间充质干细胞对小鼠急性免疫性肝损伤治疗作用的机制研究

Investigation on the therapeutic effects and the mechanism of mesenchymal stem cells on acute immune liver injury in mice

目的 探究间充质干细胞（MSC）治疗刀豆蛋白A（ConA）诱导的小鼠急性免疫性肝损伤的机制.方法 从3周龄C57BL/6小鼠的四肢致密骨片中体外分离培养MSC并鉴定其表面标记分子和成脂成骨分化能力.将15只6～7周龄C57BL/6小鼠分为对照组、MSC治疗组和PBS治疗组,每组5只.MSC治疗组经尾静脉先后注射ConA和MSC,PBS治疗组经尾静脉先后注射ConA和PBS,对照组先后两次均经尾静脉注射PBS.注射14～16 h后处死小鼠.检测外周血ALT、AST,进行肝脏Knodell评分.流式细胞分析术检测脾脏CD4＋T淋巴细胞活化比例及CD4＋T淋巴细胞中调节性T细胞（Treg）、Th1、Th2和Th17比例变化情况,并计算Th17与Treg比值.ELISA检测外周血TNF-α、IFN-γ及IL-4相关细胞因子的含量.应用独立样本t检验比较两组正态分布资料间的差异.结果 MSC治疗组的ALT、AST及Knodell评分分别为（174.2±46.9）U/L、（185.6±71.6） U/L、（3.4±1.3）分,均优于PBS治疗组[（647.0±118.0）U/L、（749.0±104.0） U/L、（5.2±0.8）分],差异均有统计学意义（t=8.33、9.98、2.55,P均＜0.05）.PBS治疗组脾脏中CD4＋T淋巴细胞活化比例为（26.10±2.17）％,CD4＋T淋巴细胞中Th1、Th2细胞所占比例为（5.81±0.79）％、（5.98±1.22）％,Th17与Treg比值为0.29±0.03,外周血中TNF-α、IFN-γ和IL-4水平分别为[（1 281.95±88.61） U/L、（1 838.66±196.91） U/L、（1 192.36±163.94） U/L],较对照组的（13.74±1.59）％、（1.35±0.17）％、（2.13±0.17）％、0.15±0.05、（21.71±2.50） U/L、（11.84±1.28） U/L、（24.46±3.96） U/L均显著升高,差异均有统计学意义（t=10.26、12.37、7.02、5.30、31.79、15.93、20.75,P均＜0.01）.MSC治疗组与PBS治疗组脾脏CD4＋T淋巴细胞活化比例差异无统计学意义[（26.20±3.09）％比（26.10±2.17）％,P＞0.05],但MSC治疗组CD4＋T淋巴细胞中Th1、Th2细胞所占比例[（1.83±0.52）％、（2.75±1.06）％],Th17与Treg比值（0.18±0.02）及其外周血中TNF-α、IFN-γ及IL-4水平[（760.71±73.19） U/L、（742.49±76.46） U/L、（825.76±101.74） U/L]均较PBS治疗组显著降低,差异均有统计学意义（t=9.45、4.48、6.41、10.14、5.56、10.22,P均＜0.01）.MSC治疗组中Th17与Treg比值与对照组差异无统计学意义（P＞0.05）.结论 MSC通过影响脾脏CD4＋T淋巴细胞亚群降低Th1、Th2细胞比例,导致外周血中后者分泌的细胞因子（TNF-α、IFN-γ和IL-4）水平下降;升高Treg比例,降低Th17比例,维持Th17与Treg平衡,进而起到治疗ConA诱导的小鼠急性免疫性肝损伤的作用.

Objective To explore the mechanisms of mesenchymal stem cells （MSC） in the treatment of concanavalin A （ConA）-induced acute immune liver injury in mice.Methods MSC were isolated and cultured from bone of the four limbs of three-week-old C57BL/6 mice.The specific surface markers were identified and osteogenic,adipogenic differentiation ability were tested.A total of 15 six to seven-week old C57BL/6 mice were divided into control group,MSC treatment group and phosphate buffer saline （PBS） treatment group,five mice in each group.The mice of MSC treatment group was injected through tail firstly with ConA and then MSC,PBS treatment group was injected through tail firstly with ConA and then PBS,control group was injected through tail with PBS twice.The mice were sacrificed in 14 to 16 hours after injection.The level of alanine aminotransferase （ALT）,aspartate transaminase （AST） in peripheral blood were detected and the pathological change in liver tissue was scored by Knodell score system.Activation rate of splenic CD4＋ T cells and the proportion changes of T hepler cell （Th）1,Th2,Th17 and regulatory T cells （Treg） were detected by flow cytometry and the ratio of Th17/Treg was calculated.The levels of tumor necrosis factor α （TNF-α）,interferon （IFN）-γ and interleukin （IL）-4 in peripheral blood were detected by enzyme linked immunosorbent assay （ELISA）.Independent-sample t test was used for comparison between groups of measurement data.Results ALT,AST and Knodell score of MSC treatment group was （174.2± 46.9） U/L,（185.6± 71.6） U/L and 3.4±1.3,respectively,which were better than those of PBS treatment group （（647.0± 118.0） U/L,（749.0± 104.0） U/L and 5.2 ±0.8,respectively）,and the differences were statically significant （t =8.33,9.98 and 2.55,all P〈0.05）.The activation rate of splenic CD4＋ T cell of PBS treatment group was （26.10±2.17） %,the proportion of Th1 and Th2 in CD4＋ T cell was （5.81±0.79） % and （5.98± 1.22）%,the ratio of Th17/Treg was 0.29±0.03,the levels of TNF-α,IFN-γ and IL4 in peripheral blood were （1 281.95±88.61） U/L,（1 838.66±196.91） U/L and （1 192.36±163.94） U/L,which were higher than those of control group （（13.74±1.59）%,（1.35±0.17）%,（2.13±0.17）%,0.15± 0.05,（21.71±2.50） U/L,（11.84±1.28） U/L and （24.46±3.96） U/L）,and the differences were statistically significant （t=10.26,12.37,7.02,5.30,31.79,15.93 and 20.75,all P〈0.01）.There was no statistically significant difference in the activation rate of splenic CD4＋T cell between MSC treatment group and PBS treatment group （（26.20±3.09）% vs （26.10±2.17）%,P〉0.05）.However,in MSC treatment group,the proportion of Th1 and Th2 in CD4＋ T cell （（1.83±0.52） % and （2.75±1.06%））,the ratio of Th17/Treg （0.18±0.02） and the levels of TNF-α,IFN-γ and IL-4 in peripheral blood （（760.71± 73.19） U/L,（742.49±76.46） U/L and （825.76±101.74） U/L） significantly decreased compared with those of PBS treatment group,and the differences were statistically significant （t=9.45,4.48,6.41,10.14,5.56 and 10.22,all P〈0.01）.There was no significant difference in the ratio of Th17/Treg between MSC treatment group and control group （P〉0.05）.Conclusions The therapeutic effects of MSC on ConA induced acute immune liver injury were through influence splenic CD4＋ T cell subsets by decreasing the proportion of Th1 and Th2 and then declining the levels of secreted cytokines such as TNF,IFN-γ and IL-4 in peripheral blood,increasing the proportion of Treg and decreasing the proportion of Th17 and keeping the balance of Th17/Treg.