摘要
目的研究腺病毒介导的IL-10基因(Ad-IL-10)转染肝星状细胞(HSC)对TNFα诱导的细胞凋亡及caspase-3表达的影响,探讨IL-10在HSC凋亡中的作用。方法体外以293细胞包装构建重组腺病毒载体系统Ad5-hIL10-IRES-EGFP。将体外培养的人HSC分为5组:空白对照组、空载组、TNFα(10 ng/mL)组、空载组+TNFα(10 ng/mL)组、Ad-IL-10+TNFα(10 ng/mL)组。荧光显微镜观察IL-10表达量;RT-PCR法测定IL-10 mRNA表达水平;TUNEL法测定HSC细胞的凋亡率;Western blot测定HSC细胞IL-10、caspase-3蛋白水平。结果成功构建Ad5-hIL10-IRES-EGFP;RT-PCR和Western blot检测显示Ad-IL-10转染3 d后HSC表达IL-10 mRNA和蛋白含量均增加,且随MOI增大而表达增强;TUNEL染色显示,TNFα诱导使HSC凋亡率显著增加,Ad-IL-10干预可使HSC凋亡率下降;Western blot检测显示,TNFα诱导后caspase-3蛋白水平显著增加,而Ad-IL-10干预可使caspase-3蛋白水平下降。结论 IL-10能抑制TNFα诱导HSC细胞发生凋亡,其信号途径涉及caspase-3。
To study the effects of adenovirus-mediated IL-10 gene on apoptosis of hepatic stellate cells induced by TNFot and the expression of caspase-3, and to investigate the relation of IL-10 and HSCs apoptosis.Methods The recombinant adenovirus vector carrying interleukin-10 marked enhanced green fluourescent protein （AdS-hIL-IO-IRES-EGFP） was constructed. Cultured human HSCs were divided into five groups： control group, empty vector control group, TNFot group, empty vector ＋ TNFot group and Ad-IL-10 ＋ TNFc~ group, respectively. The expression of IL-10 was observed by fluorescence microscope ; the level of IL-10 mRNA was assayed by RT-PCR ; the apoptosis ratio of HSCs was assayed by TUNEL; the levels of IL-10 and caspase-3 were assayed by Western blot. Results Ad5hIL-10-IRES-EGFP was established successfully. RT-PCR and Western blot analysis showed that 3 days after the transfection of Ad-IL-10, the expression of IL-10 mRNA and protein levels in HSCs were increased, and enhanced expression increased with the MOI; TUNEL staining showed that apoptosis rate of HSCs induced by TNFet increased significantly,Ad-IL-10 intervention decreased HSCs apoptosis rate; Western blot analysis showed that caspase-3 protein levels increased significantly by TNFa, Ad-IL-10 interventions enabled the caspase-3 levels descent. Conclusion IL-10 could inhibit TNFα-induced HSCs apoptosis and its signaling pathways involved in caspase-3.
出处
《胃肠病学和肝病学杂志》
CAS
2013年第1期1-5,共5页
Chinese Journal of Gastroenterology and Hepatology
基金
福建省自然科学基金(2010J01165)
福建省高校新世纪创新人才项目(NCETFJ-0714)
