重组质粒pCMV AIM-1 antisense对卵巢癌裸鼠移植瘤的抑瘤效应研究

Effect of pCMV AIM-1 antisense Constructs on Human Ovarian Cancer Xenografts Growth in Nude Mice

目的探讨pCMV AIM-1 antisence重组质粒对卵巢癌裸鼠移植瘤的体内抑瘤效应。方法(1)人卵巢癌细胞A2780转染pCMV空载体和pCMV AIM-1 antisense重组质粒,G418筛选出抗性克隆,扩增后产生的稳定转染株用于实验;(2)通过RT-PCR、Western blot检测未转染A2780细胞(对照组)、A2780转染pCMV空载体和pCMV AIM-1 antisense重组质粒三组AIM-1 m RNA和蛋白的表达;(3)将稳定转染的pCMV空载体和pCMV AIM-1 antisense重组质粒的A2780细胞注入裸鼠皮下,建立裸鼠移植瘤模型,未转染A2780细胞注入裸鼠皮下作为对照,观察三组裸鼠移植瘤生长情况,计算肿瘤重量和肿瘤体积,并进行统计对比分析;(4)SP免疫组织化学法检测移植瘤组织切片AIM-1的表达。结果 (1)A2780对照组和转染pCMV空载体组AIM-1 m RNA和蛋白表达无明显改变;而A2780转染pCMV AIM-1 antisense重组质粒组细胞AIM-1 m RNA和蛋白表达明显下降;(2)A2780对照组、A2780转染pCMV空载体组和A2780转染pCMV AIM-1 antisense重组质粒组的裸鼠肿瘤重量分别为(1.93±0.21)g、(1.90±0.39)g和(0.77±0.34)g;移植瘤体积分别为(1 011.7±235.6)mm3、(981.2±162.3)mm3和(468.7±119.6)mm3,重组质粒组与空载体组和对照组相比,差异有统计学意义(P<0.01);pCMV AIM-1 antisense重组质粒的抑瘤率达53.8%。(3)SP免疫组织化学法检测瘤体组织切片AIM-1的表达情况,见A2780对照组和转染pCMV空载体组AIM-1表达没有明显改变,而A2780转染pCMV AIM-1antisense重组质粒组AIM-1的表达明显减少。重组质粒组AIM-1蛋白表达阳性率和阳性标记指数均明显低于对照组和空载体组(P<0.01)。结论 pCMV AIM-1 antisense重组质粒对A2780移植瘤生长有明显抑制作用。

Objective To explore the effect of pCMV AIM-1 antisense constructs on human ovarian cancer xenografts growth in nude mice. Methods pCMV empty plasmids and pCMV AIM-1 antisense constructs were transfected into human ovarian cancer cells A2780, then the cells were trypsinized and expanded, and stable transfectants were generated after 400μg/ml geneticin(G418) screening. The clones carrying antisense plasmid were then selected for further experiments. RT-PCR and Western blot were used to examine the expression of AIM-1 m RNA and protein in three groups(each n=8); A2780 control group, A2780-pCMV empty plasmids and A2780-pCMV AIM-1 antisense group. Mouse tumor model was established by transferring human ovarian cancer cells A2780 or A2780-pCMV empty plasmids or A2780-pCMV AIM-1 antisense subcutaneously into the oxter of nude mice. The tumor growth, volumes and weights of subcutaneous tumor xenografts were detected. The expression of AIM-1 was detected by Streptavidinbiotin peroxidase(SP) immunohistochemical technique. Results(1)The m RNA and protein expression levels of AIM-1 were obviously silenced in A2780-pCMV AIM-1 antisense group, while no change in other two groups;(2)The tumor weight in A2780 control, A2780-pCMV empty plasmids and A2780-pCMV AIM-1 antisense groups were(1.93±0.21),(1.90±0.39) and(0.77±0.34)g, respectively; The tumor volume were(1 011.7±235.6),(981.2±162.3) and(468.7±119.6)mm3, respectively. There were significant differences in tumor weight and volume between A2780-pCMV AIM-1 antisense group and other two groups(P<0.01). The anti-tumor rate of pCMV AIM-1 antisense group could reach to 53.8%. The AIM-1 expression in A2780-pCMV AIM-1 antisense group was radically reduced, compared with A2780-pCMV empty plasmids and A2780 control groups. The positive rate and positive labeling index(PLI) of AIM-1 protein expression in A2780-pCMV AIM-1 antisense group were significantly lower than those in other two groups(P<0.01). Conclusion The pCMV AIM-1 antisense constructs suppresses the tumor xenografts growth in nude mice.