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目的:构建可诱导表达人巨细胞病毒(HCMV)UL138蛋白的稳定遗传胃癌细胞系,并评价UL138对胃癌细胞的生物学效应。方法:将p CMV-tet3G质粒转染BGC-823胃癌细胞,通过G418及荧光素酶活性检测筛选BGCTet-on细胞株。再将含有UL138外源基因的p TRE3G-UL138质粒转染稳定BGCTet-on细胞株,通过G418及潮霉素双抗筛选,获得诱导性表达UL138基因的胃癌细胞(BGC-UL138_(Tet-on))。强力霉素(DOX)诱导后,采用Western blot验证UL138蛋白表达,采用流式细胞术及CCK8试剂盒检测UL138蛋白表达对胃癌细胞的生长周期及增殖的影响。构建荷瘤裸鼠模型,体内验证UL138蛋白表达对胃癌细胞增殖的影响。结果:成功构建了DOX诱导性表达的、携带UL138基因的BGC-UL138_(Tet-on)稳转细胞株。UL138蛋白表达可显著抑制胃癌细胞增殖;流式细胞术检测显示UL138蛋白表达阻滞BGC-823细胞周期在G_1期。荷瘤裸鼠模型体内实验发现,DOX腹腔注射诱导UL138蛋白表达后,BGC-UL138_(Tet-on)移植瘤体积缩小甚至消失。结论:成功构建可诱导表达UL138蛋白的胃癌细胞株,进一步证实HCMV基因UL138的表达可在体内和体外抑制胃癌细胞的增殖,细胞实验提示细胞周期阻断在G_1期。
OBJECTIVE: To construct a stable gastric cancer cell line that can induce the expression of human cytomegalovirus (HCMV) UL138 protein and evaluate the biological effect of UL138 on gastric cancer cells. Methods: The pCMV-tet3G plasmid was transfected into BGC-823 gastric cancer cells and the BGCTet-on cell lines were screened by G418 and luciferase activity assay. The plasmid p TRE3G-UL138 containing the foreign gene of UL138 was transfected into the stable BGCTet-on cell line, and the gastric cancer cells inducing the expression of UL138 gene were obtained by screening with G418 and hygromycin double antibody (BGC-UL138_ (Tet-on) ). After DOX induction, the expression of UL138 protein was verified by Western blot. The effect of UL138 protein expression on the growth cycle and proliferation of gastric cancer cells was detected by flow cytometry and CCK8 kit. To construct a tumor-bearing nude mouse model and verify the effect of UL138 protein expression on the proliferation of gastric cancer cells in vivo. RESULTS: BGC-UL138_ (Tet-on) stable cell line carrying UL138 gene was successfully constructed. UL138 protein expression significantly inhibited the proliferation of gastric cancer cells; flow cytometry showed that UL138 protein expression blocked BGC-823 cell cycle in G_1 phase. Tumor bearing nude mice model in vivo experiments found that, DOX induced by intraperitoneal injection of UL138 protein expression, BGC-UL138_ (Tet-on) xenografts shrink or even disappear. CONCLUSION: The successful construction of a gastric cancer cell line that can induce the expression of UL138 protein further confirms that the expression of HCMV gene UL138 can inhibit the proliferation of gastric cancer cells in vitro and in vivo. Cell experiments suggest that cell cycle arrest is in G_1 phase.