Objective: To explore the improvement effect of CXC chemokine receptor 4 (Cxcr4) gene-modified bone marrow mesenchymal stem cell (BMSC)-derived exosomes on aplastic anemia (AA), and make a preliminary exploration of the mechanism.
Methods: Mouse BMSCs were isolated and cultured, then infected by recombinant lentivirus carrying Cxcr4 gene. The expression of green fluorescence was observed through fluorescence microscope, the expression of Cxcr4 mRNA was detected by real-time fluorescence quantitative PCR, and the BMSC-derived exosomes modified with Cxcr4 gene were extracted. Mouse models of AA were constructed, and control group, model group (AA), AA+BMSC group, AA+NC-BMSC group, AA+Cxcr4-BMSC group were set up. Except control group and model group, the other three groups of mice were injected 400 μl exosomes from different sources via the tail vein, after 2 weeks, the routine blood indices and the number of bone marrow nucleated cells were detected, the pathological changes of bone marrow were observed by HE staining, and the expression level of Treg cells was detected by flow cytometry.
Results: Mouse BMSCs were successfully isolated, and BMSCs with high expression of Cxcr4 and their exosomes were obtained. Compared with the control group, the number of red blood cell (RBC), white blood cell (WBC), and platelet (PLT), the hemoglobin (Hb) content and proportion of Treg cells in the peripheral blood of mice in the model group significantly decreased (P<0.01), as well as the number of bone marrow nucleated cells (P<0.01). The proliferation level of nucleated cells was low, and the medullary cavity was filled with a large number of fat cells. Compared with the model group, the number of RBC, WBC, PLT, the Hb content and proportion of Treg cells in the peripheral blood of mice in the AA+BMSC group, AA+NC-BMSC group, and AA+Cxcr4-BMSC group significantly increased (P<0.01), as well as the number of bone marrow nucleated cells (P<0.01), and pathological changes of bone marrow were improved. In addition, the number of RBC, WBC, PLT, the Hb content and proportion of Treg cells in the peripheral blood of mice in the AA+Cxcr4-BMSC group were significantly higher than those in the AA+BMSC group (P<0.01), as well as the number of bone marrow nucleated cells (P<0.01).
Conclusion: Injection of Cxcr4 gene-modified BMSC-derived exosomes has a certain improvement effect on AA mice, and the mechanism may be related to an increase of the proportion of Treg cells.
题目: Cxcr4基因修饰的BMSC来源外泌体对再生障碍性贫血的改善作用及机制研究.
目的: 探讨趋化因子受体4（Cxcr4）基因修饰的骨髓间充质干细胞（BMSC）来源的外泌体对再生障碍性贫血（AA）的改善作用，并初步探究其作用机制.
方法: 分离培养小鼠的BMSC，采用携带Cxcr4基因的重组慢病毒感染BMSC，通过荧光显微镜观察绿色荧光表达，实时荧光定量PCR检测Cxcr4 mRNA表达，并提取Cxcr4基因修饰的BMSC来源的外泌体。实验设置为对照组、模型组、AA+BMSC组、AA+NC-BMSC组、AA+Cxcr4-BMSC组，构建小鼠AA模型。除对照组和模型组外，其他三组小鼠通过尾静脉输注400 μl不同来源的外泌体，2周后检测小鼠血常规指标与骨髓有核细胞数，HE染色观察骨髓病理形态学变化，流式细胞术检测调节性T细胞（Treg）表达水平.
结果: 经鉴定成功分离到小鼠BMSC，并获得Cxcr4高表达的BMSC及其外泌体。与对照组比较，模型组小鼠外周血中红细胞、白细胞、血小板数显著减少（P<0.01），血红蛋白含量和Treg细胞比例显著下降（P<0.01），骨髓有核细胞数显著减少（P<0.01）、增生较低，骨髓腔被大量脂肪细胞填充；与模型组比较，AA+BMSC组、AA+NC-BMSC组及AA+Cxcr4-BMSC组小鼠外周血中红细胞、白细胞、血小板数显著增加（P<0.01），血红蛋白含量和Treg细胞比例显著升高（P< 0.01），骨髓有核细胞数显著增加（P<0.01），骨髓病理改变得到改善；此外，AA+Cxcr4-BMSC组小鼠外周血中红细胞、白细胞、血小板数，血红蛋白含量和Treg细胞比例较AA+BMSC组均显著升高（P<0.01），且骨髓有核细胞数也显著增加（P<0.01）.
结论: 注射Cxcr4基因修饰的BMSC来源的外泌体对AA小鼠具有一定改善作用，其机制可能与提高Treg细胞比例有关.
Keywords: CXC chemokine receptor 4; Treg cell; aplastic anemia; bone marrow mesenchymal stem cell; exosome.