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目的研究脐带沃顿胶间充质干细胞(WJCs)对重型再生障碍性贫血(SAA)患者外周血CD4+CD25+调节性T细胞及Foxp3基因表达水平的影响,探讨WJCs对SAA患者T淋巴细胞(TLCs)免疫调节作用的可能机制。方法从人脐带中分离培养WJCs,通过流式细胞术检测其表面标记并进行鉴定;采用密度梯度离心法从SAA患者外周血中分离TLCs;在植物凝集素(PHA)刺激下,将SAA患者TLCs(1×105个)进行体外培养,实验组加入不同数量级WJCs(1×103、1×104、1×105个)共培养,3 d后采用MTT法检测淋巴细胞抑制率;流式细胞仪检测实验组及正常对照组SAA患者TLCs中CD4+CD25+T细胞比例的变化,RT-PCR方法检测Foxp3基因mRNA水平的变化。结果 WJCs能明显抑制SAA患者TLCs增殖,且抑制作用与WJCs呈剂量依赖性;实验组CD4+CD25+T细胞比例以及Foxp3基因表达水平均比对照组明显增加,且与TLCs增殖水平呈负相关。结论 WJCs对SAA患者TLCs的抑制作用呈剂量依赖性,通过上调Foxp3的表达而发挥CD4+CD25+调节性T细胞的免疫调节作用可能是其机制之一。
Objective To investigate the effects of umbilical cord blood Wound-derived mesenchymal stem cells (WJCs) on the expression of CD4 + CD25 + regulatory T cells and Foxp3 in peripheral blood of patients with severe aplastic anemia (SAA). To investigate the effect of WJCs on T lymphocytes (TLCs) ) Possible mechanisms of immunomodulatory effects. Methods WJCs were isolated and cultured from human umbilical cord, and their surface markers were detected by flow cytometry and identified. TLCs were isolated from peripheral blood of patients with SAA by density gradient centrifugation. TLCs of peripheral blood from patients with SAA were stimulated by phytohemagglutinin (PHA) (1 × 105) were cultured in vitro. WJCs (1 × 103, 1 × 104 and 1 × 105) were co-cultured in experimental groups, and the inhibitory rate of lymphocytes was detected by MTT assay after 3 days. Flow cytometry The percentage of CD4 + CD25 + T cells in TLCs in experimental group and normal control group were detected by RT-PCR, and the mRNA level of Foxp3 was detected by RT-PCR. Results WJCs could significantly inhibit the proliferation of TLCs in SAA patients and the effect of WJCs in a dose-dependent manner. The proportion of CD4 + CD25 + T cells and Foxp3 expression in SAA group were significantly higher than those in control group, and negatively correlated with the proliferation of TLCs. Conclusion The inhibitory effect of WJCs on TLCs in SAA patients is dose-dependent. It may be one of the mechanisms that WJCs play an immunomodulatory role of CD4 + CD25 + regulatory T cells by up-regulating Foxp3 expression.