从胚胎干细胞诱导发育为浆细胞的体外实验研究

Induction of plasma cells from embryonic stem cells in vitro culture

目的：建立胚胎干细胞体外定向诱导分化为浆细胞的体系，为研究造血发生的基因调控及Ｂ淋巴细胞发育过程奠定基础。方法：分阶段培养，第一、二阶段使用半固体培养，分组加入细胞因子、２－巯基乙醇和胚胎细胞条件培养液，用流式细胞仪、免疫组化及Ｗｒｉｇｈｔ’ｓ染色鉴定细胞，第三阶段移入骨髓基质细胞滋养层。结果：第一阶段：加入ＳＣＦ、ＴＰＯ、ＩＬ－６、ＩＬ－３、Ｅｐｏ、和Ｇ－ＣＳＦ，４ｄ后细胞大片坏死，仅加入２－巯基乙醇可形成胚胎体。第二阶段：加入ＳＣＦ和ＴＰＯ，细胞增殖缓慢，再加入条件培养液，发育３～４ｄ可形成ＣＤ３４＋的细胞集落。第三阶段：移至骨髓基质细胞饲养层后，发育为浆细胞。结论：胚胎干细胞在体外可定向分化为浆细胞。胚胎干细胞在早期发育不需要细胞因子，胚胎细胞的条件培养液可促使其向造血细胞发育。

AIM:To find a suitable microenviroment for improved induction of plasma cells from murine embryonic stem(ES)cells,we explored the roles of cytokines and primary bone marrow stromal cells in the development of ES-D3 cells.The process was divided into three stages.First stage(0-4 day):adding SCF(stem cell-factor) ,TPO(thrombopoietin),IL-6,IL-3,erythropoietin,G-CSF into methycellulose culture,ES-D3 cells died four days later,ES-D3 cells developed into embryoid bodies and they grew well in the presence of 2-Mercaptoethanol (2-ME) only.Second stage(4-7 dyas):the embryoid bodies were trypsined,then transferred into secondary methylcellulose culture,cells grew slowly when adding SCF and TPO, but they(86%)developed quickly into the colonies of CD34 + cells in the presence of conditional medium(first stage of liquid culture,adding 2ME only).Third stage:CD34 + cells developed into CD34 - CD38 + cells when transferred onto prepared bone marrow stromal cells in the presence of IL-6 and SCF,they looked like plasma cells by cell morphology,and immunoglobulin test was positive .CONCLUSIONS:Cultured in a suitable microenviroment,ES-D3 cells can developed into plasma cells.cytokines were not needed in the early development of ES-D3 cells,conditional medium of the first stage of liquid culture could accelerate them to develop into hematopoietic cells,and the primary bone marrow stromal cells play a main role in the induction of plasma cells.