SIRT1在氢醌诱导TK6细胞凋亡中的作用

The role of SIRT1 in TK6 cell apoptosis induced by hydroquinone

目的构建TK6细胞的沉默信息调节因子1(SIRT1)基因沉默细胞株(TK6-sh SIRT1),初步探讨SIRT1在氢醌诱导TK6细胞凋亡中的作用。方法应用慢病毒介导的RNA干扰技术构建TK6细胞的SIRT1沉默细胞株,并用q PCR和Western blotting联合鉴定干扰效果,比较两种细胞的一般生物学特性(细胞形态、细胞增殖能力和细胞周期分布)。用不同浓度HQ(2.5~40μmol/L)处理TK6和TK6-sh SIRT1细胞48 h后,以CCK-8法检测细胞存活率;以流式细胞术检测细胞周期及凋亡的改变。结果成功筛选出稳定表达的人淋巴母细胞SIRT1缺陷细胞株,与TK6正常细胞株相比,TK6-sh SIRT1细胞中SIRT1在m RNA和蛋白表达水平分别下降了84.6%和94.5%,且生长速度加快了6.57 h,增殖指数增加了11.8%,差异均有统计学意义(P<0.05),但细胞形态未出现明显改变。经HQ短时间处理后:两种细胞存活率均呈剂量依赖性降低;相同染毒剂量条件下,TK6-sh SIRT1细胞的存活率均明显低于TK6细胞(P<0.05),细胞早期凋亡率高于TK6细胞(P<0.05)。结论 SIRT1缺陷可增加TK6细胞对HQ的敏感性。

Objective To construct TK6-sh SIRT1 cell lines,and preliminarily explore the role of SIRT1 in hydroquinone（HQ）-induced apoptosis. Methods SIRT1-deficient cell line of TK6 Cell was constructed using RNA interference technology. QPCR and Western blotting were used to identify the gene silencing efficiency. The biological characteristics between the two cell lines were compared, such as the cell morphology, multiplication capacity and cell cycle.Both TK6 and TK6-sh SIRT1 were exposed to HQ at different doses（2.5-40 μmol/L） for 48 h. Cell survival was measured by CCK-8. Flow cytometry was used to analyze cell cycle and apotosis rate. Results The stable SIRT1-deficient TK6 cell line was successfully screened out.Compared with the normal TK6, the expression levels of SIRT1 m RNA and protein decreased 84.6% and 94.5% respectivly,the multiplication speed shortened 6.57 h, and the PI increased 11.8% in TK6-sh SIRT1 cell line（P 〈0.05）, which revealed no significantly morphological variation.HQ caused growth inhibition in both cell lines at an obvious dose-dependent manner.At the same HQ dose, lower survival rate and higher early apoptosis rate were found in TK6-sh SIRT1 cell compared with TK6 cells（P〈0.05）. Conclusion The SIRT1-deficiency increases the sensitivity of TK6 to HQ.