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目的:探讨肿瘤抗原基因OVA66对肿瘤细胞生物学特征的影响。方法:采用脂质体法,以重组真核表达载体pcDNA3.1-OVA66转染肝癌细胞系SMMC-7721,经G418稳定筛选、克隆及扩增,采用RT-PCR、Westernblot和免疫细胞化学(ICC)染色法,检测目的基因及其蛋白的表达。用FACS检测细胞周期的变化;电镜技术观察细胞的超微结构;体外运动、侵袭实验检测细胞运动及侵袭能力;并应用基因芯片技术检测相关基因的差异表达。结果:RT-PCR检测显示,转染pcDNA3.1-OVA66的细胞中OVA66基因呈高表达。Westernblot显示,在OVA66蛋白处出现明显的条带。电镜和FACS检测表明,该基因可增强肿瘤细胞的增殖能力。体外运动、侵袭实验证实,该基因可促进肿瘤细胞的迁移、侵袭。基因芯片显示,该基因的高表达可促进Ⅰ型纤溶酶原激活物抑制剂(PAI-1)等基因的表达,影响肿瘤的侵袭和转移。结论:OVA66基因及其蛋白可促进肿瘤细胞的增殖、侵袭、转移等一系列生物学功能。
Objective: To investigate the effect of tumor antigen gene OVA66 on the biological characteristics of tumor cells. Methods: The hepatoma cell line SMMC-7721 was transfected with the recombinant eukaryotic expression vector pcDNA3.1-OVA66 by lipofectamine. The recombinant plasmid was stably selected by G418, cloned and amplified by RT-PCR, Western blot and immunocytochemistry ) Staining method to detect the expression of the target gene and its protein. The changes of cell cycle were detected by FACS. The ultrastructure of cells was observed by electron microscopy. The motility and invasion ability of cells were detected by in vitro motility and invasion assay. The differential expression of related genes was detected by gene chip technology. Results: RT-PCR showed that the OVA66 gene was highly expressed in the pcDNA3.1-OVA66 transfected cells. Western blot showed that a clear band appeared at the OVA66 protein. Electron microscopy and FACS detection showed that the gene can enhance the proliferation of tumor cells. In vitro exercise, invasion experiments confirmed that the gene can promote tumor cell migration, invasion. Gene chips showed that high expression of this gene can promote the expression of type I plasminogen activator inhibitor (PAI-1) and other genes, affecting tumor invasion and metastasis. Conclusion: OVA66 gene and its protein can promote the proliferation, invasion and metastasis of tumor cells and a series of biological functions.