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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。
Objective: To express and identify pig Fas ligand (FasL) gene in porcine chondrocytes. Methods: The fragment of FasL gene was amplified by RT-PCR, and the recombinant pGCEN-FasL retrovirus vector was constructed and transfected into PA317 cells. High-titer virus liquid was screened by G418 to infect porcine chondrocytes. The expression of FasL on chondrocytes was detected by FACS and Western blot. Results: After restriction analysis and sequencing, the pGCEN-FasL retroviral vector was successfully constructed. The expression of FasL on the surface of transfected chondrocytes was 57%. Western blot showed that there is a specific protein band at Mr 37 000, which has the effect of inducing apoptosis of Fas + cells. CONCLUSION: The recombinant FasL gene retroviral vector is successfully constructed and highly expressed FasL on the transfected chondrocytes, which provides an experimental basis for establishing immunological tolerance of allogeneic chondrocyte transplantation.