α-GalCer活化的自然杀伤T细胞对高危角膜移植后排斥反应的防治作用  

Effect ofα-GalCer-activated natural killer T cell on survival of allograft with high-risk rejection after retro- bubar injection

作　　者：宫妍 宋丽艳 孙海成 

机构地区：[1]莱阳中心医院眼科,烟台265200

出　　处：《中华实验眼科杂志》2012年第3期209-212,共4页Chinese Journal Of Experimental Ophthalmology

摘　　要：背景角膜移植排斥反应是导致角膜移植手术失败的主要原因，抑制角膜移植排斥反应的各种药物均有不良反应。研究发现自然杀伤T（NKT）细胞可致器官移植患者免疫耐受，但目前有关NKT细胞用于治疗高危角膜移植免疫反应的研究较少。目的探讨体外α-GalCer活化的NKT细胞在防治大鼠高危角膜移植免疫排斥反应中的作用。方法无菌条件下取Lewis大鼠脾脏淋巴细胞，加入质量浓度为100mg／L的α-GalCer，在RPMI1640培养基培养1周后，流式细胞仪分选出NKT细胞（密度为5×10^6个／m1）。取10只Fisher344大鼠为供体，20只Lewis大鼠为受体，受体角膜移植前1周角膜缝线诱导角膜新生血管（CNV）。将受体大鼠按照随机数字表法随机分为NKT细胞组和生理盐水组，每组各10只。Lewis大鼠行穿透角膜移植。NKT细胞组在手术结束时球后注射0．1mlNKT细胞液，生理盐水组注射相同体积的生理盐水。术后裂隙灯下观察记录角膜植片的反应情况并按照Holland的标准进行评分。术后第14天，两组各获取3只大鼠角膜植片行组织病理学检测，采用免疫组织化学法检测角膜植片中CD4＋和CD8＋T淋巴细胞的浸润情况。结果生理盐水组角膜植片平均存活时间为（7．90±1．37）d，NKT细胞组为（14．70±1．49）d，差异有统计学意义（t=10．61，P=0．00）。术后2周，生理盐水组角膜植片重度混浊水肿，大量炎性细胞浸润，新生血管长入植片，而NKT细胞组角膜植片仅轻度混浊、水肿，炎性细胞明显少于生理盐水组。免疫组织化学检测可见，生理盐水组角膜植片中大量CD4＋和CD8＋T淋巴细胞浸润，NKT细胞组中CD4＋和CD8＋T淋巴细胞明显减少。流式细胞仪检查结果表明，NKT细胞组大鼠脾脏中NKT细胞百分数为（5．67±0．25）％，明显高于生理盐水组的（1．21±0．19）％，差异有统计学意义（t=8．43，P=0．00）。�Background Corneal graft reject is a major cause of corneal transplantation failure. Although many immune-suppressing drugs have been utilized to reduce the reject response, their adverse effects on organ and tissue are still insoluble. The tolerance induction of natural killer T （NKT） cells is currently under investigation. However,the study on the application of NKT cells in high risk corneal transplantation is seldom. Objective The present study was to explore the effects of α-GalCer-aetivated NKT cells on allografts survival after high-risk corneal transplantation surgery via retro-bubar injection. Methods The lymphocytes were picked up from the spleen of SPF Lewis rats and cultured in RPMI 1640 medium with 100 mg/L α--GalCer. After one week,NKT cells were sorted by the FACSVantage system as CD161 ± TCR-cC cell from the lymphocytes with the cell densities 5×106/ml. Ten SPF Fisher344 rats were used to prepare the donor corneas, and 20 Lewis rats served as recipients. The high risk corneal transplantation models were created by corneal suturing in 20 recipient rats. Penetrating keratoplasty （PKP） was performed in the model rats. 0. 1 ml NKT cells or the same volume of normal saline solution were retro-bubarly injected at the end of surgery respectively. The corneal allografts were observed and scored based on Holland criteria at the three-day interval under the slit lamp for 30 days. Two weeks after surgery, three rats from each group were sacrificed by excessive anesthesia method and the eyeballs were obtained for histopathologieal examination. The inflammatory cell infiltration （ CD4~ and CD8~ ） in grafts was evaluated by immunochemistry and flow cytometry. The use of the animals complied with the Statement of ARVO. Results The mean survival time of the allografts was （7.90± 1.37） days in normal saline solution group and （14.70 ± 1.49） days in NKT cell group, showing a statistically significant difference between the two groups （ t = 10. 61, P = 0.00 ）. Two weeks after surgery, a

关 键 词：自然杀伤T细胞 高危角膜移植 细胞治疗 

分 类 号：R779.65[医药卫生—眼科]