利用胸腺类器官体外诱导小鼠T淋巴细胞分化

Induction of mouse T lymphocyte differentiation in vitro by thymic organoids

目的体外构建胸腺类器官,模拟胸腺组织三维结构,诱导多种来源小鼠造血干祖细胞(HSPC)向T细胞分化。方法构建表达小鼠Delta样经典缺刻基因配体1(DLL1)及绿色荧光蛋白(GFP)的逆转录病毒载体;通过逆转录病毒感染OP9小鼠骨髓间充质细胞,构建OP9-DLL1细胞系;实时荧光定量PCR和Western blot法分别检测OP9-DLL1细胞DLL1的mRNA和蛋白表达,免疫荧光细胞化学染色检测OP9-DLL1细胞DLL1的表达和分布;提取C57BL/6小鼠E13.5胎肝HSPC及骨髓HSPC,分别与OP9-DLL1细胞按适当比例混合后离心压实,置于气-液交面培养;利用荧光显微镜观察胸腺类器官生长,流式细胞术检测T细胞表面CD3、CD4、CD8、CD25、CD44、CD45、CD117、T细胞受体β(TCRβ)表达,免疫荧光组织化学染色观察造血细胞在胸腺类器官的分布。结果成功构建表达小鼠DLL1及GFP的逆转录病毒载体,逆转录病毒感染OP9细胞后,通过GFP筛选获得OP9-DLL1细胞,OP9-DLL1细胞DLL1 mRNA和DLL1蛋白表达明显增加,DLL1蛋白表达于OP9-DLL1细胞膜。诱导培养40 d内,胸腺类器官状态良好且体积逐渐增大。胸腺类器官诱导T细胞程序性分化,HSPC分化为CD3+T细胞。结论通过OP9-DLL1细胞成功构建胸腺类器官,体外诱导多种来源小鼠HSPC向T细胞分化。

Objective To induce the differentiation of hematopoietic stem progenitor cells(HSPCs) into T cell by creating thymic organoids and simulating the three-dimensional structure of thymus tissue in vitro. Methods The retroviral vector expressing the DLL1 and Green fluorescent protein(GFP) was constructed, and the OP9-DLL1 cell line was established in OP9 cells with the aid of retroviral infection. The mRNA and protein level of DLL1 in OP9-DLL1 cells was detected by quantitative real-time PCR and Western blot respectively. Immunofluorescence assay was used to detect the DLL1 protein expression and distribution in OP9-DLL1 cells. HSPCs were extracted from E13.5 fetal liver and bone marrow of C57BL/6 mouse, and mixed with OP9-DLL1 cells in an appropriate ratio respectively, then compacted by centrifuging and cultured at the air-liquid interface in medium. Fluorescence microscope was used to observe the growth of thymic organoids. Flow cytometry was used to detect the expression of T cell surface markers, including CD3, CD4, CD8, CD25, CD44, CD45, CD117 and TCRβ. Immunofluorescence cytochemical staining was used to observe the distribution of hematopoietic cells in thymic organoids. Results The retroviral vector expressing DLL1 and GFP was successfully constructed. The OP9 cells were infected with the retrovirus constructed, and OP9-DLL1 cells were obtained by GFP screening. The mRNA and protein level of DLL1 in OP9-DLL1 cells significantly increased, and DLL1 was expressed in the membrane OP9-DLL1 cells. During the 40 days of culture, the thymic organoids remained in good condition and increased gradually in volume. Thethymic organoids induced programmed differentiation of T cells, and differentiation of HSPCs into CD3+ T cells. ConclusionOP9-DLL1 cells can be used to construct thymic organoids and to induce differentiation of HSPCs into T cells in vitro.