中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2011年
11期
1010-1015
,共6页
卢建民%王会芳%李小磊%连玲艳%宋秀君
盧建民%王會芳%李小磊%連玲豔%宋秀君
로건민%왕회방%리소뢰%련령염%송수군
角膜基质细胞%树突状细胞/成熟%转化生长因子β2%前列腺素E2
角膜基質細胞%樹突狀細胞/成熟%轉化生長因子β2%前列腺素E2
각막기질세포%수돌상세포/성숙%전화생장인자β2%전렬선소E2
Corneal stroma cell%Dendritic cell/maturation%Transforming growth factor beta 2%Prostaglandin E2
背景 研究表明,位于角膜中央区的树突状细胞(DCs)完全处于未成熟状态,而位于角膜周边区的DCs则大多处于成熟状态.角膜内的DCs广泛参与多种角膜相关疾病以及角膜移植免疫排斥反应,研究角膜内DCs的成熟状态具有重要意义.目的 探讨小鼠角膜基质细胞(CSCs)是否通过分泌转化生长因子β2(TGF-β2)以及前列腺素E2( PGE2)抑制DCs的成熟.方法 获取DCs、T细胞以及CSCs培养上清液.通过酶联免疫吸附实验( ELISA)测定CSCs培养上清液以及新鲜RPM1 1640培养基内PGE2和TGF-β2的质量浓度.在DCs成熟过程中,应用TGF-β2中和抗体以及PGE2受体阻滞剂AH6809,并按照处理方式的不同分为对照组、CSCs培养上清液组、AH6809组、TGF-β2中和抗体组、AH6809 +TGF-β2中和抗体组.采用流式细胞技术检测DCs细胞表型CD11c、CD80、CD86和MHC-Ⅱ的表达情况,通过葡聚糖内吞实验检测抗原吞噬功能,并通过混合淋巴细胞反应检测刺激T细胞增生的能力.结果ELISA检测结果显示,与新鲜RPMI 1640培养基相比,CSCs培养上清液内含有较高质量浓度的TGF-β2和PGE2.与CSCs培养上清液组比较,TGF-β2中和抗体组DCs CD80、CD86和MHC-Ⅱ的表达均升高,差异均有统计学意义(P＜0.05),葡聚糖的表达降低(P＜0.05),刺激指数(SI)增大(P＜0.05);AH6809组CD86和MHC-Ⅱ的表达均升高,葡聚糖的表达降低,SI增大,差异均有统计学意义(P＜0.05)；TGF-β2中和抗体+AH6809组DCs MHC-Ⅱ的表达和SI提高,差异均有统计学意义(P＜0.05).与对照组比较,TGF-β2中和抗体+AH6809组DCs CD80、CD86的表达和SI均较低,差异均有统计学意义(P＜0.05).结论体外培养的小鼠CSCs可以通过分泌TGF-β2及PGE2抑制DCs成熟,且这两种细胞因子可发挥叠加效应.
揹景 研究錶明,位于角膜中央區的樹突狀細胞(DCs)完全處于未成熟狀態,而位于角膜週邊區的DCs則大多處于成熟狀態.角膜內的DCs廣汎參與多種角膜相關疾病以及角膜移植免疫排斥反應,研究角膜內DCs的成熟狀態具有重要意義.目的 探討小鼠角膜基質細胞(CSCs)是否通過分泌轉化生長因子β2(TGF-β2)以及前列腺素E2( PGE2)抑製DCs的成熟.方法 穫取DCs、T細胞以及CSCs培養上清液.通過酶聯免疫吸附實驗( ELISA)測定CSCs培養上清液以及新鮮RPM1 1640培養基內PGE2和TGF-β2的質量濃度.在DCs成熟過程中,應用TGF-β2中和抗體以及PGE2受體阻滯劑AH6809,併按照處理方式的不同分為對照組、CSCs培養上清液組、AH6809組、TGF-β2中和抗體組、AH6809 +TGF-β2中和抗體組.採用流式細胞技術檢測DCs細胞錶型CD11c、CD80、CD86和MHC-Ⅱ的錶達情況,通過葡聚糖內吞實驗檢測抗原吞噬功能,併通過混閤淋巴細胞反應檢測刺激T細胞增生的能力.結果ELISA檢測結果顯示,與新鮮RPMI 1640培養基相比,CSCs培養上清液內含有較高質量濃度的TGF-β2和PGE2.與CSCs培養上清液組比較,TGF-β2中和抗體組DCs CD80、CD86和MHC-Ⅱ的錶達均升高,差異均有統計學意義(P＜0.05),葡聚糖的錶達降低(P＜0.05),刺激指數(SI)增大(P＜0.05);AH6809組CD86和MHC-Ⅱ的錶達均升高,葡聚糖的錶達降低,SI增大,差異均有統計學意義(P＜0.05)；TGF-β2中和抗體+AH6809組DCs MHC-Ⅱ的錶達和SI提高,差異均有統計學意義(P＜0.05).與對照組比較,TGF-β2中和抗體+AH6809組DCs CD80、CD86的錶達和SI均較低,差異均有統計學意義(P＜0.05).結論體外培養的小鼠CSCs可以通過分泌TGF-β2及PGE2抑製DCs成熟,且這兩種細胞因子可髮揮疊加效應.
