逆转录病毒载体介导反义K-ras基因治疗胰腺癌的实验研究

Therapeutic effects of recombinant retrovirus mediated antisense K-ras gene on pancreatic cancer

目的:分离及克隆胰腺癌细胞基因组中K-ras基因片段,构建含反义K-ras癌基因逆转录病毒载体并探讨其对胰腺癌细胞增生、凋亡的影响及可能的分子机制. 方法:设计2对PCR引物,分别在上下游引物中引进BamHI 和EcoRI位点,以胰腺癌细胞株BxPC-3、PC-3基因组DNA为模板扩增K-ras基因外显子1、4及侧翼序列,将目的基因克隆入逆转录病毒载体pLXSN中,构建重组质粒,经PT-67细胞包装后获得重组逆转录病毒.将该重组逆转录病毒转染上述细胞,经G418筛选获得稳定细胞,应用MTT、流式细胞仪和免疫组化法分别研究其增生、凋亡和对胰腺癌p21蛋白表达;建立胰腺癌裸鼠移植瘤模型,观察反义K-ras基因体内治疗效果. 结果:成功构建含反义K-ras基因的重组逆转录病毒载体.反义K-ras基因抑制胰腺癌细胞增生,诱导细胞凋亡、下调K-rasmRNA和P21蛋白表达,并抑制裸鼠胰腺癌移植瘤的生长. 结论:反义K-ras基因可使胰腺癌细胞增生受到抑制,并可诱导细胞凋亡和下调K-rasmRNA和P21蛋白表达.

AIM: To isolate and clone antisense K-rasgene fragments and to clarify its effect on proliferation and apoptosis of pancreatic cancer cells and expression of K-ras mRNA and p21 protein. METHODS: Polymerase chain reaction (PCR) was used to amplify K-rasgene exon 1 and 4 as well as the flanking sequences, taking pancreatic carcinoma cell line BxPC-3 and PC-3 genomic DNA as the template. The target gene was cloned into retroviral vector pLXSN to construct the recombinant plasmid. After packaged in PT-67 cells the retrovirus was obtained. Then BxPC-3 and PC-3 cells were transfected with the recombinant retrovirus. Proliferation, apoptosis of pancreatic carcinoma cells and the expression of p21 protein were detected by MT, flow cytometry and immunohistochemistry respectively. The therapeutic effect of retrovirus on hepatocellular carcinoma was observed in mice. RESULTS: The antisense K-ras gene was successfully cloned into the vector pLXSN. Proliferation of PC-3 cells was significantly inhibited at day 1, 2, 3,4, and 5 after transfected with pLXSN-AS-exon1/4B, compared with that of non transfected cells and pLXSN-transfected group (F= 4.716, P<0.05; F= 6.914, P<0.05; F= 15.115, P<0.05; F = 16.883, P<0.05; F= 15.134, P<0.05; repectively). BxPC-3 showed no significant difference among different groups. Expression of K-ras mRNA and p21 protein in PC-3 cells obviously decreased while those in BxPC-3 cells not so obviously. Apoptotic rates of PC-3 cells transfected with pLXSN-AS-exon1 and pLXSN-AS-exon4B were significantly higher than those with pLXSN and non-transfection (10.28%, 6.7% ca 2.86%, 3.24%; P <0.01). Apoptosis of BxPC-3 cells were not significant among different groups. The size of hepatocellular carcinoma significantly decreased after treated with pLXSN-AS-exonl and pLXSN-AS-exon4B, compared with that treated with pLXSN (0.32±0.09 g, 0.352±0.05 g vs 0.60±0.09 g; P <0.01). CONCLUSION: The retrovius-mediated antisense K-ras gene can inhibit proliferation and induce apoptosis of pancreatic carcinoma cells. The mechanism may relate to down-regulating expression of K-ras mRNA and p21 protein.