运用RNA干扰(RNA interference,RNAi)技术阻断前列腺癌细胞系PC3中survivin基因的表达,并研究 survivin基因沉默后对PC3细胞产生的影响。方法:用真核转录载体pSilencerTM3．1-H1 neo构建针对survivin基因的重组真核转录载体pSilencer 3．1-SVV1、pSilencer 3．1-SVV2和pSilencer 3．1-SVV3,并用脂质体法转染前列腺癌细胞系PC3,通过RT- PCR、蛋白印迹实验检测sunrivin的表达变化,并用流式细胞术、MTT法检测转染后PC3细胞的凋亡、细胞周期、细胞生长速度、对顺铂的敏感性等芳面的变化。结果:重组载体pSilencer 3．1-SVV2和pSilencer 3．1-SVV3有效地阻断了PC3细胞中sur- vivin基因在mRNA和蛋白水平上的表达(P<0．01)。重组载体转染PC3细胞后,与对照组相比,细胞的凋亡增加了10％- 15％;细胞生长速度明显变慢,其细胞数在84 h时与对照组相比减少约30％;G1期细胞增加了10％,G2期和S期细胞减少了 5％以上。加入顺铂后,pSilencer 3．1-SVV2、pSilencer 3．1-SVV3重组质粒转染的PC3细胞的存活数下降了35％-45％,细胞凋亡则增加了10％-14％。结论:初步证明了survivin基因在前列腺癌细胞分化增殖、抗凋亡等方面所扮演的重要角色,为进一步阐明survivin基因与前列腺癌的关系以及以survivin基因为靶点的前列腺癌基因治疗研究奠定了基础。

To observe the effects of RNAi-mediated survivin gene on silencing prostate cancer cell lines PC3. Methods: Three target gene fragments were cloned into pSilencerTM3. 1-H1 neo vector separately. Three recombinant eukaryotic expression vectors, pSilencer 3. 1-SVV1, pSilencer 3.1-SVV2 and pSilencer 3. 1-SVV3 were successfully constructed, then the recombinant vectors were transfected into prostate cancer cells PC3. After PC3 cells were transfected with recombinant vectors, the interference effects were detected by RT-PCR and Western blot. The apoptosis index and cell cycle of PC3 cells were detected by Flow cytometry. The survival curve of PC3 cells treated with Platinol and the proliferation of PC3 cells were detected by MTT. Results: The results of RT-PCR and Western blot indicated that pSilencer 3. 1-SVV2 and pSilencer 3. 1-SVV3 vectors could knock down the transcription and expression of survivin gene. After PC3 cells were transfected with pSilencer 3. 1-SVV2 and pSilencer 3. 1-SVV3 vectors, the apoptosis index of PC3 cells increased by about 10% -15% , the proliferation of PC3 cells become slowly and the cells number decreased about 30% compared with control groups. The cell number during G1 phases increased 10% and during G2 and S phases the cells number decreased about 5 % . After PC3 cells treated with Platinol, the survival rate of PC3 cells transfected with pSilencer 3.1-SW2 and pSilencer 3. 1-SVV3 vectors decreased by about 35% -45% and apoptosis index increased by about 10% -14% contrast with control groups. Conclusion: The study provides basis for study the function of survivin gene, indicating the survivin may be a new target of gene therapy on prostate cancer.