中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2010年
4期
507-509
,共3页
魏少忠%吴新红%陈继红%郑新民
魏少忠%吳新紅%陳繼紅%鄭新民
위소충%오신홍%진계홍%정신민
小分子干扰RNA%前列腺癌%肿瘤坏死因子相关凋亡诱导配体%丝氨酸蛋白酶
小分子榦擾RNA%前列腺癌%腫瘤壞死因子相關凋亡誘導配體%絲氨痠蛋白酶
소분자간우RNA%전렬선암%종류배사인자상관조망유도배체%사안산단백매
siRNA%Prostate carcinoma%TNF-related apoptosis-inducing ligand%Serine protease
目的 观察肿瘤坏死因子相关凋亡诱导配体(TRAIL)对前列腺癌细胞(PC-3M)不同丝氨酸蛋白酶Omi/HtrA2表达水平的促凋亡作用.方法 构建其小分子干扰RNA(siRNA)表达载体,辅助设计Omi/HtrA2特异性siRNA序列.合成后克隆入真核表达载体psiRNA-hH1neo.脂质体法转染psiRNA-Omi/HtrA2载体至PC-3M中,检测Omi/HtrA2在PC-3M细胞中的表达及psiRNA-Omi/HtrA2对Omi/HtrA2沉默效应后的转录和表达.用原位末端转移酶标记技术检测Omi/HtrA2基因沉默后,计算不同浓度TRAIL(50、100、200、500 μg/L)下PC-3M细胞凋亡指数(AI).结果 Omi/HtrA2在PC-3M细胞中高表达.酶切和DNA测序证实siRNA基因序列正确,且准确克隆入psiR-NA-hH1neo载体中.psiRNA-Omi/HtrA2载体可特异性抑制PC-3M细胞中Omi/HtrA2的表达.不同浓度TRAIL(50、100、200、500 μg/L)对转染psiRNA-Omi/HtrA2载体后PC-3M细胞AI分别为7.23、14.87、22.65、31.78.而未转染组分别为15.28、24.17、36.33、47.76.两组之间差异有统计学意义(P<0.05).结论 Omi/HtrA2在前列腺癌细胞凋亡过程中起重要作用,TRAIL促进前列腺癌细胞的凋亡,其效果与TRAIL浓度及Omi/HtrA2表达水平相关.
目的 觀察腫瘤壞死因子相關凋亡誘導配體(TRAIL)對前列腺癌細胞(PC-3M)不同絲氨痠蛋白酶Omi/HtrA2錶達水平的促凋亡作用.方法 構建其小分子榦擾RNA(siRNA)錶達載體,輔助設計Omi/HtrA2特異性siRNA序列.閤成後剋隆入真覈錶達載體psiRNA-hH1neo.脂質體法轉染psiRNA-Omi/HtrA2載體至PC-3M中,檢測Omi/HtrA2在PC-3M細胞中的錶達及psiRNA-Omi/HtrA2對Omi/HtrA2沉默效應後的轉錄和錶達.用原位末耑轉移酶標記技術檢測Omi/HtrA2基因沉默後,計算不同濃度TRAIL(50、100、200、500 μg/L)下PC-3M細胞凋亡指數(AI).結果 Omi/HtrA2在PC-3M細胞中高錶達.酶切和DNA測序證實siRNA基因序列正確,且準確剋隆入psiR-NA-hH1neo載體中.psiRNA-Omi/HtrA2載體可特異性抑製PC-3M細胞中Omi/HtrA2的錶達.不同濃度TRAIL(50、100、200、500 μg/L)對轉染psiRNA-Omi/HtrA2載體後PC-3M細胞AI分彆為7.23、14.87、22.65、31.78.而未轉染組分彆為15.28、24.17、36.33、47.76.兩組之間差異有統計學意義(P<0.05).結論 Omi/HtrA2在前列腺癌細胞凋亡過程中起重要作用,TRAIL促進前列腺癌細胞的凋亡,其效果與TRAIL濃度及Omi/HtrA2錶達水平相關.
목적 관찰종류배사인자상관조망유도배체(TRAIL)대전렬선암세포(PC-3M)불동사안산단백매Omi/HtrA2표체수평적촉조망작용.방법 구건기소분자간우RNA(siRNA)표체재체,보조설계Omi/HtrA2특이성siRNA서렬.합성후극륭입진핵표체재체psiRNA-hH1neo.지질체법전염psiRNA-Omi/HtrA2재체지PC-3M중,검측Omi/HtrA2재PC-3M세포중적표체급psiRNA-Omi/HtrA2대Omi/HtrA2침묵효응후적전록화표체.용원위말단전이매표기기술검측Omi/HtrA2기인침묵후,계산불동농도TRAIL(50、100、200、500 μg/L)하PC-3M세포조망지수(AI).결과 Omi/HtrA2재PC-3M세포중고표체.매절화DNA측서증실siRNA기인서렬정학,차준학극륭입psiR-NA-hH1neo재체중.psiRNA-Omi/HtrA2재체가특이성억제PC-3M세포중Omi/HtrA2적표체.불동농도TRAIL(50、100、200、500 μg/L)대전염psiRNA-Omi/HtrA2재체후PC-3M세포AI분별위7.23、14.87、22.65、31.78.이미전염조분별위15.28、24.17、36.33、47.76.량조지간차이유통계학의의(P<0.05).결론 Omi/HtrA2재전렬선암세포조망과정중기중요작용,TRAIL촉진전렬선암세포적조망,기효과여TRAIL농도급Omi/HtrA2표체수평상관.
Objective To study the apoptosis of human prostate cancel cell line (PC-3M) at dif-ferent levels of Omi/HtrA2 when treated with TNF-related apoptosis-inducing ligand (TRAIL). Methods siRNA expressing vector was constructed. The expression of omi/HtrA2 in PC-3M cells was detected by u-sing reverse transcription-polymerase chain reaction (RT-PCR) techniques. According to the software of Invivogen company, Omi/HtrA2 specific siRNA sequence was designed, synthesized in vitro and cloned in-to the expression vector psiRNA-hH1neo. The constructed psiRNA-Omi/HtrA2 was transiently transfected into PC-3M cells and the inhibitory effect of psiRNA-Omi/HtrA2 on the expression of Omi/HtrA2 in PC-3M cells was detected using RT-PCR and Western blotting. The apoptesis index (AI) of PC-3M cells was determined by in situ end labeling techniques under different TRAIL concentrations (50, 100,200 and 500 μg/L). Results There was high level of the Omi/HtrA2 expression in PC 3M cells. Enzyme digestion a-nalysis and DNA sequencing confirmed the Omi/HtrA2 specific siRNA expression vector was constructed successfully. The expression of Omi/HtrA2 was down-regulated in PC-3M cells after the transfection of psiRNA-Omi/HtrA2. The AI of PC-3M cells in transfection group was 7. 23, 14. 87, 22. 65 and 31.78 re-spectively, and that in un-transfection group was 15.28, 24. 17, 36. 33 and 47. 76, respectively (P < 0. 05). Conclusion Omi/HtrA2 plays an important role in the apoptosis of PC-3M cells. TRAIL can in-duce the apoptosis of PC-3M cells concentration-dependently, and its effect was correlated with the expres-sion level of omi/HtrA2.
