腺病毒介导的野生型PTEN抑制活化肝星状细胞系HSC—T6增殖的机制  

The mechanisms of inhibitory effect of adenovirus-mediated wild-type PTEN gene on the proliferation in activated hepatic stellate cells in vitro

作　　者：郝礼森[1] 张晓岚[2,3] 王静[1] 李立文[1] 莫艳波[1] 张超[1] 姚冬梅[2,3] 姜慧卿[2,3] 

机构地区：[1]河北联合大学附属医院消化内科,河北省唐山市063000 [2]河北医科大学第二医院消化内科 [3]河北省消化病重点实验室河北省消化病研究所

出　　处：《中华肝脏病杂志》2012年第7期503-506,共4页Chinese Journal of Hepatology

基　　金：国家自然科学基金（30872513）

摘　　要：目的通过检测腺病毒介导的野生型第10号染色体缺失的磷酸酶张力蛋白同源物基因（PTEN）对体外活化肝星状细胞（HSC）细胞周期的影响，探讨过表达的野生型PTEN抑制活化HSC增殖的可能机制。方法体外培养肝星状细胞系HSC-T6，以腺病毒为载体将野生型PTEN基因瞬时转染活化HSC，实验分为空白对照组、Ad-GFP组和Ad-PTEN组。流式细胞术测定HSC细胞周期时相，Western blot及实时定量PCR检测HSC的PTEN表达量，Western blot检测HSC的细胞周期素D1（cyclin D1）和细胞周期素依赖性激酶4（CDK4）表达水平。多组间比较采用单因素方差分析，组间比较采用Tukey检验。结果携带野生型PTEN基因的腺病毒（AD-PTEN）成功转染HSC，Ad-FTEN组G0／G1期HSC细胞数（67．68%＋2．75％）较对照组（53．01％±2．37%）及Ad-GFP组（53．85％±3．08％）明显增高（F=38．59，P〈0．01）；S期HSC细胞数（14．42％±1．81％）较对照组（22．17％±1．99%）及Ad-GFP组（21．54%±1．74%）明显降低（F=18．85，P〈0．01）；G2/M期HSC细胞数（17．90%±2．70%）较对照组（24．82%±3．81％）及Ad-GFP组（24．62％±3．15％）明显降低（F=7．17，P〈0．01）。腺病毒感染HSC后72h，Ad-PTEN组cyclin D1的相对表达量（1．12±0．07）较对照组（1．45±0．05）及Ad-GFP组（1．47±0．08）明显降低（F=42．86，P〈0．01），CDK4相对表达量（1．05±0．07）较对照组（1．41±0．03）及Ad-GFP组（1．43±0．06）也明显降低（F=67．01，P〈0．01）。结论过表达的野生型PTEN通过下调cyclin D1和CDK4的表达，明显抑制体外活化HSC细胞周期的G1／S期转化，阻滞HSC细胞周期时相于G0／G1期，进而抑制其增殖。Objective Using an adenoviral vector, the wild-type PTEN gene was transduced into activated hepatic stellate cell （HSC） culcured in vitro and cell cycle markers and were detect. Thereby, the potential mechanisms of inhibitory effect of the wild-type PTEN overexpression on the proliferation in activated HSC was investigated. Methods The wild type PTEN gene was transduced into activated HSC （HSC-T6） cultured in vitro mediated by adenoviral vector. PTEN expression in HSC was measured by Western blot and Real-time fluorescent quantitation PCR. Flow cytometry （FCM） was then used to detect cell cycle phase of activated HSC. And the expressions of cyclinD1 and cyclin dependent kinase 4 （CDK4）in HSC were determined by Western blot. Results The data showed that exogenous wild type PTEN gene was successfully transduced and expressed in activated HSC cultured in vitro. The over-expression of wild type PTEN resulted in the increased number of HSC at G0/G1 phase （P〈0.01）, and the number of HSC at S phase and G2/M phase were decreased significantly, P〈0.01. Furthermore, there were decreased cyclinD1 and CDK4 expression in HSC infected with Ad-PTEN, P〈 0.01. Conclusion The over-expression of wild type PTEN inhibit transition of activated HSC in vitro from G1 to S phase and arrest cell cycle of them at G0/G1 phase via the down-regulated expressions of cyclinD 1 and CDK4, and then inhibit HSC proliferation.

关 键 词：肝硬化 细胞增殖 细胞周期 肝星状细胞.PTEN 

分 类 号：R575[医药卫生—消化系统]