分子嵌合MHC-I基因小鼠骨髓pre-T细胞的构建及体外实验研究

发布时间：2018-06-01 12:57

本文选题：分子嵌合 + MHC-I　；参考：《天津医科大学》2012年硕士论文

【摘要】：目的：构建分子嵌合MHC-I基因小鼠骨髓前体T细胞,探讨其预输注受体降低受体小鼠对供体小鼠来源T细胞刺激反应能力的影响,为研究分子嵌合诱导移植免疫耐受打下基础。方法：采用逆转录-聚合酶链式反应(RT-PCR)技术获取C57BL/6小鼠MHC-I等位基因H-2Db和H-2Kb,分别将其连入质粒pIRES,构建pIRES-H-2Db、 pIRES-H-2Kb质粒,双酶切电泳检测和测序鉴定插入序列。培养BALB/c小鼠骨髓造血干细胞,细胞因子IL-3和SCF诱导分化,流式细胞术检测前体T细胞(pre-T细胞)表面标志Thy-1.2+、CD3-。脂质体分别转染C57BL/6小鼠MHC-I等位基因H-2Db和H-2Kb入BALB/c小鼠pre-T细胞。流式细胞术检测BALB/c小鼠pre-T细胞H-2Db和H-2Kb蛋白的表达。将转染C57BL/6小鼠MHC-I等位基因的BALB/c小鼠pre-T细胞经尾静脉注射回BALB/c小鼠,饲养7天后取其脾细胞作为反应细胞,用C57BI/6小鼠的脾细胞做刺激细胞,单向混合淋巴细胞培养,改良MTT法检测其增殖效果。结果：①成功获取C57BL/6小鼠MHC-I等位基因H-2Db和H-2Kb,构建质粒pIRES-H-2Db和pIRES-H-2Kb,双酶切鉴定插入序列大小正确,并经测序与Genbank上H-2Db和H-2Kb序列完全一致。②成功体外培养BALB/c小鼠骨髓pre-T细胞,流式细胞术检测Thy-1.2+、CD3-细胞的比例可达64.8%。③脂质体成功转染C57BL/6小鼠MHC-I等位基因H-2Db,流式细胞术检测H-2Db蛋白表达为14.6%,与未转染组(0.1%)和转染空质粒组(0.3%)相比有明显升高(p0.05)；转染C57BL/6小鼠MHC-I等位基因H-2Kb,流式细胞术检测H-2Kb蛋白表达为14.8%,与未转染组(0.1%)和转染空质粒组(0.3%)相比亦有明显升高(p0.05)。④单向混合淋巴细胞培养显示,共注射组的刺激指数(SI)(0.764±0.074)比空质粒组SI(0.983±0.081)和未转染组S1(0.994±0.142)明显下降(P0.01)；共注射组与转染质粒pIRES-H-2Db组(0.859±0.085)、转染质粒pIRES-H-2Kb (0.860±0.097)相比,SI值差异有统计学意义(p0.05)；转染质粒pIRES-H-2Db组、转染质粒pIRES-H-2Kb组与未注射组和转染空质粒组分别比较,差异亦有统计学意义(p0.05),而未转染组与转染空质粒组的S1值差异无统计学意义(O0.05)。结论：①成功构建C57BL/6小鼠MHC-I等位基因H-2Db和H-2Kb真核表达质粒。②细胞因子诱导可体外收获BALB/c小鼠骨髓pre-T细胞。③C57BL/6小鼠MHC-I等位基因H-2Db和H-2Kb成功体外转染至BALB/c小鼠pre-T细胞,且转染后可稳定表达。④体外成功获得分子嵌合MHC-I等位基因受体小鼠pre-T细胞,其能降低受体对异基因供体来源T细胞刺激的反应能力。
[Abstract]:Aim: to construct precursor T cells from bone marrow of mice with molecular chimeric MHC-I gene, and to investigate the effect of preinfusion receptor on the stimulating response of donor mice to T cells derived from donor mice, so as to lay a foundation for the study of molecular chimerism in inducing transplantation immune tolerance. Methods: the MHC-I alleles H-2Db and H-2Kb of C57BL/6 mice were obtained by reverse transcription-polymerase chain reaction (RT-PCR) technique. The plasmid pIRES-H-2Db, pIRES-H-2Kb was constructed, and the inserted sequence was identified by double digestion electrophoresis and sequencing. Bone marrow hematopoietic stem cells of BALB/c mice were cultured and differentiation was induced by cytokines IL-3 and SCF. The surface marker Thy-1.2 CD3-was detected by flow cytometry. The MHC-I allele H-2Db and H-2Kb of C57BL/6 mice were transfected into pre-T cells of BALB/c mice by liposome respectively. The expression of H-2Db and H-2Kb in pre-T cells of BALB/c mice was detected by flow cytometry. The pre-T cells of BALB/c mice transfected with MHC-I allele of C57BL/6 mice were injected into the tail vein of BALB/c mice. The spleen cells of BALB/c mice were taken as reaction cells after feeding for 7 days. The splenocytes of C57BI/6 mice were used as stimulators and mixed lymphoid fine cells in one way. Cell culture and modified MTT method were used to detect the proliferation effect. Results the MHC-I alleles H-2Db and H-2Kb of C57BL/6 mice were successfully obtained at 1: 1. The plasmids pIRES-H-2Db and pIRES-H-2Kb were constructed. The size of the inserted sequences was confirmed by double enzyme digestion, and the pre-T cells of BALB/c mice bone marrow were successfully cultured in vitro by sequencing with H-2Db and H-2Kb sequences on Genbank. The MHC-I allele H-2Db of C57BL/6 mice was successfully transfected with liposome 64.8.3 by flow cytometry, and the expression of H-2Db protein by flow cytometry was 14.6%, which was significantly higher than that of untransfected group (0.1%) and empty plasmid group (0.3%). The MHC-I allele H-2Kb was transfected into C57BL/6 mice, and the expression of H-2Kb protein was detected by flow cytometry. Compared with the control group (0.1%) and the empty plasmid group (0.3%), the expression of H-2Kb protein was also significantly increased in unidirectional mixed lymphocyte culture. The stimulation index (SI) of the co-injection group was 0.764 卤0.074) significantly lower than that of the blank plasmid group (SI(0.983 卤0.081) and the untransfected group (S 1N 0.994 卤0.142), and the SI value of the co-injection group and the transfected plasmid pIRES-H-2Db group was 0.859 卤0.085 and 0.860 卤0.097, respectively, and the SI value of the transfected plasmid pIRES-H-2Db group was significantly higher than that of the control group. Compared with the control group and the blank plasmid group, the difference was also significant (p 0.05), but there was no significant difference in S1 value between the untransfected group and the empty plasmid group. Conclusion the successful construction of H-2Db and H-2Kb eukaryotic expression plasmids of MHC-I alleles H-2Db and H-2Kb in C57BL/6 mice can induce the successful transfection of MHC-I alleles H-2Db and H-2Kb into BALB/c mouse pre-T cells in vitro, and the MHC-I allele H-2Db and H-2Kb of BALB/c mouse bone marrow pre-T cells can be harvested in vitro. After transfection, the mouse pre-T cells with chimeric MHC-I alleles were successfully obtained after transfection, which could reduce the ability of the receptor to respond to the stimulation of allogeneic donor derived 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】：2012
【分类号】：R392

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