猪FasL基因在猪软骨细胞上的表达和鉴定  

Expression of FasL gene in porcine chondrocytes

作　　者：王树军[1] 王颖[1] 张惠珍[1] 金姝[1] 沈天伟[1] 葛瑜[1] 葛海良[1] 

机构地区：[1]上海第二医科大学上海市免疫学研究所,上海200025

出　　处：《细胞与分子免疫学杂志》2005年第2期133-136,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家重点基础研究发展规划(973)项目(No.G1999054300)

摘　　要：　目的: 实现猪Fas配体(FasL)基因在猪软骨细胞中表达和鉴定。方法: 用RT- PCR扩增猪FasL基因片段, 构建重组pGCEN-FasL逆转录病毒载体, 并转染PA317细胞。经G418筛选获得高滴度的病毒液, 感染猪软骨细胞, 应用FACS和Westernblot检测软骨细胞上FasL的表达。结果: 经酶切分析、测序证明, 成功地构建pGCEN- FasL逆转录病毒载体。转染的软骨细胞表面FasL的表达率为 57%。Westernblot显示, 在Mr为 37 000处有 1条特异性蛋白带, 具有诱导Fas+细胞凋亡的作用。结论: 成功地构建重组猪FasL基因的逆转录病毒载体, 并在转染的软骨细胞上高表达具有生物学活性的FasL, 为建立同种异体软骨细胞移植的免疫耐受提供了实验依据。AIM: To express the Fas ligand(FasL) gene in porcine chondrocytes. METHODS: The porcine FasL gene fragment was amplified by RT-PCR and then inserted into the pGCEN retroviral vector. The recombinant vector was transfected into packaging cells PA317 which were then screened with G418. Supernatant of screened PA317 cells containing high titer recombinant virus was used to infect porcine chondrocytes. Expression of FasL in chondrocytes was analyzed by FACS and Western blot. RESULTS: The recombinant pGCEN-FasL retroviral expression vector was successfully constructed as shown by restriction enzyme digestion analysis and DNA sequencing. FasL was expressed in 57% of infected chondrocytes. Western blot analysis also confirmed the expression of FasL in chondrocytes. The expressed FasL could induce apoptosis of Fas+ cells. CONCLUSION: The recombinant pGCEN-FasL retroviral vector has been successfully constructed and FasL with biological activity was highly expressed in porcine chondrocytes, which lays the foundation for allogenic chondrocytes transplantation.

关 键 词：软骨细胞 FASL基因 逆转录病毒载体 表达 猪 

分 类 号：R392.12[医药卫生—免疫学]