融合蛋白IL6/IL2在髓系-NK起始细胞(ML-IC)分析体系中对NK细胞分化、增殖的影响

Effects of FPIL6/IL2on the Differentiation and Proliferation of Lymphoid in the ML-IC Assay

目的建立完善的造血干细胞体外培养及分析体系，用来研究比长期培养起始细胞（ＬＴＣ－ＩＣ）更为原始的造血干祖细胞。在此基础上，研究更适合于ＮＫ细胞的分化、培养介质。方法构建并表达ＦＰＩＬ６／ＩＬ２，采用髓系－ＮＫ起始细胞体外培养、分析体系，通过观察培养后单细胞是否能同时向髓系及ＮＫ细胞分化，来判断此单细胞是否为髓系－ＮＫ起始细胞，即髓系－淋系起始细胞（ｍｙｅｌｏｉｄ－ｌｙｍｐｈｏｉｄ ｉｎｉｔｉａｔｉｎｇ ｃｅｌｌ，ＭＬ －ＩＣ）。髓系检测指标是以祖细胞造血集落形成（ＣＦＣ）法检测ＬＴＣ－ＩＣ。ＮＫ细胞的检测指标是以ＦＡＣＳ法检测ＣＤ５６＋ ＮＫ细胞集落。通过观察培养后ＮＫ细胞集落数目的变化，研究ＦＰＩＬ６／ＩＬ２对ＮＫ－ＩＣ分化、增殖的影响。结果经过起始４周的培养后，实验组中（２５．７５±５．６８）％的细胞能够在一个或更多的二级培养体系中产生功能性ＮＫ－ＩＣ细胞，而对照组为（６．８１±１．９７）％，经过６～７周的培养后，实验组共检测出１０２个ＮＫ－ＩＣ细胞，而对照组为３３个，实验组较对照组明显扩增。结论髓系－ＮＫ起始细胞体外培养、分析体系能同时定量及定性研究造血干细胞的分化及增殖。ＦＰＩＬ６／ＩＬ２能明显促进?

Objective To develop an in vitro assay that allows the culture and identification of a single hu man bone marrow progenitor closely related to hematopoietic stem cell,which is more primitive than LTC-IC,and to find an efficient culture conditions for NK-IC expansion.Methods Fusion protein IL6/IL2was reconstructed and expressed in E.coli DH5α.ML -IC was determined by watching if the single cell can give rise to secondary progenitors with both LTC-IC and NK-IC characteristics.LTC-IC frequency was determined by the CFC clonogenic methylcellulose assay.NK-IC frequency was determi -ned by phenotyping CD56positive NK cells.The effect of FPIL6/IL2on the expansion of NK-IC was examined by comparing the colony number of NK cells before and after the culture.Results After the initial4-week expansion culture,we showed that(25.75±5.68)%of freshly sorted Lin-/34+/DRdim cells were able to generate functional NK-IC in one or more secondary FPIL6/IL2cult-ures,whereas(6.81±1.97)%in the control.A total of102NK-IC cells were present when were cultured for6～7weeks in FPIL6/IL2expansion medium,which was much higher than the33NK-IC cells in the control.Conclusion ML -IC assay will prove useful to assess a very primitive hematopo-ietic cell with multilineage generative capacity.FPIL6/IL2is capable of initiating and promoting NK-IC expansion greatly in ex vivo cultures in terms of net-conservation and netproliferation.