顺铂诱导肾小管上皮细胞程序性死亡的机理

Mechanism of programmed cell death of renal proximal tubular cells induced by cisplation

目的:探讨顺铂导致细胞凋亡的发生部位和诱导细胞凋亡的信号传导途径,为更好地预防铂类药物对肾脏的毒副作用奠定基础。方法:将含目的基因的Bcl-2(Bcl-acta、Bcl-cb5及Bcl-nt)的pcDNA3.1(+)/Hygromycin质粒及pEGFP-C3质粒,通过共转染方式导入肾小管上皮细胞(RPTC)。并将细胞分为对照组、Bcl-acta组、Bcl-cb5组及Bcl-nt组。细胞经顺铂处理后,应用显微共聚焦和免疫荧光及免疫印记技术,分析各种突变的Bcl-2基因对细胞Bax激活和线粒体细胞色素C释放的保护作用,并进行Hoechst33258染色观察细胞凋亡,综合分析顺铂诱导细胞凋亡的信号传导途径。结果:转染Bcl-cb5组细胞色素C释放百分率为35.74%,明显高于Bcl-nt组(18.7%)和Bcl-acta组(24.6%),Bcl-cb5组细胞色素C释放的细胞数与Bcl-nt组及Bcl-acta组比较明显增多(P<0.05)。未经转染Bcl-2的细胞和经过转染Bcl-cb5的细胞均可见明显的Bax激活和细胞凋亡。而经Bcl-acta和Bcl-nt转染后再进行顺铂处理,24h后比Bcl-cb5基因转染组和对照组的细胞凋亡率明显降低(P<0.05)。结论:转染Bcl-acta和Bcl-nt基因组RPTC抵抗凋亡的能力明显高于转染Bcl-cb5基因组,转染Bcl-cb5基因能够使所表达的蛋白在内质网上定位,但不能发挥抗凋亡的作用。说明RPTC抵抗顺铂诱导的细胞凋亡的主要作用是通过线粒体途径而不是通过内质网起作用的。

Objective To study the signal pathway of apoptosis of renal proximal tubular cells （RPTC） caused by cisplatin for prevention of toxic effects of cisplatin on kidney. Methods pcDNA3.1/Hygromycin vector and pEGFP-C3 vector including Bcl-2 （Bcl-acta, Bcl-cb5 and Bcl-nt） were co-transfected in RPTC. After treated with cisplatin, Bax was activated, cytochrom C release and apoptosis were analyzed with confocal microscope and immunofluoresence technique. Apoptotic cells stained with Hoechst33258 were also counted and statistically analyzed. Results The percent of cytochrom C release （35.74%） in Bcl-cb5 transfected group was higher than those in Bcl-nt group （18.7%） and Bcl-acta group （24.6M） （P〈0.05）. More Bax activation and apoptosis were found in control group and Bcl-cb5 group. The results of apoptotic cell counting showed more apoptosis in Bcl-cb5 group and control group was observed compared with Bcl-acta group and Bcl-nt group （P〈0.05）. Conclusion Bclacta and Bcl-nt gene can protect RPTC from apoptosis induced by cisplatin in mitochondrial pathway. It means that cisplatin-induced RPTC apoptosis mainly is in mitochondrial signal pathway but not in endoplasmic reticulum （ER）.