OX40L在小鼠B16黑素瘤细胞中表达及对活化淋巴细胞凋亡影响

目的:构建OX40L的真核表达载体,分析其在B16细胞中的表达,研究OX40L对活化T淋巴细胞凋亡的影响.方法:以小鼠C57BL/6脾脏的cDNA为模板,PER扩增小鼠OX40L基因,构建真核表达载体pVAX1-OX40L,转染B16细胞后,荧光染色检测OX40L的表达;利用AnnexinV-PE凋亡试剂盒,检测B16黑素瘤细胞-淋巴细胞体外混合培养中对活化淋巴细胞凋亡的影响.结果:成功构建OX40L的真核表达载体,并转染B16细胞中,流式细胞术和激光共聚焦显微术均可检测到OX40L表达于B16细胞表面.淋巴细胞体外混合培养显示,表达OX40L的B16细胞组活化淋巴细胞的凋亡率为6.57%,而空质粒转染组为17.24%.结论:OX40L能表达于B16细胞表面,并能显著抑制活化淋巴细胞凋亡.

Expression of OX40L gene in B16 melanoma cells and its effect on the apoptosis of activated lymphocytes

Aim:To construct the eukaryotic expression vector for OX40L and analyze its expression in the transfected B16 cells, and to study its effect on the apoptosis of activated lymphocytes. Methods: U-sing C57BL/6 mice spleen cDNA as a template, the OX40L gene was amplified by PCR, and then the eukaryotic expression vector pVAX1-OX40L was constructed. The expression of mouse OX40L gene in B16 cells transfected with this vector was detected by fluorescence staining. Meanwhile, the apoptosis of the activated lymphocytes in tumor cell-mixed lymphocyte culture (MLC) was analyzed by AnnexinV-PE staining. Results: The eukaryotic expression vector for OX40L was successfully constructed and transfected into B16 cells. Flow cytometry and confocal microscopy showed that OX40L was expressed on the cell sur-face of B16 cells. MLC results revealed that the apoptosis ratio of OX40L gene-modified group was 6.57% while that of empty plasmid transfected group was 17.24%. Conclusion: OX40L could be expressed on B16 cell surface, and the apoptosis of activated lymphocytes could be significantly inhibited by OX40L.