中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
Chinese Journal of Experimental Ophthalmology
2015年
10期
870-875
,共6页
白伶伶%张灵君%郑慧%王梅艳%东莉洁%李筱荣%张晓敏
白伶伶%張靈君%鄭慧%王梅豔%東莉潔%李篠榮%張曉敏
백령령%장령군%정혜%왕매염%동리길%리소영%장효민
间充质干细胞%自身免疫性疾病/化学方法诱导%葡萄膜炎/预防和控制%辅助性T细胞1/免疫%辅助性T细胞17/免疫%调节性T淋巴细胞/免疫%抗原递呈细胞%动物模型
間充質榦細胞%自身免疫性疾病/化學方法誘導%葡萄膜炎/預防和控製%輔助性T細胞1/免疫%輔助性T細胞17/免疫%調節性T淋巴細胞/免疫%抗原遞呈細胞%動物模型
간충질간세포%자신면역성질병/화학방법유도%포도막염/예방화공제%보조성T세포1/면역%보조성T세포17/면역%조절성T림파세포/면역%항원체정세포%동물모형
Mesenchymal stem cells%Autoimmune diseases/chemically induced%Uveitis/prevention & control%T helper cell 1/immunology%T helper cell 17/immunology%T-lymphocytes,regulatory/immunology%Antigen presenting cells%Disease models,animal
背景 我们前期研究发现,间充质干细胞(MSCs)可以有效治疗大鼠实验性自身免疫性葡萄膜炎(EAU),减轻组织损害,但其具体作用机制仍在研究中.目的 研究MSCs对大鼠EAU模型中T细胞亚群和抗原递呈细胞(APCs)的影响.方法 收集6只清洁级4～6周龄Wistar雄性大鼠双侧股骨、胫骨骨髓,采用贴壁培养法纯化Wistar大鼠骨髓MSCs.采用随机数字表法将12只清洁级Lewis雌性大鼠分为MSCs组和PBS组,每组6只.于Lewis大鼠单后足及背部皮下注射200μl含30μg光感受器间维生素A类结合蛋白(IRBP) 1177-1191多肽片段R16及完全弗氏佐剂(CFA)的乳化液以建立EAU模型,造模后于裂隙灯显微镜下观察大鼠眼部炎症表现.造模后9～11d,MSCs组大鼠每日经尾静脉注射密度为5×106/ml的MSCs悬液1 ml;PBS组大鼠以同样的方法注射等容积的PBS.造模后15d,分离各组大鼠脾脏和引流淋巴结中的T细胞及APCs,采用流式细胞仪检测各组大鼠脾脏和引流淋巴结中γ干扰素(IFN-γ)阳性CD4+T细胞、白细胞介素-17(IL-17)阳性CD4+T细胞和叉头状螺旋转录因子p3(Foxp3)阳性CD4+T细胞的比例,以评估辅助性T细胞1(Th1)、Th17和调节性T细胞(Treg)细胞亚群的作用;依据各组T细胞与APCs共培养的方式不同分为PBS共培养组、PBS-MSCs交叉培养组、MSCs-PBS培养组和MSCs共培养组,分别加入不同质量浓度(0.3、1.0、10.0 μg/ml)的R16抗原进行刺激,无R16抗原刺激的细胞作为空白对照,采用5-溴脱氧尿嘧啶核苷(BrdU)法测定各组大鼠T细胞吸光度(A)值,计算T细胞增生指数.结果 造模后11、12、13和14d,MSCs组大鼠眼前节炎症评分均明显低于PBS组,差异均有统计学意义(t=3.825、5.100、4.250、3.400,均P＜0.05).与PBS组大鼠比较,MSCs组大鼠脾脏和淋巴结中IFN-γ+ CD4+T细胞比例均明显下降,差异均有统计学意义(t=5.651、4.376,均P＜0.05);MSCs组大鼠脾脏和引流淋巴结中IL-17+CD4+T细胞比例均明显下降,差异均有统计学意义(t=3.300、4.925,均P＜0.05),Foxp3+ CD4+T细胞的比例均明显升高,差异均有统计学意义(t=-5.172、-2.825,均P＜0.05).PBS共培养组大鼠脾脏T细胞增生指数随着R16抗原质量浓度的增加而升高,在各自质量浓度(0.3、1.0和10.0 μg/ml)R16刺激条件下,MSCs共培养组T细胞增生指数较PBS共培养组明显下降,差异均有统计学意义(P=0.027、0.000、0.000);在R16质量浓度为1.0 μg/ml和10.0 μg/ml条件下,MSCs-PBS交叉培养组和PBS-MSCs交叉培养组T细胞增生指数均明显低于PBS共培养组,差异均有统计学意义(1.0μg/ml R16:P=0.001、0.000;10.0μg/ml R16:P=0.000、0.000).结论 MSCs可通过同时抑制EAU大鼠体内抗原特异性T细胞和APCs的功能以及上调Treg细胞比例来发挥对大鼠EAU的治疗作用.
