白细胞介素-10基因转染肝星状细胞对细胞凋亡及信号转导和转录活化因子-1表达的影响

Effects of adenovirus-mediated interleukin-10 gene on apoptosis of hepatic stellate cells induced by tumor necrosis factor α and the expression of signal transducer and activator of transcription-1

目的观察腺病毒介导的白细胞介素-10（IL-10）基因（Ad—IL-10）转染肝星状细胞（HSC）对肿瘤坏死因子-α（TNF-α）诱导的细胞凋亡及信号转导和转录活化因子-1（STAT-1）表达的影响。方法以293细胞包装构建重组腺病毒载体系统Ad5-hiL10-IRES-增强型绿色荧光蛋白（EGFP）。将体外培养的人HSC分为5组：空白对照组、空载组、TNF-α（10μg／L）组、空载组＋TNF-α（10μg/L）组、Ad—IL—10＋TNF—α（10μg／L）组。流式细胞仪检测HSC细胞的凋亡比例；Westernblot检测HSC细胞STAT-1及其磷酸化蛋白水平。结果成功构建Ad5-hlL-10-IRES-EGFP；流式细胞仪检测显示TNF—α诱导使HSC凋亡率显著增加，Ad—IL-10干预可使HSC凋亡率下降（P〈0．05）；其凋亡率分别为：空白组0．010±0．001，TNF—α组0．230±0．090，空载组0．210±0．080，IL-10组0．070±0．030。Western blot检测显示，TNF-α诱导后STAT-1磷酸化蛋白水平显著增加，而Ad-IL-10干预可使两者水平下降（P〈0．01）；各组STAT—P／STAT-1吸光度值分别为：空白组0．028±0．006，空载组0．025±0．007，TNF—α组0．261±0．085，空载对照组0．254±0．065，IL-10组0．029±0．009。结论IL—10能抑制TNF—α诱导HSC细胞发生凋亡，其信号途径与STAT-1有关。

Objective To study the effects of adenovirns-mediated intedeukin-10（IL-10 gene on apoptosis of hepatic stellate cells （HSCs） induced by tumor necrosis factor-α （TNF-α） and the expression of signal transducer and activator of transcription-1 （ STAT-1 ） , and to investigate the relation of IL-10and apoptosis of HSCs. Methods The recombinant adenovirus vector carrying IL-10marked enhanced green fluourescent protein [ Ad5-hIL-10IRES-enhanced green fluorescent protein（EGFP） ] was constructed. Cultured human HSCs were divided into five groups： control group, empty vector control group, TNF-α group, empty vector ＋ TNF-α group and Ad-IL-10＋ TNF-α group. The apoptosis rate of HSCs was assayed by flow cytometry. The levels of STAT-1 and STAT-1 phosphorylation were assayed by Western blotting. Results AdS-hIL-10IRES-EGFP was established successfully. Flow eytometry showed that apoptosis rate of HSCs induced by TNF-α was increased significantly, and Ad-IL-10intervention decreased apoptosis rate of HSCs （P 〈0. 05）. The apoptosis rate was as follows： control group 0. 010 ±0. 001, TNF-α group 0. 230 ±0. 090, empty vector group 0. 210 ± 0. 080, Ad-IL-10＋ TNF-α group 0. 070 ± 0. 030, respectively. Western blotting analysis showed that STAT-1 and STAT-1 phosphorylation protein levels were increased significantly by TNF-α, and Ad-IL-10interventions could reduce the STAT-1 levels （ P 〈 0. 01 ）. The STAT-P/STAT-1 Awas as follows ： control group 0. 028 ± 0. 006, empty vector group 0. 025 ± 0. 007, TNF-α group 0. 261 ± 0. 085, empty vector ＋ TNF-α group 0. 254±0. 065, Ad-IL-10＋ TNF-α group 0. 029 ± 0. 009, respectively. Conclusion IL-10could inhibit TNF-α-induced HSCs apoptosis and its signaling pathways involved in STAT-1.