【摘要】 目的构建仅定位在脂筏内或外的tmTNF-α突变体,研究脂筏与tmTNF-α生物学功能的关系。方法通过重组PCR,构建C-47A-tmTNF-α-pIRES2-EGFP,cav-tmTNF-α-pIRES2-EGFP重组质粒。采用激光共聚焦观察定位、蔗糖密度梯度离心法提取脂筏、MTT比色法检测胞毒。结果获得了预期突变,且无任何其它点突变,移码及缺失突变的突变体。tmTNF-α部分定位在脂筏内,效应细胞的胞毒与tmTNF-α定位在脂筏内外无关,而破坏靶细胞脂筏结构导致胞毒效应下降,且与ICAM-1相关。结论效应细胞的胞毒与脂筏无关,而靶细胞与脂筏密切相关,该研究有助于进一步认识tmTNF-α介导的生物学功能与脂筏间的关系。更多还原

【Abstract】 To study the relationship between lipid rafts and tmTNF-α,we established overlapping PCR methods for constructing a mutant named cav-tmTNF-αwhich locate only in lipid rafts together with another mutant C-47A-tmTNF-αwhich only out of lipid rafts. And then the mutants were inserted into vector pIRES2-EGFP.It was confirmed that there were no other point mutations,frame-shift or deletion mutations but the expected ones.For analysis of the function,recombined plasmids were detected by confocal microscope,sucrose density gradient centrifugation,and MTT colorimetric.The results showed that part of tmTNF-αwere localized in lipid rafts,and were related to ICAM-1.One the other hand,whatever tmTNF-αwas inside or outside of the lipid rafts,the cytotoxicity to target cell was unaffected. But destruction of the target cell lipid rafts resulted in declined cytotoxicity of tmTNF-α.This study might provide potential information on the bio-function for tmTNF-α.更多还原