分泌mRANTES趋化因子的小鼠肝癌细胞的建立及其体内致瘤性的初步研究

Establishment of A Mouse Hepatocellular Carcinoma Cell Line That Produces mRANTES Chemokines and the Tumorigenecity of mRANTES Transfected Hepal-6

目的:建立分泌mRANTES的小鼠肝癌细胞,并观察其体内致瘤性。方法:mRANTEScDNA被克隆入pBabepuro逆转录病毒载体,构建的重组逆转录病毒载体pBabepuromRANTES转染包装细胞,嘌呤酶素抗性细胞培养上清感染小鼠肝癌细胞系Hepa1-6,免疫组化检测Hepa1-6、Hepa1-6mRANTES的mRANTES蛋白的表达。绘制Hepa1-6mRANTES与Hepa1-6的生长曲线观察细胞的生长。琼脂糖凝胶打孔法体外观察Hepa1-6mRANTES分泌蛋白对小鼠脾细胞的趋化作用。观察Hepa1-6mRANTES体内致瘤性。结果:构建了重组逆转录病毒载体pBabepuromRANTES,Hepa1-6不表达mRANTES,Hepa1-6mRANTES表达mRANTES蛋白。Hepa1-6mRANTES与Hepa1-6的生长曲线基本一样。Hepa1-6mRANTES分泌了对小鼠脾细胞有趋化作用的分子。Hepa1-6mRANTES体内致瘤性降低。结论:Hepa1-6mRANTES能产生mRANTES,mRANTES不改变细胞体外生长状况,Hepa1-6mRANTES产生的mRANTES有趋化活性,mRANTES能使Hepa1-6mRANTES致瘤性降低。

Objective:To establish a mouse hepatocellular carcinoma that can produce mRANTES and to evaluate the possibility of cancer gene therapy by mMIP -1.Methods:mRANTES cDNA was cloned into retrovirus vector pBabe puro and pBabe puro mRANTES was constructed,then pBabe puro -mRANTES was used to transfect packaging cells.The anti -puromycin cells were proliferated and the supernatant was used to infect hepal-6.The anti-puromycin clone(hepal-6mRANTES)and hepal-6were analysed for the expression of mMIP-1-protein by immunohistochemistry.The growth curves of hepal-6and hepal-6mRANTES were drawn.The chemotaxis of mRANTES produced by hepal-6mRANTES to mouse spleen cells was observed on agarose gel.C57B/L mouse was inoculated with the tumor cells and the tumorigenecity was studied.Results:Recombinant retrovirus vector pBabe puro mRANTES with mRANTES cDNA was constructed.Hepal-6could not produce mRANTES protein,while hepal-6mRANTES could produce mRANTES protein.The growth curves of hepal -6and hepal -6 mRANTES showed no difference.The chemotaxis of mRANTES produced by hepal-6mRANTES to mouse spleen cells was observed.The tumorigenecity was reduced.Conclusion:In this study,a mouse hepatocellular carcinoma hepal-6mRANTES is established,and it show that mRANTES can affect the tumorigenecity of hepal-6.