miR-1-5p修饰脐带间充质干细胞对系统性红斑狼疮T淋巴细胞亚群的免疫调节

Immunomodulatory effects of umbilical cord-mesenchymal stem cells modified by miR-1-5p on T lymphocyte subsets in systemic lupus erythematosus

背景:近年来有很多关于基因修饰间充质干细胞治疗疾病的研究,包括自身免疫性疾病的治疗。已发现多种miRNA在自身免疫性疾病中异常表达,其中miR-1在多发性肌炎中呈低表达,miR-1-3p是与系统性红斑狼疮易感基因相关的miRNA之一。目的:研究miR-1-5p修饰脐带间充质干细胞对系统性红斑狼疮T淋巴细胞亚群的免疫调节作用。方法:选取疾病活动期30-60岁系统性红斑狼疮女性患者和健康体检者各8例,实时荧光定量PCR检测两组外周血单个核细胞中的miR-1-5p表达水平;无菌条件下采集足月顺产或剖宫产新生儿脐带进行脐带间充质干细胞的分离培养,利用脂质体法,按浓度梯度为5,7,10,15 nmol/L的Red Fluorescent Control miRNA分别转染脐带间充质干细胞24,48,72 h,流式细胞术检测不同浓度梯度和时间梯度下miR-1-5p转染效率;密度梯度离心法分离系统性红斑狼疮患者外周血单个核细胞,与miR-1-5p转染的脐带间充质干细胞共培养,分为miR-1转染脐带间充质干细胞与外周血单个核细胞共培养组,miRNA阴性对照转染脐带间充质干细胞与外周血单个核细胞共培养组,未转染脐带间充质干细胞与外周血单个核细胞共培养组,外周血单个核细胞单独培养组。共培养48 h,实时荧光定量PCR检测各组外周血单个核细胞中IL-17A、Foxp3、IFN-γ、IL-4基因的相对表达量;流式细胞术检测各组外周血单个核细胞中Th17、Treg、Th1、Th2细胞的分化情况。结果与结论:①与健康对照组相比,miR-1-5p在系统性红斑狼疮患者外周血单个核细胞中的表达显著下调(P<0.05);②当miR-1-5p浓度为10 nmol/L、转染时间为48 h时,转染效率最高;③与外周血单个核细胞组相比,miR-1-5p转染脐带间充质干细胞与外周血单个核细胞共培养组Foxp3的表达增加,Treg细胞比例显著升高,差异有显著性意义(P<0.05);与外周血单个核细胞组相比,其他3个共培养组Th17/Treg比值在基因表达水平上是下降的,差异有显著性意(P<0.01),但流式检测各组Th17/Treg细胞比值,差异无显著性意义(P>0.05);与外周血单个核细胞组相比,miR-1-5p转染脐带间充质干细胞与外周血单个核细胞共培养组Th1/Th2比值在基因表达水平上是下降的,差异有显著性意义(P<0.05),但流式检测各组Th1/Th2细胞比值,差异无显著性意义(P>0.05);④结果显示,miR-1-5p修饰的脐带间充质干细胞对系统性红斑狼疮Treg细胞具有免疫调节作用,过表达miR-1-5p可以促进系统性红斑狼疮患者外周血单个核细胞中Treg细胞的分化和增殖。miR-1-5p通过上调Treg细胞表达调节Th17/Treg的免疫失衡,同时也调节Th1/Th2的免疫失衡。

BACKGROUND:In recent years,there were lots of researches on the treatment of diseases by gene modified mesenchymal stem cells,including the treatment of autoimmune diseases.Various miRNAs have been found to be abnormally expressed in autoimmune diseases,among which miR-1 is low-expressed in polymyositis,and miR-1-3p is one of the miRNAs related to systemic lupus eythematosus susceptibility genes.OBJECTIVE:To investigate the immunoregulatory effect of miR-1-5p modified umbilical cord-mesenchymal stem cells on T lymphocyte subsets in systemic lupus erythematosus.METHODS:The expression level of miR-1-5p in the two groups was detected by real-time fluorescence quantitative PCR in eight patients aged 30-60 years with active systemic lupus erythematosus and eight healthy subjects.Umbilical cord-mesenchymal stem cells were isolated and cultured from umbilical cord of newborns delivered at term or cesarean section under aseptic conditions.Using the liposome method,the umbilical cord-mesenchymal stem cells were transfected with Red Fluorescent Control with a concentration gradient of 5,7,10,and 15 nmol/L for 24,48,and 72 hours.Transfection efficiency of miR-1-5p was determined by flow cytometry at different concentration gradients and time gradients.Peripheral blood mononuclear cells in systemic lupus erythematosus patients were isolated by density gradient centrifugation and co-cultured with umbilical cord-mesenchymal stem cells which were transfected with miR-1-5p,and divided into four groups:umbilical cord-mesenchymal stem cells transfected with miR-1 and co-cultured with peripheral blood mononuclear cells of systemic lupus erythematosus patients,umbilical cord-mesenchymal stem cells transfected with miRNA negative control and co-cultured with peripheral blood mononuclear cells of systemic lupus erythematosus patients,umbilical cord-mesenchymal stem cells co-cultured with peripheral blood mononuclear cells of systemic lupus erythematosus patients,and peripheral blood mononuclear cells of systemic lupus erythematosus patients without treatment group.The coculture was conducted for 48 hours.The relative expression levels of IL-17A,Foxp3,IFN-γand IL-4 genes in each group were detected by real-time fluorescence quantitative PCR.Flow cytometry was used to detect the differentiation of Th17,Treg,Th1 and Th2 cells in peripheral blood mononuclear cells of each group.RESULTS AND CONCLUSION:(1)miR-1-5p was significantly down-regulated in peripheral blood mononuclear cells of systemic lupus erythematosus patients compared with healthy controls(P<0.05).(2)The transfection efficiency was highest when the concentration of miR-1-5p was 10 nmol/L and the transfection time was 48 hours.(3)Compared with the peripheral blood mononuclear cells of systemic lupus erythematosus patients without treatment group,the expression of Foxp3 in umbilical cord-mesenchymal stem cells transfected with miR-1-5p and co-cultured with peripheral blood mononuclear cells of systemic lupus erythematosus patients group was increased;the proportion of Treg cells was significantly increased;and the differences were statistically significant(P<0.05).Compared with the peripheral blood mononuclear cells of systemic lupus erythematosus patients without treatment group,Th17/Treg ratio decreased in the other three co-culture groups,and the difference was statistically significant(P<0.01).Th17/Treg cell ratio detected by flow cytometry in each group was not significantly different(P>0.05).Compared with the peripheral blood mononuclear cells of systemic lupus erythematosus patients without treatment group,the Th1/Th2 ratio in the umbilical cord-mesenchymal stem cells transfected with miR-1-5p and co-cultured with PBMC of systemic lupus erythematosus patients group decreased,and the difference was statistically significant(P<0.05).However,the Th1/Th2 cell ratio of each group detected by flow cytometry was not significantly different(P>0.05).(4)The results showed that the umbilical cord-mesenchymal stem cells modified by miR-1-5p had an immunoregulatory effect on Treg cells of systemic lupus erythematosus,and overexpression of miR-1-5p could promote the differentiation and proliferation of Treg cells of systemic lupus erythematosus patients.miR-1-5p regulates the immune imbalance of Th17/Treg and Th1/Th2 by up-regulating the expression of Treg cells.