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

doi:10.3971/j.issn.1000-8578.2015.11.006

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

摘要

摘要: 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 mRNA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对照组转染pCMV空载体组AIM-1 mRNA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 mRNA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。

Abstract: 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 mRNA 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 mRNA 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.

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