探讨高表达肿瘤抗原基因OVA66对WISH细胞生物学特征的影响

目的 分析转染OVA66基因WISH细胞的生物学特征变化,探讨该基因的功能.方法 采用脂质体方法将重组真核表达载体pcDNA3.1-OVA66转染正常人羊膜细胞系WISH,经G418稳定筛选、克隆和扩增.采用RT-PCR、Western blotting和免疫荧光等方法检测目的基因及其蛋白的表达,通过电镜分析细胞超微结构,体外侵袭实验检测细胞生长特性和侵袭能力, 软琼脂克隆形成实验检测细胞的克隆性生长能力,并应用基因芯片技术检测相关基因差异表达.结果 RT-PCR检测显示,转染pcDNA3.1-OVA66细胞OVA66基因呈高表达;Western bloting显示OVA66蛋白处出现明显的条带;电镜检测表明该基因可明显增强WISH细胞增殖能力;体外侵袭实验显示,该基因可增强WISH细胞的侵袭转移能力;软琼脂克隆形成实验表明OVA66基因可以促进单克隆性生长能力;基因芯片提示,该基因高表达影响某些基因的异常表达.结论 提示OVA66基因高表达可促进细胞的增殖,利于肿瘤的侵袭和转移.

Effects of the over-expression of tumor antigen gene OVA66 on biological characteristics of WISH cells

Objective To analyze the biological characteristics of OVA66 gene-transfected WISH cell and to explore the function of the gene. Methods The eukaryotic expression vector pcDNA3.1-mock and recombined vector pcDNA3.1-OVA66 were transfected into normal amnionic cell line WISH by LipofectAMINETM 2000. After stable selection and clone proliferation of cells by G418, the expression of OVA66 gene and protein was detected by RT-PCR, Western blotting, and immunofluorescence assay (IFA). The ultrastructure was observed by electron microscope. Experiments on cell migration and invasion were performed in vitro. The colony grow ability of the gene-transfected cell was detected by Soft agar colony formation assay. The OVA66 associated genes were analyzed by genechip after gene transfection. Results In WISH cell transfected with pcDNA3.1-OVA66, OVA66 gene were highly expressed at mRNA levels and OVA66 protein content was highly increased as compared with control cells. Electron microscope indicated that OVA66 gene promoted the growth and proliferation of WISH cell. In vitro experiments on cell migration and invasion showed that this gene enhanced migrative and invasive action of WISH cells. Soft agar colony formation assay demonstrated the OVA66 gene could promote the grow ability as single clone. The result of genechip showed that over-expression of the OVA66 gene could affect abnormal expression of some other genes. Conclusion Over-expression of OVA66 gene cells suggest that the OVA66 gene can enhance cell proliferation and promote invasion and metastasis of tumor cells.