人内皮抑素基因转染原代成人黑色素瘤细胞

Human endostatin gene transfected adult skin melanoma cells

目的:研究人内皮抑素(hum an endostatin,hEndo)基因转染的原代黑色素瘤细胞的体外和体内生物学特性。方法:先构建重组人内皮抑素的真核表达载体pcDNA3.1-hEndo,将人内皮抑素基因导入原代成人黑色素瘤细胞,检测转基因细胞hEndo蛋白的表达、分泌和抑制细胞增殖活性,并验证转基因细胞在体外和裸鼠体内的生长特性。结果:载体pcDNA3.1-hEndo经酶切后获得5.4 kb和624 bp条带,电转后G418筛选获得克隆;RT-PCR检测到转染细胞表达hEndomRNA,W estern b lot检测到转染细胞分泌hEndo蛋白,MTT表明转基因细胞上清可抑制细胞增殖;裸鼠体内生长实验显示转基因黑色素瘤生长速度明显低于对照组(P<0.05),肿瘤组织RT-PCR示hEndo基因稳定表达。结论:转hEndo基因原代黑色素瘤细胞可有效、稳定地表达有生物学活性的hEndo,hEndo能抑制瘤细胞在动物体内的生长速度。

Objective To study the biological characteristics of human endostatin （hEndo） gene transfected adult skin melanoma cells in vitro and in vivo. Methods The plasmid pcDNA3.1 （-）-hEndo was transfected into adult skin melanoma cells by electroporation, and then the stable clones were selected with G418. The transcription and expression of hEndo gene in the transfected melanoma cells were verified by RT-PCR and agarose gel electrophoresis analysis and Western blot. The biological activities of hEndo protein were investigated by MTT in vitro. Stable clones expressing endostatin were subcutaneously injected into the right flank of BALB/c-nu/nu mice of 4-6 weeks old. Then the growth of transduced tumors in vivo was investigated. Results The bands of 624 bp and 5.4 kb were identified from digested plasmid pcDNA3.1 （-）-hEndo. The stable clones were selected with G418 after the eletroporation, the expression of hEndo mRNA was verified by RT-PCR, and Western blot displayed the expression product of hEndo was about 20 kD in the transfected melanoma cells. MTT showed that the conditioned medium of melanoma cells transduced with recombination human endostatin expression vector could inhibit the proliferation of human umbilical vein endothelial cells in vitro. The growth of transduced cells in vivo showed that transfected melanoma cells grew in vivo at a slower rate than the control cells （P〈0.05）. RT-PCR showed that endostatin expressed in the transduced tumors. Conclusion Adult skin melanoma cells in vitro transfected with exogenetic hEndo gene can express and secrete active hEndo,and inhibit the growth of transduced tumors in vivo.