反义K-ras基因对胰腺癌细胞增殖和凋亡的影响

目的 探讨反义K-ras癌基因对胰腺癌细胞增殖和凋亡及Fas/FasL、Bcl-2、Bax表达的影响.方法 将重组逆转录病毒载体pLXSN转染包装细胞PT-67,获得重组逆转录病毒.在细胞和动物水平将该重组逆转录病毒分别感染PC-3和BxPC-3胰腺癌细胞,经G418筛选获得稳定细胞系,应用MTT、流式细胞仪和免疫组化法分别研究其增殖、凋亡及其相关基因表达情况.结果 成功制备含反义K-ras基因的重组逆转录病毒.感染含反义K-ras基因重组病毒后,胰腺癌PC-3细胞和移植瘤(有K-ras基因第12密码子点突变GGT→GTT)增殖受到抑制,G<,0/1>期细胞增加而S期细胞明显减少,细胞凋亡增加.免疫组化显示含反义K-ras癌基因逆转录病毒感染后PC-3细胞Bax/Bcl-2比值升高,Fas表达上调.结论 反义K-ras基因可使胰腺癌细胞及移植瘤增殖受到抑制,并可通过上调Bax/Bcl一2比值和(或)促进Fas表达诱导细胞凋亡.

Effect of antisense K-ras gene on proliferation and apoptosis of pancreatic carcinoma cells

Objective To evaluate the effect of antisense gene targeted to the oncogene K-ras on pancreatic carcinoma cell proliferation, apoptosis and expression of Fas/FasL, Bcl-2 and Bax genes.Methods The recombinant retroviral vector pLXSN was transfected into packaging cell line PT-67.Human pancreatic carcinoma cell lines BxPC-3 and PC-3 were infected with the recombinant retrovirus and followed by selection with G418. Cell proliferation,apoptosis and the expression of related genes were assessed by MTT,flow cytometry and immunohistochemistry (IHC). Results The recombinant retrovirus encoding antisense K-ras gene was constructed successfully. The proliferation of PC-3 cells and the transplanted tumor in nude mice (with the point mutation GGT to GTT at codon 12 of K-ras gene) were inhibited,the proportion of G0/1 phase cells increased and the S phase cells decreased signif-icantly while the apoptosis of cells increased. Meanwhile,the increase of Bax/Bcl-2 ratio and the up-regulation of Fas expression of retroviruses infected PC-3 were detected by IHC assay. Conclusion The antisense K-ras gene can inhibit the proliferation of human pancreatic carcinoma cells linetransplanted tumors in nude mice and induce apoptosis by up-regulating the ratio of Bcl-2/Bax and/or promoting the expression of Fas.