中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2012年
3期
209-212
,共4页
自然杀伤T细胞%高危角膜移植%细胞治疗
自然殺傷T細胞%高危角膜移植%細胞治療
자연살상T세포%고위각막이식%세포치료
Natural killer T cell%High-risk corneal transplantation%Cell therapy
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100 mg/L的α-GalCer,在RPMI 1640培养基培养1周后,流式细胞仪分选出NKT细胞(密度为5×106个/ml).取10只Fisher 344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0.1 ml NKT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).结论 α-GalCer活化的NKT细胞球后注射可以明显延长大鼠高危角膜移植植片的存活时间,为角膜移植排斥反应的防治提供了新的手段.
揹景 角膜移植排斥反應是導緻角膜移植手術失敗的主要原因,抑製角膜移植排斥反應的各種藥物均有不良反應.研究髮現自然殺傷T(NKT)細胞可緻器官移植患者免疫耐受,但目前有關NKT細胞用于治療高危角膜移植免疫反應的研究較少. 目的 探討體外α-GalCer活化的NKT細胞在防治大鼠高危角膜移植免疫排斥反應中的作用. 方法 無菌條件下取Lewis大鼠脾髒淋巴細胞,加入質量濃度為100 mg/L的α-GalCer,在RPMI 1640培養基培養1週後,流式細胞儀分選齣NKT細胞(密度為5×106箇/ml).取10隻Fisher 344大鼠為供體,20隻Lewis大鼠為受體,受體角膜移植前1週角膜縫線誘導角膜新生血管(CNV).將受體大鼠按照隨機數字錶法隨機分為NKT細胞組和生理鹽水組,每組各10隻.Lewis大鼠行穿透角膜移植.NKT細胞組在手術結束時毬後註射0.1 ml NKT細胞液,生理鹽水組註射相同體積的生理鹽水.術後裂隙燈下觀察記錄角膜植片的反應情況併按照Holland的標準進行評分.術後第14天,兩組各穫取3隻大鼠角膜植片行組織病理學檢測,採用免疫組織化學法檢測角膜植片中CD4+和CD8+T淋巴細胞的浸潤情況.結果 生理鹽水組角膜植片平均存活時間為(7.90±1.37)d,NKT細胞組為(14.70±1.49)d,差異有統計學意義(t=10.61,P=0.00).術後2週,生理鹽水組角膜植片重度混濁水腫,大量炎性細胞浸潤,新生血管長入植片,而NKT細胞組角膜植片僅輕度混濁、水腫,炎性細胞明顯少于生理鹽水組.免疫組織化學檢測可見,生理鹽水組角膜植片中大量CD4+和CD8+T淋巴細胞浸潤,NKT細胞組中CD4+和CD8+T淋巴細胞明顯減少.流式細胞儀檢查結果錶明,NKT細胞組大鼠脾髒中NKT細胞百分數為(5.67±0.25)％,明顯高于生理鹽水組的(1.21±0.19)％,差異有統計學意義(t=8.43,P=0.00).結論 α-GalCer活化的NKT細胞毬後註射可以明顯延長大鼠高危角膜移植植片的存活時間,為角膜移植排斥反應的防治提供瞭新的手段.
배경 각막이식배척반응시도치각막이식수술실패적주요원인,억제각막이식배척반응적각충약물균유불량반응.연구발현자연살상T(NKT)세포가치기관이식환자면역내수,단목전유관NKT세포용우치료고위각막이식면역반응적연구교소. 목적 탐토체외α-GalCer활화적NKT세포재방치대서고위각막이식면역배척반응중적작용. 방법 무균조건하취Lewis대서비장림파세포,가입질량농도위100 mg/L적α-GalCer,재RPMI 1640배양기배양1주후,류식세포의분선출NKT세포(밀도위5×106개/ml).취10지Fisher 344대서위공체,20지Lewis대서위수체,수체각막이식전1주각막봉선유도각막신생혈관(CNV).장수체대서안조수궤수자표법수궤분위NKT세포조화생리염수조,매조각10지.Lewis대서행천투각막이식.NKT세포조재수술결속시구후주사0.1 ml NKT세포액,생리염수조주사상동체적적생리염수.술후렬극등하관찰기록각막식편적반응정황병안조Holland적표준진행평분.술후제14천,량조각획취3지대서각막식편행조직병이학검측,채용면역조직화학법검측각막식편중CD4+화CD8+T림파세포적침윤정황.결과 생리염수조각막식편평균존활시간위(7.90±1.37)d,NKT세포조위(14.70±1.49)d,차이유통계학의의(t=10.61,P=0.00).술후2주,생리염수조각막식편중도혼탁수종,대량염성세포침윤,신생혈관장입식편,이NKT세포조각막식편부경도혼탁、수종,염성세포명현소우생리염수조.면역조직화학검측가견,생리염수조각막식편중대량CD4+화CD8+T림파세포침윤,NKT세포조중CD4+화CD8+T림파세포명현감소.류식세포의검사결과표명,NKT세포조대서비장중NKT세포백분수위(5.67±0.25)％,명현고우생리염수조적(1.21±0.19)％,차이유통계학의의(t=8.43,P=0.00).결론 α-GalCer활화적NKT세포구후주사가이명현연장대서고위각막이식식편적존활시간,위각막이식배척반응적방치제공료신적수단.
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-activated NKT cella 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-α+ 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 histopathological 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,all the allografts showed the severe opacity with lots of new blood vessels and edema in normal saline solution group.However,the corneal grafts were clear in NKT cell group.Abundant CD4+ and CD8+T lymphocytes were seen in the allografts in normal saline solution group,but the inflammatory cells were obviously less in NKT cell group.The percentage of NKT cells in the spleen was (5.67±0.25)％ in NKT cell group and ( 1.21±0.19)％ in normal saline solution group ( t =8.43,P =0.00 ). Conclusions α-GalCer-activated NKT cells can prolong the survival time of allografts in high-risk corneal transplantation.Retro-bubar injection of α-GalCer-activated NKT cells probably is a new approach to the prevention of the rejection of corneal transplantation.
