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目的:构建分子嵌合主要组织相容性复合体(MHC)-Ⅰ基因小鼠骨髓造血干细胞,并探讨其诱导脾脏T细胞对异基因小鼠树突状细胞(DC)反应的机制。方法:密度梯度法分离培养BALB/c小鼠骨髓造血干细胞。构建携带C57BL/6小鼠MHC-Ⅰ基因慢病毒载体(病毒感染组),携带无意义基因慢病毒载体(阴性对照组)。分别感染BALB/c小鼠骨髓造血干细胞,构建分子嵌合细胞。分别取病毒感染组、阴性对照组及未加入病毒的空白对照组造血干细胞输注BALB/c小鼠后7d,获取脾脏T淋巴细胞,分别与C57BL/6小鼠DC进行混合淋巴细胞培养,测定刺激指数。结果:成功体外分选及培养BALB/c小鼠骨髓造血干细胞。病毒感染组C57BL/6小鼠MHC-Ⅰ蛋白表达率可达98.17%。单向混合淋巴细胞培养结果显示,C57BL/6小鼠DC对输注病毒感染组细胞后BALB/c小鼠脾脏T细胞刺激指数明显降低(P<0.01)。结论:输注分子嵌合MHC-Ⅰ基因造血干细胞后,小鼠脾脏T细胞对异基因小鼠DC反应明显减低。
OBJECTIVE: To construct a molecular chimeric MHC-I mouse bone marrow hematopoietic stem cells and to explore the mechanism of its induction of dendritic cells on dendritic cells (DCs) in allogeneic mice. Methods: BALB / c mouse bone marrow hematopoietic stem cells were isolated and cultured by density gradient method. Construction of lentiviral vector carrying MHC-I gene of C57BL / 6 mice (virus infected group) and carrying lentiviral vector without sense gene (negative control group). BALB / c mouse bone marrow hematopoietic stem cells were infected respectively to construct molecular chimeric cells. Seven days after the hematopoietic stem cells were transfused into BALB / c mice, the splenic T lymphocytes were obtained from the virus-infected group, the negative control group and the control group without the addition of virus, respectively. Mixed lymphocytes were cultured with C57BL / 6 mouse DC respectively. Stimulation index. Results: BALB / c mouse bone marrow hematopoietic stem cells were successfully sorted and cultured in vitro. The expression of MHC-Ⅰ protein in C57BL / 6 mice infected with virus was up to 98.17%. The results of one-way mixed lymphocyte culture showed that the stimulation of C57BL / 6 mouse DCs significantly decreased the splenic T cell stimulation index of BALB / c mice (P <0.01). CONCLUSION: DC responses of splenic T lymphocytes to allogeneic mice were significantly reduced after chimeric MHC-Ⅰ gene transfused into hematopoietic stem cells.