PCDGF反义RNA真核表达载体的构建、转染及其对卵巢癌细胞增殖能力的抑制效应

目的:检测特异性封闭高度恶性人卵巢癌细胞株Sw626、A2780中畸胎瘤源性生长因子(PC -cell -devivedGrowthFactor,PCDGF)的表达,并观察其对增殖能力的抑制效应。方法:用RT -PCR技术扩增PCDGF基因cDNA保守序列,经pGEM -TEasy载体克隆后双酶切,反向插入哺乳动物真核表达载体pcDNA3 1 ( +)构建反义PCDGF核酸载体。并通过脂质体转染法转染人卵巢癌细胞株Sw626、A2780,用RT- PCR和Westernblot技术检测转染前后PCDGFmRNA和蛋白的表达,MTT实验检测其增殖能力的变化。结果:PCDGF反义核酸载体转染株的mRNA和蛋白水平明显低于对照组,Sw626的抑制率分别为50%、72%;A2780分别为53%、70%。同时增殖能力亦明显降低,其抑制率Sw626及A2780分别为71%、73%。结论:PCDGF反义核酸在高度恶性人卵巢癌细胞株Sw626及A2780中起了特异性封闭作用。为进一步研究PCDGF的分子生物学特性、致瘤机理及今后利用反义PCDGF核酸载体治疗卵巢癌提供了实验和理论模型。

Construction and transfection of the eukaryotic expression vector of PCDGF antisense RNA and its role in the growth inhibition of ovarian cancer cells

Objective:To observe the antiproliferation effect in Sw626 and A2780 cell lines by inhibiting PCDGF expression specifically.Methods:The conservative region of PCDGF gene amplified by RT-PCR,and then was cloned into pGEM-T Easy vector.After the recombinant plasmid was certified by DNA sequencing,the conservative region of PCDGF gene was inserted into pcDNA3.1(+)vector in an antisense oritation.Then the eukaryotic expression vector for PCDGF antisense RNA was transfected into most malignant ovarian cancer cell lines by lipofectamine.The expression levels of PCDGF before and after transfection were detected by RT-PCR and Western-blot assay.And MTT assay was used to evaluate the changes of proliferation capability.Results: The expression levels of PCDGF mRNA and protein decreased sharply in anti-sense PCDGF transfected cells.The inhibitory rates were 50% in mRNA level and 72% in protein level in A2780,and 53% and 70% in Sw626,respectively.At the same time,the proliferation in hibition rates were 71% and 73% in A2780 and Sw626,respectively.Conclusion:The eukaryotic expression vector for PCDGF antisense RNA may play an important role in the specific antiproliferation of ovarian cancer cell lines.The present study provides a good experimental model for the further study of molecular characteristic of PCDGF.