目的探讨RNA干扰(RNAi)N—Ras基因增强人肝癌MHCC-97细胞的放射敏感性。方法通过构建干扰N-Ras基因siRNA,采用免疫组织化学法、实时荧光定量RT—PCR、Westernblot、MTT法观察RNAi前后肝癌MHCC97-H细胞RNA水平、蛋白水平、生长情况等变化,照射后用细胞克隆计数实验检测放射敏感性变化。结果MHCC97-H细胞株RNAi后mRNA表达抑制率为96.9%±0.159%,RNAi前后差异有统计学意义(t=40.377,P〈0.05),蛋白表达抑制率为89.8%±0.012%,RNAi前后差异有统计学意义(t=31.595,P〈0.05),免疫组织化学显示90%表达被抑制,细胞生长抑制率为21.9%,RNAi前后差异均有统计学意义(F=4.63,P〈0.05)。MHCC97-H细胞RNAi后增敏比(SER)=1.15。结论RNAi肝癌MHCC97一H细胞N—Ras基因可以达到放射增敏的目的。
Objective To investigate the radiosensitivity of silencing N-Ras by RNA interference in hepatoma carcinoma cell MHCC-97. Methods N-Ras RNA interference (RNAi) vector was constructed by using pcDNA 6.2-GW/EmGFP-mir plamid. The RNAi effect was detected by RT-PCR, Western bolt,immunohistochemisty and MTT method. Survival curve for each cell line were obtained by measuring the clone forming abilities of irradiated cell populations. Results After silencing the N-Ras by RNAi,The expression level of N-Ras mRNA,N-Ras protein, immunohistochemisty were decreased 96.9% ±0.159%(t=40.377,P〈0.05),89.8% ±0.012%(t=31.595,P〈0.05), 90%, respectively, and The survival of hepatoma carcinoma cell MHCC-97 line were inhibited 21.9% ( F = 4.63, P 〈 0. 05 ). Which have significant difference in statistics. The SER of hepatoma carcinoma cell MHCC-97 line after interference was 1.15. Conclusions RNAi targeting silence N-Ras may increase the radiosensitivity of hepatoma carcinoma cell MHCC-97 line.