目的:通过CK8/18阳性胸腺上皮细胞与人脐血单个核细胞在Transwell板的共培养,探讨CK8/18阳性TEC对T细胞增殖分化的影响.方法:用胶原酶消化法分离纯化胸腺上皮细胞、免疫组化鉴定分离纯化的TEC,采用密度梯度离心法分离获得脐血单个核细胞,并采用免疫磁珠分选CD34+细胞,将TEC种植培养于Transwell双层培养板上层,使其均匀平铺于上层板底部,再将分选后的细胞加入上层小室,经过48小时的共培养,流式细胞术检测进入下室细胞的表型变化.结果:分离纯化的TEC经免疫组化鉴定CK8/18阳性.TEC与脐血单个核细胞共培养后,CD3+CD4-CD8-双阴性、CD3+CD4+CD8-单阳性细胞显著增加,CD3+CD4+CD8+双阳性细胞亚群细胞减少,CD8单阳性细胞增加不明显;CD45RA阳性细胞比率无显著变化,CD45RO阳性细胞比率显著增加.结论:CK8/18阳性TEC能选择促进脐血单个核细胞CD4+T细胞和CD45RO+细胞增殖.%Objective :To study the effects of CK8/18 positive thymic epithlial cell (TEC ) on T cells proliferation and differentiation , we established a model by co-cultured CK8/18 positive TEC and cord blood mononuclear cells (CBMC ) in transwell co-culture system .Methods :Thymic epithelial cells were obtained through collagenase digestion culture and identified with TEC keratin (CK ) and CK8/18 antigen . CD34+ cells were collected and purified from CBMC by means of imunomagnetic beads on MiniMACS device .The original culture of CK8/18 positive TEC was put into transwell upper chamber (pore size 5 μm) ,and then the CD34+ cells were added to transwell upper chamber until the PC membrane were completely covered by TEC ,when the cells into the lower chamber after 48 hours were detected with flow cytometry .Results :The TEC were anti-CK-positive and anti-CK8/18 staining positive .Compared with the control ,the CD3+ CD4- CD8 double negative cells ,CD3+ CD4 + CD8- single and CD45RO+ cells that entered the transwell lower chamber increased significantly ( P＜0 .01 ) in transwell co-culture system .Conclusion :CK8/18 positive thymic epithelial cells can promote CBMC CD4+ single-positive T cells and CD45RO+ cells proliferation .
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