中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2010年
6期
810-812
,共3页
热休克蛋白70%精原细胞%RNA干扰%脱噬作用
熱休剋蛋白70%精原細胞%RNA榦擾%脫噬作用
열휴극단백70%정원세포%RNA간우%탈서작용
HSP70%Spermiogonium%siRNA%Apoptosis
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.
目的 探討熱休剋蛋白(HSP)70-2基因沉默對精原細胞的影響及其機製.方法 構建HSP70-2特異的短髮夾RNA(shRNA)質粒載體,併轉染大鼠精原細胞;應用逆轉錄-聚閤酶鏈反應(RT-PCR)檢測HSP70-2 mRNA錶達,Western blot檢測HSP70-2、Cyclin DI、bcl-2和box蛋白錶達,應用流式細胞術分析細胞週期和凋亡.結果 HSP70-2基因沉默組的HSP70-2蛋白錶達量為0.93±0.13,明顯低于陰性對照組1.31±0.10(P<0.01);流式細胞術檢測結果顯示,與轉染Scrambled的陰性對照組比較,轉染pGenesil-1-HSP70-2重組質粒組的PC3細胞在G1期齣現明顯細胞週期阻滯和細胞凋亡,G1期阻滯細胞比例為(80.09±2.37)%,凋亡率為(7.44±2.41)%,而陰性對照組分彆為(61.85±2.31)%和(1.09±0.57)%(P<0.05).與陰性對照組比較,轉染後細胞bcl-2、Cyclin D1錶達水平顯著降低,而bax錶達水平升高.結論 HSP70-2基因沉默能誘導精原細胞凋亡,其機製可能是通過Cyclin D1蛋白調節細胞週期併通過上調box蛋白錶達促進細胞凋亡.
목적 탐토열휴극단백(HSP)70-2기인침묵대정원세포적영향급기궤제.방법 구건HSP70-2특이적단발협RNA(shRNA)질립재체,병전염대서정원세포;응용역전록-취합매련반응(RT-PCR)검측HSP70-2 mRNA표체,Western blot검측HSP70-2、Cyclin DI、bcl-2화box단백표체,응용류식세포술분석세포주기화조망.결과 HSP70-2기인침묵조적HSP70-2단백표체량위0.93±0.13,명현저우음성대조조1.31±0.10(P<0.01);류식세포술검측결과현시,여전염Scrambled적음성대조조비교,전염pGenesil-1-HSP70-2중조질립조적PC3세포재G1기출현명현세포주기조체화세포조망,G1기조체세포비례위(80.09±2.37)%,조망솔위(7.44±2.41)%,이음성대조조분별위(61.85±2.31)%화(1.09±0.57)%(P<0.05).여음성대조조비교,전염후세포bcl-2、Cyclin D1표체수평현저강저,이bax표체수평승고.결론 HSP70-2기인침묵능유도정원세포조망,기궤제가능시통과Cyclin D1단백조절세포주기병통과상조box단백표체촉진세포조망.
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.
