摘要
目的观察凋亡相关基因bcl-2高表达抑制过氧化氢诱导肾小管上皮细胞凋亡的作用。方法构建含有人bcl-2cDNA的逆转录病毒真核表达载体PLXSN，脂质体法将重组质粒转染PA317细胞，G418筛选阳性克隆，鉴定后浓缩收集病毒上清，浓缩病毒液，感染人肾小管上皮细胞株HK-2，Westernblot检测bcl-2mRNA表达。5mmol／L过氧化氢（H2O2）诱导细胞凋亡后，流式细胞仪检测细胞凋亡发生变化。结果流式细胞仪分析显示5mmol／LH2O2成功诱导肾小管上皮细胞凋亡，bcl-2逆转录病毒感染肾小管上皮细胞后，bcl-2蛋白水平呈高表达，HKbcl-2组细胞凋亡数量（12．41±3．46）较HK-2组（19．62±4．20）显著减少（P〈0．05）。而转染空载体的对照组无明显变化（19．62±4．20，P〈0．05）。结论bcl-2蛋白高表达显著抑制氧化剂诱导的肾小管上皮细胞凋亡。
Objective To investigate the inhibitory effects of the bcl-2 expression on hydrogen peroxide （ H2O2 ） -induced apoptosis of renal tubular epithelial cells. Methods A recombinant retroviral vector PLbcl-2SN was constructed and transfected to packaging cell line PA317 by lipofectine. The G418 resistant colonies were selected, and the supernatants of the colony cultures were used to infect the human kidney epithelial cell line HK-2 ,which was named as HK-2bcl-2. Western blot was employed to detect the bcl-2 expression. The apoptosis of HK-2 and HK-2bcl-2 cells was induced by H2O2. The changes in the number of apoptotic ceils were assessed by flow cytometry. Results Five mmol/L H2O2 induced apoptosis of HK-2 cells successfully. After bcl-2 retroviral superuatants were infected into the renal epithelial cells HK-2 ,the bcl-2 expression in the PLbcl-2SN transfected HK-2bcl-2 cells was increased significantly as compared with control group （P 〈 0.05 ）. The number of apoptotic ceils in HKbcl-2 group was significantly reduced as compared with HK-2 group [ （ 12.41 ±3.46） vs （ 19.62 ± 4.20）, P 〈 0.05 ]. Conclusion Upregulation of bcl-2 protein can suppress the oxidant-induced apoptosis of renal epithelial cells effectively.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2009年第3期363-365,共3页
Chinese Journal of Experimental Surgery
