自然杀伤T细胞结膜下注射防治大鼠角膜移植免疫排斥反应的初步研究  

The effects of conjunctival injection with NKT cells on inhibiting rat corneal transplantation rejection

作　　者：刘颖[1] 接英[1] 潘志强[1] 武宇影[1] 

机构地区：[1]首都医科大学附属北京同仁医院眼科中心 北京市眼科研究所,100730

出　　处：《眼科》2007年第3期175-178,共4页Ophthalmology in China

基　　金：国家自然科学基金(30271385和30471862);教育部新世纪人才计划资助

摘　　要：目的探讨自然杀伤T细胞（NKT）对大鼠角膜移植免疫排斥反应的防治作用。设计实验性研究。研究对象8只Fisher 344大鼠为供体，16只Lewis大鼠为受体。方法无菌取Lewis大鼠脾脏中淋巴细胞，RPMI1640培养基体外培养三周后，流式细胞仪分选出NKT细胞（浓度5×10^4／ml）。取8只Fisher 344大鼠为供体，16只Lewis大鼠为受体行穿透性角膜移植术。将受体大鼠随机分为治疗组和对照组，每组各8只。治疗组在手术结束时结膜下注射0．1ml上述浓度的NKT细胞，对照组注射相同体积生理盐水。术后观察记录植片的存活情况，术后第10天，每组2只大鼠取材，行组织病理学、免疫组织化学和流式细胞仪检测。主要指标角膜植片平均存活时间，组织病理学及免疫组织化学染色检查淋巴细胞浸润情况。结果对照组角膜植片平均存活时间为（8．00±1．58）天，NKT细胞治疗组为（26．00±1．34）天。组织病理学检查可见对照组大鼠角膜植片重度水肿，角膜基质纤维板层排列紊乱，大量炎性细胞浸润，新生血管长入植片。而治疗组植片仅表现为轻度水肿，少量炎性细胞浸润。免疫组织化学染色可见对照组植片中大量CD4＋和CD8＋淋巴细胞浸润，治疗组中仅见少量CD4＋和CD8＋淋巴细胞浸润。流式细胞仪检查结果表明在治疗组脾脏中NKT细胞为（3．90±0．32）％，明显高于对照组（1．85±0．21）％；外周血中治疗组NKT细胞为（1．34±0．12）％，对照组为（3．59±0．23）％。结论NKT细胞结膜下注射可延长角膜移植片存活时间，为防治角膜移植免疫排斥反应提供新的思路。Objective To explore the effects of conjunctival injection with NKT cells on inhibiting corneal transplantation immune rejection in rat model. Design Experimental study. Participants Fisher344 rats were used as donors and Lewis rats as recipients. Methods The lymphocytes were taken from the spleen of the Lewis rats and cultured in RPMI1640 medium. After three weeks, NKT cells were sorted by the FACSVantage system as CD161 ＋ TCR-α＋ cell from the lymphocytes： For corneal transplantation, Fisher344 rats were used as donors and Lewis rats as recipients. The recipient rats were divided into two groups. One was conjunctival injected with 0.1ml NKT cells at concentration of 5×10^4/ml at the end of keratoplasty. The group control received saline buffer of the same volume. All aUografts were observed and scored for thirty days. Ten days after surgery, two rats in each group were sacrificed for histopathological and immunological evaluation. Main Outcome Measures Mean survival time of the allografts, histopathological and immunological evaluation on lymphocytes. Results The mean survival time of the allografts was 8.00±1.58 d in the group control while 26.00±1.34 d in the group treatment. Ten days after surgery, all the allografts were rejected in the group control with severe opacity and edema. But in the group treatment, almost all the allografts were not rejected, but with slight opacity and edema. There were abundant CD4＋ and CD8＋ lymphocytes infiltration in the allografts of the group control, but significant reduction in the group treatment. The percentage of NKT cells in the spleen was （3.90±0.32）% in the experiment group, while（ 1.85±0.21 ）% in the control group. The percentage of NKT cells in the blood was（1.34±0.12）% in the experiment group, while （3.59±0.23）% in the control group. Conclusions Conjunctival injection with NKT cells could prolong the allografts survival in corneal transplantation, which suggested a new therapy to treat high-risk corneal transplantation.

关 键 词：自然杀伤T细胞 角膜移植 免疫耐受 

分 类 号：R779.65[医药卫生—眼科]