【摘要】 目的研究血管内皮细胞系人脐静脉内皮细胞(ECV304)表达的HLAG1分子对同种异体T细胞增殖的作用。方法采用脂质体介导的DNA转染技术,将构建的真核表达质粒pcDNA3HLAG1转染ECV304,用免疫荧光法检测其表达的HLAG1分子。将表达HLAG1的ECV304与同种异体T细胞进行混合细胞培养后,用四甲基偶氮唑(MTT)比色法检测ECV304表达HLAG1对外周血T细胞增殖和抗原特异性细胞毒T淋巴细胞(CTL)杀伤活性的影响。结果ECV304转染pcDNA3HLAG1后可稳定表达HLAG1分子;转染空质粒pcDNA3和转染pcDNA3HLAG1的T细胞的刺激指数分别为(1.59±0.41)和(1.33±0.46),二组比较,差异有统计学意义(P<0.05);细胞毒实验显示,抗原特异性CTL对转染空质粒pcDNA3和转染pcDNA3HLAG1的ECV304的杀伤率分别为(64.81±3.07)%和(52.33±4.48)%,二组比较,差异有统计学意义(P<0.05)。结论HLAG1分子可以抑制同种异体T细胞的增殖和CTL的细胞毒作用。更多还原

【Abstract】 Objective To study the inhibitory effects of HLA-G1 expressed in ECV304 on the proliferation of allogeneic T cells.Methods The recombinant plasmid pcDNA3-HLA-G1 which contained a full-length cDNA of HLA-G1 was constructed, and the ECV304 cell was transfected with the pcDNA3-HLA-G1 by using the lipofectin transfection. The expressed HLA-G1 on the cell surface was checked by specific monoclonal antibody (G11E5) with indirect immunofluorescence assay and FCM. The HLA-G1 expressed in ECV304 was used as stimulator in co-culture with allogeneic T cells, to perform allogeneic T cell proliferation assay and to generate ECV304-specific alloreactive CTL. The proliferation of T cells and the CTL’s cytotoxicity against ECV304 were tested by the MTT method.Results The expression of HLA-G1 on the surface of the ECV304 was verified with the immunofluorescent staining of the pcDNA3-HLA-G1 transfected cells. The proliferation intensity of allogeneic T cells was significantly decreased after the HLA-G1 expressed in ECV304, as the stimulation index of co-culture of allogeneic T cells with plasmid pcDNA3 transfected ECV304 was 1.59±0.41, meanwhile it was 1.33±0.46 to pcDNA3-HLA-G1 transfected ECV304, with the difference being significant (P< 0.05). The cytotoxicity of allogeneic CTL was also inhibited by the HLA-G1 expressed on the targets, indicated by the specific lysis of specific CTL against pcDNA3-HLA-G1 transfected ECV304, which was (52.33±4.48) %, meanwhile the specific lysis against plasmid pcDNA3 transfected ECV304 was (64.81±3.07) %, with the difference being significant (P< 0.05). Furthermore, this inhibition was blocked by addition of HLA-G specific mAb (G11E5).Conclusions The expression of HLA-G1 in the ECV-304 cells can inhibit the proliferation and cytotoxicity of allogeneic T cells. Our results support the hypothesis that HLA-G1 bears the tolerance-inducing effects in allogeneic T cell reaction. It is hopeful that HLA-G1 provides an alternative tool to treat autoimmune diseases and to induce and maintain tolerance to allografts.更多还原