猪主动脉内皮细胞株的建立和鉴定  

Establishment and identification of porcine aorta endothelial cell lines

作　　者：王建锋[1] 李霄[1] 韩炜[1] 张卓超[1] 姜楠[1] 张勇[1] 李伟民[1] 曹阳[1] 王德益[1] 窦科峰[1] 

机构地区：[1]第四军医大学西京医院肝胆胰脾外科,西安710032

出　　处：《中华实验外科杂志》2013年第2期265-267,F0003,共4页Chinese Journal of Experimental Surgery

基　　金：基金项目：国家高技术研究发展计划（863计划）资助项目（2012AA021005）

摘　　要：目的建立猪主动脉内皮细胞株，为以猪为供体的异种移植研究提供必要的细胞水平研究平台。方法分离健康幼猪主动脉内皮细胞并用携带链霉素抗性和猿猴病毒40大T抗原（SV40LT）基因的慢病毒转染细胞。检测SV40LT基因和仪一1，3-半乳糖转移酶（a-1，3-GT）基因和仪-1，3-半乳糖抗原表达。最后测定2类细胞与人血清发生反应的能力。结果聚合酶链反应（PCR）结果显示，SV40LT基因在转染后细胞内高表达（P〈0．05）。原代细胞倍增时间约为21h，而细胞株倍增时间约18h。2类细胞均具有吞噬低密度脂蛋白（LDL）和表达血管性假性血友病因子（vWF）因子特性（P〉0．05）。a-1，3-GT基因和a-1，3-半乳糖抗原在2类细胞内表达均无差异[荧光强度分别为（498．2±3．5）和（500．6±3．2），P〉0．05]。与人血清共培养时，随血清浓度升高，2类内皮细胞的增殖能力下降、细胞凋亡率增加，并均可结合人IgM抗体、c3和C5b-9（P〉0．05）。结论猪动脉内皮细胞株具有与原代内皮细胞相同的形态结构和表型特征，并具有和人血清发生免疫反应的能力。Objective To establish pig aortic endothelial cell lines for providing necessary cell model for xenotransplantation research. Methods The cell lines were established following the transfection of primary endothelial cells isolated from the aortas of the immature pig with lentivirus carrying genes for ne- omycin resistance and Simian Virus 40 Large T （SV40LT） antigen. The SV40LT mRNA, a-1,3-galacto- syhransferase （a-1,3-Gal） mRNA and ot-l,3-galactose were detected. At last, the response between the cells and human serum was tested. Results The expression of the SVZIOLT mRNA in cell lines were very high （P 〈0. 05）. Doubling time of the primary cells and the cell lines was about 21 h and 18 h, respective- ly. The cells also displayed endothelial cell characteristics since they endocytosed acetylated low density lipo- proteins and steadily continuously expressed the yon Willebrand factor （ P 〉 0. 05 ）. The high expression of ct-1,3-Gal mRNA and a-1,3-galactose [the fluorescence intensity was （498.2±3.5） and （500. 6 ±3. 2）, respectively ] had no difference in the two types of cells （P 〉 0. 05 ）. When co-incubation with human serum, human natural antibodies bound to the two types of cells. Conclusion These results suggest that pig endothe- lial cell lines retained their original morphological structure and pbenotypic characterization, and also had a- bility to respond to human serum.

关 键 词：猪 内皮细胞 异种移植 

分 类 号：R[医药卫生]