分化抗原5C5在人免疫细胞的表达

Expression of differentiation antigen 5C5 on human immune competent cells

目的 明确 5C5蛋白为分化抗原 ,检测其在人免疫细胞的表达 ,以及它的信号传导作用。方法 用 5C5蛋白免疫小鼠 ,制备抗 5C5蛋白的单抗。用反义技术制备 5C5蛋白表达抑制的 3D5细胞。用免疫荧光染色和Western印迹确定 5C5单抗的特异性。用胞膜蛋白免疫沉淀和流式细胞术确定其分化抗原的性质。用双色免疫荧光染色流式细胞术检测 5C5分化抗原在多种人免疫细胞的表达。用Fura 3 AM试剂作探针 ,用激光共聚焦显微镜观察胞内Ca2 + 浓度的变化。结果 制备了抗 5C5蛋白的特异性单抗。用 5C5单抗作探针 ,从 3D5细胞膜免疫沉淀到 1条相对分子质量 (Mr)为 19× 10 3的蛋白带 ,并在活 3D5细胞见阳性免疫荧光染色。 5C5分化抗原在人周围血CD19+ 、CD14 + 、CD4 + 、CD8+ 和CD5 6 + 细胞的表达率分别为 (33.4 7± 7.9) %、(86 .13± 6 .7) %、(15 .79± 7.1) %、(18.11±12 .77) %和 (11.76± 7.4 7) % ,在人免疫细胞株的表达率分别为 5 8.0 % (Nalm6 )、72 .7% (3D5 )、5 3.6 %(BJAB)、6 1.0 % (Daudi)、6 .9% (SKW6 )、6 .3% (Jurkat)、5 .8% (Peer)、2 1.3% (K5 6 2 )和 39.1% (U937)。3D5细胞在 5C5单抗刺激下 ,胞内Ca2 + 浓度逐渐增高 ,可达 2倍左右 ,对照小鼠Ig则无此作用。结论5C5蛋白为一个分化抗原 ,在人周?

Objective To confirm that 5C5 protein is a differentiation antigen. To detect expression of differentiation antigen 5C5 on human immune competent cells. To examine signaling activity of differentiation antigen 5C5. Methods McAb 5C5 was developed by immunization of mouse with 5C5 protein. 3D5 cells with inhibition of 5C5 protein expression was developed by antisense technique. The specificity of McAb 5C5 was measured with Western blot and flowcytometry. The nature of differentiation antigen was detected by immunoprecipitation on intact cell and flowcytometric assay on live cell with McAb 5C5 as a probe. Expression of 5C5 differentiation antigen on human immune competent cells was assayed by means of double colour staining and flowcytometry. The intracellular Ca 2+ concentration was detected using Fura 3 as a probe. Results McAbs specifically to 5C5 protein were prepared. With McAb 5C5 as a probe a protein band with molecular weight 19kD was immunoprecipitated from intact 3D5 cell and positive staining was observed on live 3D5 cell. The percentage of 5C5 differentiation antigen on CD14 +, CD19 +, CD4 +, CD8 + and CD56 + cell from human peripheral mononuclear cells was 86.13%±6.7%, 33.47%±7.9%, 15.79%±7.1%, 18.11%±12.77% and 11.76%±7.47% respectively. The percentage of 5C5 differentiation antigen on human immune cell lines was 58.0% (Nalm6), 72.7% (3D5), 53.6% (BJAB), 61.0% (Daudi), 6.9% (SKW6), 6.3% (Jurkat), 5.8% (Peer), 21.3% (K562) and 39.1% (U937). The Ca 2+ concentration in 3D5 cell increased gradually under stimulation of McAb 5C5. It reached a 2 fold increase at maximum. Control mouse IgG had no effect on intracellular Ca 2+ concentration. Conclusion 5C5 protein is a differentiation antigen. 5C5 differentiation antigen is heavily expressed on CD14 + cells, at medium on CD19 + cells, and weakly on CD4 +, CD8 + and CD56 + cells. 5C5 differentiation antigen has signaling function. [