分子嵌合MHC-I基因小鼠骨髓造血干细胞模型的建立及体外实验研究

发布时间：2019-03-22 11:29

【摘要】： 目的：构建分子嵌合造血干细胞,体外探讨它对T细胞增殖能力的影响,为研究分子嵌合诱导移植免疫耐受奠定基础。方法：①采用RT-PCR方法获取供体C57BL／6小鼠MHC-I基因H-2Db,通过双酶切、定向克隆技术将H-2Db亚克隆入逆转录病毒表达质粒pMSCVneo,通过Lipofectamine 2000与辅助质粒共转染293T细胞以获得携带H-2Db基因的重组逆转录病毒。②应用淋巴细胞分离液密度梯度分离法分选受体BALB／c小鼠骨髓造血干细胞。③将携带H-2Db的逆转录病毒转染造血干细胞并优化转染条件,同时设置未转染组,转染空质粒组；以荧光实时定量PCR和流式细胞术分别在mRNA水平和蛋白水平检测转染后各组造血干细胞H-2Db表达情况。④受体BALB／c小鼠输注造血干细胞后7天,取脾细胞做为反应细胞,与来自供体C57BL／6小鼠脾细胞的刺激细胞行单向混合淋巴细胞培养,MTT法检测各组T细胞增殖情况。结果：①成功获取H-2Db基因,构建的质粒pMSCVneo-H-2Db经限制性双酶切检测和测序分析,结果表明质粒构建成功。②质粒pMSCVneo-H-2Db经包装后,获得了携带H-2Db基因的逆转录病毒颗粒。③成功体外分选及培养受体BALB／c小鼠骨髓造血干细胞,经流式细胞术检测CD34阳性细胞可达(35.60±2.19)%。④与未转染组和转染空质粒组相比,转染构建质粒组H-2Db基因mRNA水平和蛋白水平表达均显著升高,差异有统计学意义(P0.01)；而未转染组与转染空质粒组之间差异无统计学意义(P0.05)。⑤单向混合淋巴细胞培养显示,转染构建质粒组刺激指数较未转染组和转染空质粒组明显降低(P0.01),差异有统计学意义；而未转染组与转染空质粒组之间刺激指数差异无统计学意义(P0.05)。结论：①淋巴细胞分离液密度梯度分离法可成功从小鼠骨髓细胞中分离造血干细胞。②携带H-2Db基因的逆转录病毒载体可高效稳定转染受体BALB／c小鼠骨髓造血干细胞,且感染后H-2Db基因可稳定表达。③构建的分子嵌合造血干细胞模型可以显著抑制T细胞增殖能力,为进一步研究分子嵌合诱导移植免疫耐受打下实验基础。
[Abstract]:Aim: to construct molecular chimeric hematopoietic stem cells and to explore its effect on T cell proliferation in vitro, so as to lay a foundation for the study of molecular chimerism induced transplantation immune tolerance. Methods: 1 MHC-I gene of donor C57BL/6 mice was obtained by RT-PCR method. H-2Db was subcloned into retrovirus expression plasmid pMSCVneo, by restriction endonuclease digestion and directed cloning technique. Recombinant retrovirus carrying H-2Db gene was obtained by co-transfection of Lipofectamine 2000 with helper plasmid 293T cells. (2) Bone marrow hematopoietic stem cells of recipient BALB/c mice were separated by density gradient separation of lymphocyte separation solution. 3. The retrovirus carrying H-2Db was transfected into hematopoietic stem cells and the transfection conditions were optimized. At the same time, the non-transfected group was transfected into empty plasmid group. The expression of H-2Db in hematopoietic stem cells was detected at mRNA level and protein level by fluorescence real-time quantitative PCR and flow cytometry, respectively. Spleen cells were taken as response cells 7 days after infusion of hematopoietic stem cells in 4-receptor BALB/c mice. One-way mixed lymphocyte culture was performed with stimulated cells from donor C57BL/6 mouse spleen cells. The proliferation of T cells in each group was detected by MTT method. Results: 1 the H-2Db gene was successfully obtained, and the constructed plasmid pMSCVneo-H-2Db was detected by restriction double enzyme digestion and sequenced. The results showed that the plasmid was constructed successfully. 2 after packaging, the plasmid pMSCVneo-H-2Db was successfully constructed. The retrovirus particles carrying H-2Db gene were obtained. 3. Bone marrow hematopoietic stem cells of recipient BALB/c mice were successfully isolated and cultured in vitro. The percentage of CD34 positive cells was (35.60 卤2.19)% by flow cytometry. 4 compared with the untransfected group and the transfected empty plasmid group, the expression of mRNA and protein of H-2Db gene in the transfected plasmid group was significantly higher than that in the control group. The difference was statistically significant (P0.01); There was no significant difference between the non-transfected group and the transfected empty plasmid group (P0.05). (5) the stimulation index of the transfected constructed plasmid group was significantly lower than that of the non-transfected group and the transfected empty plasmid group (P0.01), and there was no significant difference between the untransfected group and the transfected empty plasmid group (P0.05). The difference was statistically significant. There was no significant difference in stimulation index between the untransfected group and the transfected empty plasmid group (P0.05). Conclusion: 1Hematopoietic stem cells can be successfully isolated from mouse bone marrow cells by density gradient method of lymphocyte separation. 2 retroviral vector carrying H-2Db gene can efficiently and stably transfect hematopoietic stem cells into bone marrow stem cells of recipient BALB/c mice. 3 the molecular chimeric hematopoietic stem cell model could significantly inhibit the proliferation of T cells, which laid the experimental foundation for further study of molecular chimerism induced transplantation tolerance. 3. The expression of H-2Db gene was stable after infection. 3 the molecular chimeric hematopoietic stem cell model could significantly inhibit the proliferation of T cells.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R-332;R392

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