배경 연구표명,위우각막중앙구적수돌상세포(DCs)완전처우미성숙상태,이위우각막주변구적DCs칙대다처우성숙상태.각막내적DCs엄범삼여다충각막상관질병이급각막이식면역배척반응,연구각막내DCs적성숙상태구유중요의의.목적 탐토소서각막기질세포(CSCs)시부통과분비전화생장인자β2(TGF-β2)이급전렬선소E2( PGE2)억제DCs적성숙.방법 획취DCs、T세포이급CSCs배양상청액.통과매련면역흡부실험( ELISA)측정CSCs배양상청액이급신선RPM1 1640배양기내PGE2화TGF-β2적질량농도.재DCs성숙과정중,응용TGF-β2중화항체이급PGE2수체조체제AH6809,병안조처리방식적불동분위대조조、CSCs배양상청액조、AH6809조、TGF-β2중화항체조、AH6809 +TGF-β2중화항체조.채용류식세포기술검측DCs세포표형CD11c、CD80、CD86화MHC-Ⅱ적표체정황,통과포취당내탄실험검측항원탄서공능,병통과혼합림파세포반응검측자격T세포증생적능력.결과ELISA검측결과현시,여신선RPMI 1640배양기상비,CSCs배양상청액내함유교고질량농도적TGF-β2화PGE2.여CSCs배양상청액조비교,TGF-β2중화항체조DCs CD80、CD86화MHC-Ⅱ적표체균승고,차이균유통계학의의(P＜0.05),포취당적표체강저(P＜0.05),자격지수(SI)증대(P＜0.05);AH6809조CD86화MHC-Ⅱ적표체균승고,포취당적표체강저,SI증대,차이균유통계학의의(P＜0.05)；TGF-β2중화항체+AH6809조DCs MHC-Ⅱ적표체화SI제고,차이균유통계학의의(P＜0.05).여대조조비교,TGF-β2중화항체+AH6809조DCs CD80、CD86적표체화SI균교저,차이균유통계학의의(P＜0.05).결론체외배양적소서CSCs가이통과분비TGF-β2급PGE2억제DCs성숙,차저량충세포인자가발휘첩가효응.
Background Researches demonstrated that dendritic cells(DCs) are uniformly immature in the central cornea but mature in the peripheral region of cornea.So an important question is which factor impact the maturation of DCs,especially in terms of corneal transplant rejection and the known roles of DCs in the development and persistence of some corneal diseases.Objective This study aimed to examine whether corneal stroma cells (CSCs) inhibit DCs maturation through secreting transforming growth factor beta 2 (TGF-β2) and prostaglandin E2 (PGE2).Methods DCs,T cells and CSCs were isolated and cultured from clean BALB/c and C57BL/6 mice.The level of PGE2 and TGF-β2in CSCs culture supernatant and the fresh RPMI 1640 medium were then analyzed by enzyme linked immunosorbent assay (ELISA).During the DCs maturation stage,the neutralizing TGF-β2 antibody and the EP2 receptor antagonist AH6809 were added in the CSCs culture supernatant respectively.According to the different treatment,cultured cells were assigned to different groups as follows:control group,CSCs culture supernatant group,AH6809 group,TGF-β2 antibody group,AH6809 +TGF-β2 antibody group.Subsequently,the cellular surface markers for DCs,including CD11c,CD80,CD86,and MHC- Ⅱ,were analyzed by flow cytometry.The capability of stimulating the proliferation of T lymphocytes was evaluated by allogeneic mixed lymphocyte reactions,and the function of endocytosis was assessed by fluorescein isothiocyanate-dextran(FITC) uptake.Results The data of ELISA showed a higher concentration of TGF-β2 and PGE2 in murine CSCs culture supernatant than in the fresh RPMI 1640 medium.Compared with the CSCs culture supernatant group,the expression of CD80,CD86,and MHC- Ⅱ was up-regulated ( P ＜ 0.05 ),the expression of dextran was down-regulated ( P ＜ 0.05 ),and the stimulate index was increased( P＜ 0.05 ) in the TGF-β2 antibody group; the expression of CD86,and MHC-Ⅱ was up-regulated (P＜0.05),the expression of dextran was down-regulated ( F =13.740,P =0.006 ),and the stimulate index was increased(P＜0.05) in the AH6809 group;the expression of MHC-Ⅱ was up-regulated and the stimulate index was increased with statistical difference in interaction(P＜0.05 ) in the AH6809+TGF-β2 antibody group.Compared with the control group,the expression of CD80 and CD86,and the stimulate index was still lower(P＜0.05 ).Conclusions TGF-β2 and PGE2 contribute to the inhibitory effects on DCs maturation mediated by murine CSCs in vitro and further have additive effect on the immunosuppression of DCs.
