逆转录病毒介导的p16基因对人卵巢癌细胞系生长抑制作用

摘要

目的研究逆转录病毒介导的p16基因对人卵巢癌细胞系CAOV3生长与致瘤性的抑制作用。rn方法用脂质体介导法将外源野生型p16基因转染不表达p16的人卵巢癌细胞系CAOV3，对转染后细胞DNA、RNA和蛋白进行分析并观察其生物学行为变化。rn结果外源性野生型p16基因已整合入细胞并获稳定表达，表达有外源p16基因的细胞生长速度、集落形成率及裸鼠致瘤性均有部分抑制。细胞周期分析可见G1期增加，S期下降。电镜下超微结构出现生长抑制特征。rn结论 p16基因在卵巢癌发生、发展过程中起重要作用，为用p16基因转染对卵巢癌患者进行基因治疗提供了实验依据。%Objective　To study the inhibitory effects of retrovirus-mediated p16 gene on the human ovarian cancer cell line CAOV3.rnMethods　The recombinant eukaryotic expression vector pDOR-p16 containing exogenous human wt-p16 cDNA and vector with neomycin resistance gene only were introduced into a CAOV3 cell line which does not express p16 endogenously by lipofectamine-mediated gene transfection. By using polymerase chain reaction amplification, mRNA in situ hybridization and immunocytochemistry, the clones obtained were tested for their efficiency of transfection and effects of vector expression. Their biologic behavior was observed further.rnResults　Exogenous wt-p16 was transferred into CAOV3 cells successfully and permanent expression was obtained. The growth rate of the transfected CAOV3 cells in regular medium and soft agar was inhibited, and the tumorigenicity in nude mice showed that two of four mice failed to form tumors, and the others developed tumors 7 to 14 days later than mice of the contrast group. The percentage of phase G1 cells increased and that of phase S cells decreased. Under electron microscope, the ultrastructural changes of the cells revealed necrosis and growth retardation.rnConclusions　The p16 gene plays an important role in the generation and development of ovarian carcinoma. This study might provide experimental evidence for gene therapy in human ovarian cancer.