针对K-ras基因点突变的反义寡核苷酸对人胰腺癌Patu 8988细胞的抑制作用

Inhibitory effects of antisense oligodeoxynuleotide specific to K-ras point mutation on human pan- creatic cancer cell Patu 8988

目的检测人胰腺癌 Patu 8988细胞 K-ras 基因点突变形式,并观察针对该点突变的硫代反义寡核苷酸在体内外对 Patu 8988细胞增殖和凋亡的影响。方法顺序特异引物聚合酶链反应(PCR-SSP)法和基因测序检测 Patu 8988细胞 K-ras 点突变形式,根据点突变形式设计并合成硫代反义寡核苷酸(K-ras mutation ASODN)作用 Patu 8988,通过噻唑蓝(MTT)比色法检测细胞生长情况;流式细胞术(FCM)检测 K-ras 蛋白表达和细胞凋亡;逆转录-聚合酶链反应(RT-PCR)检测K-ras mRNA 表达水平;以 Patu 8988细胞建立裸鼠胰腺癌模型观察 K-ras mutation ASODN 在体内的抗肿瘤效果。结果 PCR-SSP 法和基因测序检测表明 Patu 8988细胞存在 K-ras 基因第12密码子点突变,其突变方式为 GGT→GTT。体外实验表明 K-ras mutation ASODN 组较正义寡核苷酸(SODN)组、随机寡核苷酸(RODN)组和空白对照组可明显抑制 Patu 8988细胞生长(P<0.01),并呈时间-剂量效应关系;转染48 h 后,K-ras mutation ASODN 组的 K-ras 蛋白和 mRNA 表达程度较SODN 组、RODN 组和对照组均明显降低(P<0.01),而细胞凋亡明显增多(P<0.05)。体内实验表明 K-ras mutation ASODN 较 SODN 组、RODN 组和对照组能有效抑制 BALB/C 裸鼠人胰腺癌的生长(P<0.01)。结论针对 K-ras 基因第12密码子点突变的反义寡核苷酸可明显抑制人胰腺癌 Patu8988细胞生长,促进细胞凋亡,其机制可能是通过下调 K-ras 蛋白和 K-ras mRNA 表达而起作用。

Objective To detect K-ras point mutation and its style at codon 12,and to investigate the effects of antisense oligodeoxynucleotide specific to K-ras gene mutation on proliferation and apoptosis of human pancreatic cancer Patu 8988 cells in vitro and in vivo. Methods K-ras gene point mutation and its style at codon 12 of Patu 8988 cell were detected by using PCR-SSP and sequence analysis. According to the mutation style,antisense oligodeoxynucleotide （K-ras mutation ASODN） specific to K-ras point mutation at codon 12 was designed and synthesized. After treatment of Patu 8988 cells with K-ras-mutation ASODN,the proliferation of Patu 8988 cells, the expression of K-ras protein,the apoptosis and the expres- sion of K-ras mRNA were detected by MTr assay, flow cytometry （FCM） and RT-PCR, respectively. The antitumor activity of K-ms mutation ASODN was evaluated in BALB/c nude mice bearing pancreatic canc- er inoculated with Patu 8988 cells. Results The results of PCR-SSP and sequence analysis showed that the Patu 8988 cells had point mutation at codon 12 and the mutation style was GGT→GTr. In vitro, K-ras mutation ASODN could apparently inhibit the growth of Patu 8988 cells （ in a certain degree of dose-dependent and time-dependent manner） ,induce apoptosis and decrease the expression of K-ms protein and K-ras mRNA in contrast with SODN, RODN and control groups. In tumor bearing mice, K-ras mutation ASODN showed a significantly inhibitory effect on the growth of transplanted pancreatic cancer, resulting in a statistically significant difference compared with SODN,RODN and control groups （P 〈 0.01 ）. Conclusion K-ras mutation ASODN could significantly inhibit the growth of Patu 8988 cells and induce their apoptosis via reinforcing specific down-zegulation of K-ras protein and K-ras mRNA expression.