内源性同步重建骨髓放射性损伤受体免疫自稳机制的研究

As we know, it was well demonstrated that the delayed and lower grade reconstitution of immune homeostasis played the key role in the graft-verse-host disease(GVHD) of transplant recipients after bone marrow radiation injury, and nowadays, unspecific immunity inhibitors were still the main measures to prevent GVHD, but which often bring adverse side-effects such as progressively depress recipient immune function. The previous research including our lab work proved that CD4+CD25+FoxP3+ T regulatory cells（Treg） had definite effect on maintaining the immune homeostasis and preventing GVHD after bone marrow transplantation, but it has shown lower rates in peripheral blood and lower proliferation characteristics, as well as delayed reconstitution. In this study, we establish the animal model on allogeneic bone marrow transplant(allo-BMT) after radiation injury, use TGF-β as endogenous expansion revulsant factor, and gene transfer donor bone marrow stromal cells by constructing the lentiviral vector of TGF-β, then mix with donor bone marrow and transplant them to radiation-pretreated recipient, in order to induce a higher proportion of CD4+CD25+FoxP3+ T regulatory cells proliferation than that in normal, and to increase the relative ratio and absolute number of CD4+CD25+FoxP3+ T regulatory cells in the outputted mature blood cells. By observing the changes of the related indexes for bone marrow radiation injury with recipient immune homeostasis and hematopoietic reconstruction, we analyze the effect of endogenous expansion of CD4+CD25+FoxP3+ T regulatory cells and its effect on preventing GVHD. We try to illustrate the immunologic molecular mechanism of CD4+CD25+FoxP3+ T regulatory cells acting on B lymphocytes, and accordingly hope this study could contribute to providing new ideas for the controlment of immune disorder diseases and graft rejection.

骨髓放射性损伤后，移植受体免疫自稳机制重建滞后和低下是GVHD发生的主要原因。非特异性免疫抑制剂阻抗GVHD时存在加重受体免疫低下和阻抗时机被动等缺陷。国内外和我们前期研究表明：CD4+CD25+FoxP3+Treg具备维持免疫自稳和阻抗GVHD效能，但其本身低比例低增殖且重建滞后。本项目拟建立骨髓放射性损伤allo-BMT模型，并以TGF-β内源性扩增诱导Treg细胞，构建携带TGF-β的慢病毒载体，转染供体骨髓基质细胞后与移植骨髓混合，然后移植给放射预处理小鼠，内源性诱生CD4+CD25+Treg细胞增殖，同步提高成熟血细胞中Treg细胞相对比例。并观察骨髓放射性损伤与受体免疫自稳和造血重建相关指标变化，分析CD4+CD25+FoxP3+Treg细胞内源性诱生与对GVHD的阻抗效应。初步阐明Treg细胞作用于B淋巴细胞的分子免疫机制，为临床干预移植排斥和免疫紊乱疾病调控提供新思路。

骨髓放射性损伤后，移植受体免疫自稳机制重建滞后和低下是GVHD发生的主要原因。CD4+CD25+FoxP3+Treg具备维持免疫自稳和阻抗GVHD效能，但其本身低比例低增殖且重建滞后。有研究表明接受BMSCs治疗的allo-HSCT患者外周血CD4+CD25+FoxP3+Treg细胞含量明显上升，BMSCs可通过上调Treg细胞并修复胸腺损伤诱导免疫耐受发挥抗GVHD的作用。维持BMSCs的数量对于免疫细胞的分化成熟及免疫功能的恢复和重建意义重大，研究BMSCs放射性损伤机制可为临床干预移植排除和免疫紊乱提供新思路。本研究建立了小鼠放射性损伤模型，通过芯片发现一系列差异表达的circRNA，利用生物学软件预测mmu-circRNA_016901的应答元件，发现其与miRNA1249-5p有结合位点，采用GO和KEGG软件对miRNA1249-5p进行分析，发现miRNA1249-5p可影响Akt的表达，进一步可调节BMSCs内的自噬水平。通过基因过表达和基因干扰技术在细胞水平上调节mmu-circRNA_016901的表达，并检测自噬相关蛋白Akt、LC3II和Beclin1，进一步确定其变化会影响其对应的一系列靶基因的表达以及细胞的自噬水平。研究阐明了mmu-circRNA_016901在小鼠BMSCs辐射损伤过程中的作用以及其机制，为临床辐射损伤治疗提供新的靶点。