中华消化杂志
中華消化雜誌
중화소화잡지
Chinese Journal of Digestion
2015年
2期
104-109
,共6页
董魁%张续乾%郭海英%王伟强%李文文%王邦茂%刘文天
董魁%張續乾%郭海英%王偉彊%李文文%王邦茂%劉文天
동괴%장속건%곽해영%왕위강%리문문%왕방무%류문천
间质干细胞%伴刀豆蛋白A%肝疾病%治疗%T淋巴细胞亚群
間質榦細胞%伴刀豆蛋白A%肝疾病%治療%T淋巴細胞亞群
간질간세포%반도두단백A%간질병%치료%T림파세포아군
Mesenchymal stem cells%Concanavalin A%Liver disease%Therapy%T-lymphocyte subsets
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.
目的 探究間充質榦細胞(MSC)治療刀豆蛋白A(ConA)誘導的小鼠急性免疫性肝損傷的機製.方法 從3週齡C57BL/6小鼠的四肢緻密骨片中體外分離培養MSC併鑒定其錶麵標記分子和成脂成骨分化能力.將15隻6～7週齡C57BL/6小鼠分為對照組、MSC治療組和PBS治療組,每組5隻.MSC治療組經尾靜脈先後註射ConA和MSC,PBS治療組經尾靜脈先後註射ConA和PBS,對照組先後兩次均經尾靜脈註射PBS.註射14～16 h後處死小鼠.檢測外週血ALT、AST,進行肝髒Knodell評分.流式細胞分析術檢測脾髒CD4+T淋巴細胞活化比例及CD4+T淋巴細胞中調節性T細胞(Treg)、Th1、Th2和Th17比例變化情況,併計算Th17與Treg比值.ELISA檢測外週血TNF-α、IFN-γ及IL-4相關細胞因子的含量.應用獨立樣本t檢驗比較兩組正態分佈資料間的差異.結果 MSC治療組的ALT、AST及Knodell評分分彆為(174.2±46.9)U/L、(185.6±71.6) U/L、(3.4±1.3)分,均優于PBS治療組[(647.0±118.0)U/L、(749.0±104.0) U/L、(5.2±0.8)分],差異均有統計學意義(t=8.33、9.98、2.55,P均＜0.05).PBS治療組脾髒中CD4+T淋巴細胞活化比例為(26.10±2.17)％,CD4+T淋巴細胞中Th1、Th2細胞所佔比例為(5.81±0.79)％、(5.98±1.22)％,Th17與Treg比值為0.29±0.03,外週血中TNF-α、IFN-γ和IL-4水平分彆為[(1 281.95±88.61) U/L、(1 838.66±196.91) U/L、(1 192.36±163.94) U/L],較對照組的(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均顯著升高,差異均有統計學意義(t=10.26、12.37、7.02、5.30、31.79、15.93、20.75,P均＜0.01).MSC治療組與PBS治療組脾髒CD4+T淋巴細胞活化比例差異無統計學意義[(26.20±3.09)％比(26.10±2.17)％,P＞0.05],但MSC治療組CD4+T淋巴細胞中Th1、Th2細胞所佔比例[(1.83±0.52)％、(2.75±1.06)％],Th17與Treg比值(0.18±0.02)及其外週血中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]均較PBS治療組顯著降低,差異均有統計學意義(t=9.45、4.48、6.41、10.14、5.56、10.22,P均＜0.01).MSC治療組中Th17與Treg比值與對照組差異無統計學意義(P＞0.05).結論 MSC通過影響脾髒CD4+T淋巴細胞亞群降低Th1、Th2細胞比例,導緻外週血中後者分泌的細胞因子(TNF-α、IFN-γ和IL-4)水平下降;升高Treg比例,降低Th17比例,維持Th17與Treg平衡,進而起到治療ConA誘導的小鼠急性免疫性肝損傷的作用.
목적 탐구간충질간세포(MSC)치료도두단백A(ConA)유도적소서급성면역성간손상적궤제.방법 종3주령C57BL/6소서적사지치밀골편중체외분리배양MSC병감정기표면표기분자화성지성골분화능력.장15지6～7주령C57BL/6소서분위대조조、MSC치료조화PBS치료조,매조5지.MSC치료조경미정맥선후주사ConA화MSC,PBS치료조경미정맥선후주사ConA화PBS,대조조선후량차균경미정맥주사PBS.주사14～16 h후처사소서.검측외주혈ALT、AST,진행간장Knodell평분.류식세포분석술검측비장CD4+T림파세포활화비례급CD4+T림파세포중조절성T세포(Treg)、Th1、Th2화Th17비례변화정황,병계산Th17여Treg비치.ELISA검측외주혈TNF-α、IFN-γ급IL-4상관세포인자적함량.응용독립양본t검험비교량조정태분포자료간적차이.결과 MSC치료조적ALT、AST급Knodell평분분별위(174.2±46.9)U/L、(185.6±71.6) U/L、(3.4±1.3)분,균우우PBS치료조[(647.0±118.0)U/L、(749.0±104.0) U/L、(5.2±0.8)분],차이균유통계학의의(t=8.33、9.98、2.55,P균＜0.05).PBS치료조비장중CD4+T림파세포활화비례위(26.10±2.17)％,CD4+T림파세포중Th1、Th2세포소점비례위(5.81±0.79)％、(5.98±1.22)％,Th17여Treg비치위0.29±0.03,외주혈중TNF-α、IFN-γ화IL-4수평분별위[(1 281.95±88.61) U/L、(1 838.66±196.91) U/L、(1 192.36±163.94) U/L],교대조조적(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/L균현저승고,차이균유통계학의의(t=10.26、12.37、7.02、5.30、31.79、15.93、20.75,P균＜0.01).MSC치료조여PBS치료조비장CD4+T림파세포활화비례차이무통계학의의[(26.20±3.09)％비(26.10±2.17)％,P＞0.05],단MSC치료조CD4+T림파세포중Th1、Th2세포소점비례[(1.83±0.52)％、(2.75±1.06)％],Th17여Treg비치(0.18±0.02)급기외주혈중TNF-α、IFN-γ급IL-4수평[(760.71±73.19) U/L、(742.49±76.46) U/L、(825.76±101.74) U/L]균교PBS치료조현저강저,차이균유통계학의의(t=9.45、4.48、6.41、10.14、5.56、10.22,P균＜0.01).MSC치료조중Th17여Treg비치여대조조차이무통계학의의(P＞0.05).결론 MSC통과영향비장CD4+T림파세포아군강저Th1、Th2세포비례,도치외주혈중후자분비적세포인자(TNF-α、IFN-γ화IL-4)수평하강;승고Treg비례,강저Th17비례,유지Th17여Treg평형,진이기도치료ConA유도적소서급성면역성간손상적작용.
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.
