慢病毒载体介导Nanog小分子干扰核糖核酸抑制人胃癌裸鼠移植瘤的生长

Lentiviral vector-mediated Nanog short-hairpin RNA inhibits the growth of human gastric carcinoma xenograft in nude mice

目的构建对Nanog基因有干扰作用的慢病毒载体,探讨其在人胃癌细胞SGC-7901裸鼠移植瘤模型中的表达,检测其对裸鼠移植瘤生长的影响。方法将前期细胞实验验证有效的siRNA序列构建于慢病毒载体中,制备携带shRNA-Nanog的慢病毒,同时构建胃癌裸鼠移植瘤模型。以lentivirus-shRNA-Nanog感染的裸鼠作为实验组(目的组),以无关序列慢病毒感染的裸鼠(空载体组)及未感染的裸鼠(未感染组)作为对照组,测量肿瘤瘤体体积及质量的变化;活体荧光成像观察总荧光强度及转移情况,Western blot法检测移植瘤组织中Nanog蛋白的表达情况;TUNEL法观察其对皮下移植瘤细胞凋亡的影响。结果经基因测序鉴定,Nanog基因shRNA重组慢病毒表达载体构建成功。活体荧光成像显示感染27 d后,目的组小鼠肿瘤总荧光强度明显低于空载体组和未感染组,裸鼠移植瘤瘤体积及瘤质量小于未感染组及空载体组;Western blot法显示目的组Nanog蛋白表达较对照组明显降低;TUNEL结果显示目的组凋亡指数较对照组明显增大,而空载体组与未感染组比较,差异无统计学意义。结论成功构建了Nanog基因shRNA表达载体并包装成慢病毒颗粒,在体内感染能明显抑制肿瘤生长。

Objective To construct lentiviral vector that interferes with Nanog, investigate its expression in human gastric cancer cell SGC-7901-transplanted nude mice, and explore the effect of shRNA-Nanog transfection on the growth of the xenograft in mice. Methods Lentivirus carrying shRNA-Nanog was prepared by incorporating previously validated siRNA into Nanog gene specific lentiviral vectors. The models of human gastric cancer transplantation were constructed in nude mice. The mouse models were randomly divided into lentivirus-shRNA-Nanog transfection group （experimental group）, GFP infection group （empty vector group） and uninfected group （control group）. The tumor volume and mass changes were measured. Small animal in vivo imaging was employed to examine the fluorescent intensity and tumor metastasis. The Nanog protein expression was determined by Western blot analysis. The apoptosis of transinfected tumor cells was analyzed by TUNEL method. Results The gene sequencing demonstrated that the recombinant lentivirus carrying shRNA-Nanog was successfully established. In vivo imaging showed that 2？ days after transfection, the total fluorescent intensity in the experimental group was significantly lower than that in the empty vector group or the uninfected group. The xenograft volume and mass in the experimental group decreased significantly as compared with those in the empty vector group or the uninfected group. Western blotting indicated that the expression of Nanog protein in the experimental group was significantly lower than that in the empty vector or uninfected group. TUNEL results revealed that the apoptosis rate in the experimental group was significantly higher than that in the other two groups. No statistically significant difference was found between the empty vector group and the uninfected group. Conclusion We successfully established Nanog gene-shRNA expression vector and capsulated it as lentivirus particles, which could inhibit the growth of xenograft in nude mice.