揹景 我們前期研究髮現,間充質榦細胞(MSCs)可以有效治療大鼠實驗性自身免疫性葡萄膜炎(EAU),減輕組織損害,但其具體作用機製仍在研究中.目的 研究MSCs對大鼠EAU模型中T細胞亞群和抗原遞呈細胞(APCs)的影響.方法 收集6隻清潔級4～6週齡Wistar雄性大鼠雙側股骨、脛骨骨髓,採用貼壁培養法純化Wistar大鼠骨髓MSCs.採用隨機數字錶法將12隻清潔級Lewis雌性大鼠分為MSCs組和PBS組,每組6隻.于Lewis大鼠單後足及揹部皮下註射200μl含30μg光感受器間維生素A類結閤蛋白(IRBP) 1177-1191多肽片段R16及完全弗氏佐劑(CFA)的乳化液以建立EAU模型,造模後于裂隙燈顯微鏡下觀察大鼠眼部炎癥錶現.造模後9～11d,MSCs組大鼠每日經尾靜脈註射密度為5×106/ml的MSCs懸液1 ml;PBS組大鼠以同樣的方法註射等容積的PBS.造模後15d,分離各組大鼠脾髒和引流淋巴結中的T細胞及APCs,採用流式細胞儀檢測各組大鼠脾髒和引流淋巴結中γ榦擾素(IFN-γ)暘性CD4+T細胞、白細胞介素-17(IL-17)暘性CD4+T細胞和扠頭狀螺鏇轉錄因子p3(Foxp3)暘性CD4+T細胞的比例,以評估輔助性T細胞1(Th1)、Th17和調節性T細胞(Treg)細胞亞群的作用;依據各組T細胞與APCs共培養的方式不同分為PBS共培養組、PBS-MSCs交扠培養組、MSCs-PBS培養組和MSCs共培養組,分彆加入不同質量濃度(0.3、1.0、10.0 μg/ml)的R16抗原進行刺激,無R16抗原刺激的細胞作為空白對照,採用5-溴脫氧尿嘧啶覈苷(BrdU)法測定各組大鼠T細胞吸光度(A)值,計算T細胞增生指數.結果 造模後11、12、13和14d,MSCs組大鼠眼前節炎癥評分均明顯低于PBS組,差異均有統計學意義(t=3.825、5.100、4.250、3.400,均P＜0.05).與PBS組大鼠比較,MSCs組大鼠脾髒和淋巴結中IFN-γ+ CD4+T細胞比例均明顯下降,差異均有統計學意義(t=5.651、4.376,均P＜0.05);MSCs組大鼠脾髒和引流淋巴結中IL-17+CD4+T細胞比例均明顯下降,差異均有統計學意義(t=3.300、4.925,均P＜0.05),Foxp3+ CD4+T細胞的比例均明顯升高,差異均有統計學意義(t=-5.172、-2.825,均P＜0.05).PBS共培養組大鼠脾髒T細胞增生指數隨著R16抗原質量濃度的增加而升高,在各自質量濃度(0.3、1.0和10.0 μg/ml)R16刺激條件下,MSCs共培養組T細胞增生指數較PBS共培養組明顯下降,差異均有統計學意義(P=0.027、0.000、0.000);在R16質量濃度為1.0 μg/ml和10.0 μg/ml條件下,MSCs-PBS交扠培養組和PBS-MSCs交扠培養組T細胞增生指數均明顯低于PBS共培養組,差異均有統計學意義(1.0μg/ml R16:P=0.001、0.000;10.0μg/ml R16:P=0.000、0.000).結論 MSCs可通過同時抑製EAU大鼠體內抗原特異性T細胞和APCs的功能以及上調Treg細胞比例來髮揮對大鼠EAU的治療作用.
