PTEN基因重组腺病毒对人气道平滑肌细胞增殖的影响及其作用机制

Effect of recombinant adenovirus carrying PTEN gene on the proliferation of human airway smooth muscle cells in vitro and the mechanisms

目的观察携带野生型PTEN基因的重组腺病毒(Ad-PTEN)体外转染人气道平滑肌细胞(HASMC)后的表达,研究其对HASMC增殖的影响,并对其作用机制作初步的探讨。方法利用pAdxsi载体系统构建携带PTEN基因的腺病毒载体,体外转染HASMC,荧光显微镜观察Ad-PTEN的转染效率。RT-PCR及Western blotting检测PTEN在HASMC中的表达。采用MTS/PMS法检测细胞增殖,流式细胞仪测定细胞周期分布。Western blotting检测Akt及p-Akt的表达,RT-PCR检测P21mRNA的表达。结果外源性野生型PTEN基因经腺病毒介导成功转入HASMC,当感染复数MOI为100时,体外转染效率高达98%以上。Ad-PTEN感染HASMC后,能有效地引起细胞内过度表达PTEN。上调PTEN表达能显著提高G0/G1期细胞的比例,抑制细胞的增殖。上调PTEN表达能显著降低p-Akt及提高P21的表达水平。结论成功构建了过度表达PTEN的原代培养HASMC模型,该模型能有效地引起细胞内过度表达PTEN。上调PTEN表达能有效抑制HASMC的增殖,可能是通过对PI3K/AKt信号通路的抑制及上调P21的表达而起作用的。

Objective To observe the effect of exogenous phophatase and tensin homolog deleted on chromosone 10 （PTEN） gene transfer via recombinant adenoviruses on the proliferation of human airway smooth muscle cells （HASMCs） in vitro and investigate the possible mechanisms. Methods With a recombinant adenovirus vector containing PTEN （Ad-PTEN） constructed using the pAdxsi system, PTEN gene was transiently transfected into HASMCs and the transfection efficiency was determined by fluorescence microscope. RT-PCR and Western blotting were performed to detect the expression of PTEN mRNA and protein in the infected cells. MTS/PMS assay was used to analyze the proliferation of HASMCs, and the cell cycle changes of the transfected cells were evaluated by flow cytometry with PI staining. The expression levels of Akt and p-A kt proteins were detected by Western blotting, and P21 mRNA expression determined by RT-PCR. Results The recombinant adenovirus Ad-PTEN showed a wild-type PTEN gene transfer efficiency of 98% at the multiplicity of infection （MOI） of i 00. RT-PCR and Western blotting showed that infection with the recombinant adenovirus resulted in PTEN overexpression in the HASMCs, causing also increased ratio of G0/Gl cells and proliferation inhibition of the ASMCs. The overexpression of PTEN significantly decreased the expression level of p-Akt but increased P21 mRNA expression. Conclusion The recombinant adenovirus containing PTEN can be successfully transfected into HASMCs cultured in vitro, resulting in PTEN overexpression at both the mRNA and protein levels. PTEN overexpression can efficiently inhibit the proliferation of HASMCs possibly through the PI3K/PKB/AKt and P21 pathways.