天津医药

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分子嵌合MHC-Ⅰ基因小鼠骨髓造血干细胞输注降低脾脏T细胞对异基因小鼠DC反应的研究

闫永嘉1,付蔚华2,高莹1,朱理玮1   

  1. 1. 天津医科大学总医院
    2. 天津医大总医院普外科
  • 收稿日期:2011-08-03 修回日期:2011-11-30 出版日期:2012-02-15 发布日期:2012-02-15
  • 通讯作者: 付蔚华

The Research on The Mechanism of Mouse Bone Marrow Hematopoietic Stem Cells with Chimeric MHC-I Gene Reduce T Lymphocyte Response to Allogeneic Dendritic Cells

  • Received:2011-08-03 Revised:2011-11-30 Published:2012-02-15 Online:2012-02-15

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摘要： 【摘要】 目的 构建分子嵌合MHC-Ⅰ基因小鼠骨髓造血干细胞，并探讨其诱导T细胞对异基因小鼠DC无反应的机理。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，感染BalB/C小鼠骨髓造血干细胞，构建分子嵌合细胞。输注Balb/C小鼠后7天，获取脾脏T淋巴细胞，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，流式细胞术检测CD34阳性率可达（34.64±2.76）%。病毒载体感染BalB/C小鼠骨髓造血干细胞3天，实时定量PCR检测，C57BL/6小鼠MHC-Ⅰ基因mRNA水平表达量比空白对照组增加657倍，比阴性对照组增加666倍。流式细胞学检测病毒感染组C57BL/6小鼠MHC-Ⅰ蛋白表达率可达98.17%，较空白对照组及阴性对照组均有明显升高。单向混合淋巴细胞培养结果显示，C57 BL/6小鼠DC对输注分子嵌合细胞后BalB/C小鼠脾脏T细胞刺激指数明显降低。(P<0.01) 结论 成功构建了过表达慢病毒载体。应用过表达慢病毒载体可以成功构建分子嵌合细胞。将分子嵌合细胞输注小鼠后，其脾脏T细胞对异基因小鼠DC反应明显减低。

关键词: MHC-Ⅰ基因, 骨髓造血干细胞, 慢病毒载体, 免疫耐受

Abstract: [Abstract]Objective: To construct the mouse bone marrow hematopoietic stem cells with chimeric MHC-I gene. To explore the mechanism of it inducing T lymphocyte response to allogeneic dendritic cells. Methods: mouse bone marrow hematopoietic stem cells were collected with the lymphocyte separation medium (ficoll), by density gradient separation. Identified hematopoietic stem cell (HSC) were infected by the constructed lentiviral vectors. After infection, the molecular chimerism was successfully constructed. The ability of molecular chimerism reduce T lymphocyte response to allogeneic dendritic cells was observed through MLC. Results: Using ficoll, the Balb/C mouse bone marrow HSCs were successfully separated. The rate of CD34+ cells was up to (34.64±2.76)%. 3 days after lentivirus infection, H2-K1 gene expression at mRNA level in Balb/C mouse hematopoietic stem cells increased 657-fold than that of the non-infect group, increased 666-fold than that of the control group. The H2-K1 expression at mRNA level between control group and non-infect group had no significant difference (P>0.05). Flow cytometry analysis indicated that H-2Kd expressed in 98.17% cells. The result of MLC demonstrates that the index of stimulation to T lymphocyte significantly decrease. (P<0.01) Conclusion: The lentiviral vector carrying C57 mice`s MHC-I gene H2-K1 was successfully constructed. Balb/C mouse bone marrow hematopoietic stem cells can be successfully infected by the lentiviral vector. After infection, a stable and long-term expression of H2-K1 was achieved and H-2Kb protein was expressed. The molecular chimerism could reduce T lymphocyte response to allogeneic dendritic cells.

Key words: MHC-I gene, bone marrow hematopoietic stem cell, lentiviral vectors, immune tolerance