배경 아문전기연구발현,간충질간세포(MSCs)가이유효치료대서실험성자신면역성포도막염(EAU),감경조직손해,단기구체작용궤제잉재연구중.목적 연구MSCs대대서EAU모형중T세포아군화항원체정세포(APCs)적영향.방법 수집6지청길급4～6주령Wistar웅성대서쌍측고골、경골골수,채용첩벽배양법순화Wistar대서골수MSCs.채용수궤수자표법장12지청길급Lewis자성대서분위MSCs조화PBS조,매조6지.우Lewis대서단후족급배부피하주사200μl함30μg광감수기간유생소A류결합단백(IRBP) 1177-1191다태편단R16급완전불씨좌제(CFA)적유화액이건립EAU모형,조모후우렬극등현미경하관찰대서안부염증표현.조모후9～11d,MSCs조대서매일경미정맥주사밀도위5×106/ml적MSCs현액1 ml;PBS조대서이동양적방법주사등용적적PBS.조모후15d,분리각조대서비장화인류림파결중적T세포급APCs,채용류식세포의검측각조대서비장화인류림파결중γ간우소(IFN-γ)양성CD4+T세포、백세포개소-17(IL-17)양성CD4+T세포화차두상라선전록인자p3(Foxp3)양성CD4+T세포적비례,이평고보조성T세포1(Th1)、Th17화조절성T세포(Treg)세포아군적작용;의거각조T세포여APCs공배양적방식불동분위PBS공배양조、PBS-MSCs교차배양조、MSCs-PBS배양조화MSCs공배양조,분별가입불동질량농도(0.3、1.0、10.0 μg/ml)적R16항원진행자격,무R16항원자격적세포작위공백대조,채용5-추탈양뇨밀정핵감(BrdU)법측정각조대서T세포흡광도(A)치,계산T세포증생지수.결과 조모후11、12、13화14d,MSCs조대서안전절염증평분균명현저우PBS조,차이균유통계학의의(t=3.825、5.100、4.250、3.400,균P＜0.05).여PBS조대서비교,MSCs조대서비장화림파결중IFN-γ+ CD4+T세포비례균명현하강,차이균유통계학의의(t=5.651、4.376,균P＜0.05);MSCs조대서비장화인류림파결중IL-17+CD4+T세포비례균명현하강,차이균유통계학의의(t=3.300、4.925,균P＜0.05),Foxp3+ CD4+T세포적비례균명현승고,차이균유통계학의의(t=-5.172、-2.825,균P＜0.05).PBS공배양조대서비장T세포증생지수수착R16항원질량농도적증가이승고,재각자질량농도(0.3、1.0화10.0 μg/ml)R16자격조건하,MSCs공배양조T세포증생지수교PBS공배양조명현하강,차이균유통계학의의(P=0.027、0.000、0.000);재R16질량농도위1.0 μg/ml화10.0 μg/ml조건하,MSCs-PBS교차배양조화PBS-MSCs교차배양조T세포증생지수균명현저우PBS공배양조,차이균유통계학의의(1.0μg/ml R16:P=0.001、0.000;10.0μg/ml R16:P=0.000、0.000).결론 MSCs가통과동시억제EAU대서체내항원특이성T세포화APCs적공능이급상조Treg세포비례래발휘대대서EAU적치료작용.
Background Our previous studies found that mesenchymal stem cells (MSCs) can ameliorate experimental autoimmune uveitis (EAU) and reduce tissue impairment.Its mechanism is still pending.Objective This study was performed to investigate the effects of MSCs on T cell subsets and antigen presenting cells (APCs) in EAU rats.Methods MSCs were isolated from bone marrow of six male Wistar rats and cultured by plastic adherence method.Twelve female Lewis rats were assigned randomly into MSCs group and PBS group.EAU rat model was induced by immunization with 200 μl emulsion containing 30 μg interphotoreceptor retinoid-binding protein (IRBP) 1177-1191 polypeptide fragment R16 and complete Freund adjuvant (CFA).The eye manifestations of the rats were observed and scored under the slit lamp microscope after modeling.The R16-immunized rats were treated intravenously with 5×106/ml MSCs for 3 consecutive days from day 9 to 11 after modeling in the MSCs group,and the equivalent volume of PBS was used with the same way in the PBS group.Fifteen days after modeling,the spleens and draining lymph nodes were collected to evaluate the proportion of interferon-γ (IFN-γ) positive CD4+ T cells,interleukin-17 (IL-17)positive CD4+ T cells and forkhead helix transcription factor p3 (Foxp3) positive CD4+ T cells by flow cytometry.The T cells and APCs from the different groups were cocultured and divided into PBS cocultured group,MSCs cocultured group, PBS-MSCs cross-cultured group and MSCs-PBS cross-cultured group under the stimulation of R16 at the concentration of 0.3,1.0 or 10.0 μg/ml, and the proliferation indexes of the T cells in different groups were assayed by 5-bromodeoxyuridine (BrdU) Elisa kit.The use of experimental animals complied with the regulations on the management of experimental animals promulgated by the national science and technology commission.Results The ocular surface inflammatory scores of 11,12,13 and 14 days after modeling in the MSCs group were significantly lower than that in the PBS group (t=3.825,5.100,4.250,3.400, all at P＜0.05).Compared with the PBS group, the proportions of IFN-γ positive CD4+ T cells in spleen and draining lymph notes were considerably decreased in the MSCs group (t =5.651,4.376, both at P＜0.05) , so were the IL-17+ CD4+ T cells (t =3.300,4.925, both at P＜0.05).However,the proportions of Foxp3 + CD4+ T cells in spleen and draining lymph notes were statistically raised in the MSCs group compared with the PBS group (t =-5.172,-2.825,both at P＜0.05).The proliferation index of T cells increased with the rise of R16 dose in the PBS cocultured group, and the proliferation indexes were all declined in the MSCs cocultured group compared with the PBS cocultured group under the stimulation of 0.3,1.0 and 10.0 μg/ml of R16 (P =0.027,0.000,0.000).In addition, significant reduces of proliferation indexes of T cells were seen in the PBS-MSCs cross-cultured group and MSCs-PBS cross-cultured group in comparison with the PBS cocultured group when stimulated by 1.0 μg/ml and 10.0 μg/ml R16 (1.0 μg/ml R16 : P =0.001,0.000;10.0 μg/ml R16:P=0.000,0.000).Conclusions MSCs can ameliorate EAU by inhibiting the functions of antigen-specific T cells and APCs and up-regulating T regulatory cells in EAU rats.
