促进胚胎干细胞来源造血干/祖细胞移植后细胞免疫功能恢复

Generation of hematopoietic stem/progenitor cells with property of strengthened cell mediated immunity from an embryonic stem cell line

目的 :体外诱导胚胎干细胞分化为造血干 /祖细胞过程中 ,增加成熟T淋巴细胞的含量 ,以促进其重建致死量照射小鼠的造血功能后免疫功能的早期重建。方法 :胚胎干细胞在含甲基纤维素的培养基中自由分化形成胚胎体 ,分化第 6d添加造血生长因子 ,同时添加胸腺肽 ,流式细胞仪检测分化细胞中CD3 4 + 的造血干 /祖细胞和CD+ 3 的成熟T淋巴细胞含量 ,最后将分化细胞注射入致死量照射小鼠体内 ,观察 6 0d ,以移植物抗宿主病 (GVHD)发病率作为T淋巴细胞免疫功能的指标 ,用PCR检测Sry反映移植细胞在宿主体内的存活。结果 :分化第 13d ,未加胸腺肽 ,CD+ 3 的成熟T淋巴细胞含量仅 10 5 2 % ,重建造血后无GVHD发生 ;添加胸腺肽 ,CD+ 3 的成熟T淋巴细胞含量升高达 2 2 93% ,重建造血后GVHD发病率 10 0 %。结论 :胚胎干细胞体外分化为造血干 /祖细胞过程中 ,添加胸腺肽 ,能增加CD+ 3 的成熟T淋巴细胞含量 ,体内重建造血后细胞免疫功能恢复较快。

AIM: To induce lymphoid stem cells and/or T-cell precursors to diffe rentiate into functional mature T lymphocyte, and to increase the surface marker of T lymphocytes such as CD + 3, while embryonic stem(ES) cells differentiate d into hematopoietic stem/progenitor cells(HSPCs) in vitro . When they were i njec ted into lethally irradiated mice, these differentiated cells had the advantage in immune reconstitution. METHODS: Embryonic stem cells formed e mbryoid bodies(EBs) in the medium containing methycellulose, hematopoietic growt h factors(HGFs) was added to the culture system on the 6th day, thymopeptide was added at the same time. Flow cytometry were performed to detect the surface mar ker CD 34 and CD 3 of the differentiated cells. Finally the differentiated cells were injected into lethally irradiated mice, 60 days later, the incidence rate of graft versus host disease(GVHD) was taken as the mark of cell mediated immunity, PCR was performed to detect the sex determining region of the Y-chromo some(Sry) in bone marrow cells and spleen cells of the survival host female mice . RESULTS: The percentage of CD + 3 T lymphocytes was 10.52% a nd the incidence rate of GVHD was 0% on the 13th day, but they respectively rose up to 22.93% an d 100% if thymopeptide was added in the procedure of inducing ES cells to differ entiate into HSPC in vitro . CONCLUSION: The quantity of CD + 3 T lymphocytes increased in medium containing thymopeptide when ES cells differe ntiated into CD 34 + HSPC.