蛛网膜下腔出血后,继发慢性脑血管痉挛分子机制中有重要的调节作用,可作为基因治疗的靶点之一。

Adenovirus-mediate-PKGIa suppresses proliferation of cerebral arterial smooth muscle cell after subarachnoid hemorrhage

Objectives To study observe the proliferation of cerebral arterial smooth muscle cell (CASMC) induced by bloody cerebrospinal fluid(BCSF) and intevention by adenovirus-mediate-pKGIa (Ad-PKGIa) in rats.and to investigate the potential regulative role of PKGIa gene in molecular mechanism of cerebral vasospasm after subarachnoid hemorrhage (SAH).Methods Pure CASMC were cultured by tissue-sticking methods.RT-PCR and Western blot were used to examine the PKGIa mRNA and protein expression after CASMC were transfected by Ad-PKGIa.The changes of CASMC proliferation were determined by MTT and ~3H-TdR incorporation method. Results Ad-PKGIa could transfect CASMC and was highly expressed.BCSF could stimulate the proliferation of CASMC,increase ~3H-TdR incorporation from (6374.4±545.3) to (9256.6 ± 587.3,P < 0.05) and increase the absorbance value of MTT from (0.4 ± 0.1) to (0.6 ± 0.1, P < 0.05),these changes could be blocked after CASMC was transfected by Ad-PKGIa.Conclusions The results suggest that PKGIa signaling pathway might play an important role in the cerebral vasospasm after SAH.PKGIa gene might be a target for gene therapy.