应用OP9/OP9-DL1阐明小鼠8.5天胚胎的淋系潜能

Clarification of Lymphoid Potential in Mouse E8.5 Embryos by OP9/OP9-DL1 Coculture

胚胎时期淋巴细胞的发生位点一直存在争议。为明确能自主产生淋系的解剖部位,本研究选取小鼠8.5天胚胎的卵黄囊(胚外)和脏壁层(胚内体系)作为实验对象,此时血液循环尚未建立。用集落形成实验检测脏壁层和卵黄囊的造血集落,用OP9共培养检测脏壁层和卵黄囊的B淋巴细胞,用三步法检测脏壁层和卵黄囊的T细胞生成。结果表明:在标准的集落形成实验中,两者均能产生典型的造血集落,但其类型有差异。通过OP9共培养发现:脏壁层可产生B淋巴细胞,它们表达B220、CD19和IgM。采用三步法在OP9-DL1上诱导脏壁层至第16天,可产生CD44-/CD25+幼稚T细胞前体以及CD4+/CD8-单阳性T细胞。然而,上述共培养体系均不能诱导卵黄囊产生B和T淋巴细胞。结论:与卵黄囊相比,脏壁层具有较强的体外淋系分化潜能。今后,应用新的模式动物有望获得更可靠的体内证据,以解答淋系起源这一关键科学问题。

The anatomical location of lymphocyte ontogeny in the developing mouse embryo remains controversial.To define the site that can generate lymphocytes de novo,the intraembryonic splanchnopleura（SP） and extraembryonic yolk sac（YS） at 8.5 days postcoitum,when systemic circulation is not established,were investigated. The results indicated that in standard colony forming assay,the cells from both splanchnopleura and yolk sac formed typical myeloerythroid colonies,but their types were distinct.When cocultured with the OP9,the splanchnopleura produced B cells expressing B220,CD19 and surface IgM.Using a three-step culture protocols with the OP9 expressing Delta-like 1 as feeders,the splanchnopleura produced immature T precursor cells（CD44-/CD25＋） and more mature single positive T cells（CD4＋/CD8-） after 16 days of incubation.However,the yolk sac failed to generate B and T lymphocytes under identical conditions.It is concluded that prior to linked embryonic circulation,the splanchnopleura other than the yolk sac had robust lymphoid potential in vitro.In the future,more reliable evidence from novel model animals will ultimately delineate the embryonic origin of lymphocytes in vivo.