CAJ | 학술논문

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-PTEN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％±3.0％)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通过下凋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.
목적 통과검측선병독개도적야생형제10호염색체결실적린산매장력단백동원물기인(PTEN)대체외활화간성상세포(HSC)세포주기적영향,탐토과표체적야생형PTEN억제활화HSC증식적가능궤제. 방법 체외배양간성상세포계HSC-T6,이선병독위재체장야생형PTEN기인순시전염활화HSC,실험분위공백대조조、Ad-GFP조화Ad-PTEN조.류식세포술측정HSC세포주기시상,Western blot급실시정량PCR검측HSC적PTEN표체량,Western blot검측HSC적세포주기소D1 (cyclin D1)화세포주기소의뢰성격매4(CDK4)표체수평.다조간비교채용단인소방차분석,조간비교채용Tukey검험. 결과 휴대야생형PTEN기인적선병독(AD-PTEN)성공전염HSC,Ad-PTEN조G0/G1기HSC세포수(67.68％±2.75％)교대조조(53.01％±2.37％)급Ad-GFP조(53.85％±3.0％)명현증고(F=38.59,P＜0.01);S기HSC세포수(14.42％±1.81％)교대조조(22.17％±1.99％)급Ad-GFP조(21.54％±1.74％)명현강저(F=18.85,P＜ 0.01);G2/M기HSC세포수(17.90％±2.70％)교대조조(24.82％±3.81％)급Ad-GFP조(24.62％±3.15％)명현강저(F=7.17,P＜0.01).선병독감염HSC후72h,Ad-PTEN조cyclin D1적상대표체량(1.12±0.07)교대조조(1.45±0.05)급Ad-GFP조(1.47±0.08)명현강저(F=42.86,P＜0.01),CDK4상대표체량(1.05±0.07)교대조조(1.41±0.03)급Ad-GFP조(1.43±0.06)야명현강저(F=67.01,P＜ 0.01).결론 과표체적야생형PTEN통과하조cyclin D1화CDK4적표체,명현억제체외활화HSC세포주기적G1/S기전화,조체HSC세포주기시상우G0/G1기,진이억제기증식.
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 PCP.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.