摘要
目的 探讨热休克蛋白(HSP)70-2基因沉默对精原细胞的影响及其机制.方法 构建HSP70-2特异的短发夹RNA(shRNA)质粒载体,并转染大鼠精原细胞;应用逆转录-聚合酶链反应(RT-PCR)检测HSP70-2 mRNA表达,Western blot检测HSP70-2、Cyclin DI、bcl-2和box蛋白表达,应用流式细胞术分析细胞周期和凋亡.结果 HSP70-2基因沉默组的HSP70-2蛋白表达量为0.93±0.13,明显低于阴性对照组1.31±0.10(P〈0.01);流式细胞术检测结果显示,与转染Scrambled的阴性对照组比较,转染pGenesil-1-HSP70-2重组质粒组的PC3细胞在G1期出现明显细胞周期阻滞和细胞凋亡,G1期阻滞细胞比例为(80.09±2.37)%,凋亡率为(7.44±2.41)%,而阴性对照组分别为(61.85±2.31)%和(1.09±0.57)%(P〈0.05).与阴性对照组比较,转染后细胞bcl-2、Cyclin D1表达水平显著降低,而bax表达水平升高.结论 HSP70-2基因沉默能诱导精原细胞凋亡,其机制可能是通过Cyclin D1蛋白调节细胞周期并通过上调box蛋白表达促进细胞凋亡.
Objective To investigate the effects and mechanisms of heat shock protein (HSP) 70-2 short hairpin RNA (shRNA ) on the rat spermiogonium cells. Methods The recombinant plasmid series of HSP70-2-targeted short hairpin RNA (shRNA) were constructed by pGenesil-1 plasmids vector, then transfected into rat spermiogonium cells. The HSP70-2 expression in spermiogonium cells was detected by reverse transcription-polymerase chain reaction ( RT-PCR) and Western blotting. Apoptosis related proteins Cyclin D1, bcl-2, and bax expression was detected in the transfected cells. Flow cytometry was used to observethe cell cycle and apoptosis. Results HSP70-2 protein expression in the pGenesil-HSP70-2 treated group was significantly lower than that in the control group (0. 93 ±0. 13 vs 1. 31 ±0. 10,P〈0.01). The results of flow cytometry revealed that in the pGenesil-1-HSP70-2 recombinant plasmid transfection group, the ratio of G1 phase arrest cells was (80. 09 ± 2. 37) % and the apoptotic rate was (7. 44 ± 2.41) % , while in the control group those were (61. 85 ± 2. 31) % and(1.09±0.57)% respectively ( P 〈 0. 05). In the pGenesil-1-HSP70-2 recombinant plasmid transfection group, the expression of Cyclin D1 and bcl-2 was decreased, and that of Bax increased. Conclusion HSP70-2-shRNA could significantly induce spermiogonium cell apoptosis probably by down-regulating the bcl-2 and Cyclin D1 expression, and up-regulatingthe Bax expression.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2010年第6期810-812,共3页
Chinese Journal of Experimental Surgery