稳定表达肝癌相关抗原HAb18G/CD147成纤维细胞株的建立及其意义

目的:探讨将HAb18G全长cDNA转染成纤维细胞NIH/3T3的可行性及其意义.方法:将含有肝癌相关抗原HAb18G全长cDNA的真核表达载体pcDNA3.0/HAb18G利用脂质体介导法转染小鼠成纤维细胞NIH/3T3,G418筛选建立稳定表达阳性克隆细胞株t3T3;利用流式细胞仪、间接免疫荧光染色方法鉴定目的蛋白转染情况;RT-PCR琼脂糖电泳、明胶酶谱分别从mRNA和蛋白水平检测转染前后HAb18G刺激3T3细胞分泌基质金属蛋白酶(MMP-2、MMP-9)的情况.结果:利用脂质体介导法将HAb18G全长cDNA转染入成纤维细胞NIH/3T3,G418筛选,流式细胞仪分析、间接免疫荧光染色证实成功建立了稳定表达HAb18G的阳性克隆株;RT-PCR琼脂糖电泳证实t3T3细胞MMP-2、MMP-9 mRNA转录水平较未转染前增多;明胶酶谱表明t3T3分泌MMPs能力较未转染前增强.结论:HAb18G全长cDNA可以稳定转染成纤维细胞,并能从核酸和蛋白水平诱导基质金属蛋白酶高表达,具有其独特的生物学意义.

Construction of mouse fibroblast NINH/3T3 strain stably expressing HAb18g/CD147 and its implication

AIM: To observe the possibility and implication of stable transfection of full length HAb18G cDNA into NIH/3T3 cell line. METHODS: The eukaryotic expression vector pcDNA3.0 containing human full length HAb18G cDNA and empty vector pcDNA3.0 were introduced respectively into NIH/3T3 fibroblast cells by liposome mediated gene transfection method. NIH/3T3 HAb18G was selected by G418 and the positive clone strain t3T3 was gained. The expression of HAb18G in t3T3 was identified by flow cytometry and indirect immunoflourescence staining and the effects on 3T3 excreting MMPs after transfecting were studied by gelatin zymography and RT PCR. RESULTS: Flow cytometry and indirect immunoflourescence staining showed that HAb18G was stably transfected into NIH/3T3 cells and agarose gel electrophoresis showed that the RT PCR products of MMP 2 and MMP 9 mRNA increased after transfection. The gelatin zymography confirmed that the ability of fibroblast to excrete MMPs was enhanced after transfection. CONCLUSION: Successfully constructed fibroblast strain with stable HAb18G expression can supply a cell model in studying the mechanism of MMPs excretion stimulating HAb18G.