穿透角膜移植术后虹膜睫状体血管通透性变化
천투각막이식술후홍막첩상체혈관통투성변화
Diversity of vascular permeability in iris and ciliary body after penetrating keratoplasty
  • 万方数据
    中华实验眼科杂志 중화실험안과잡지
    CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
    2013年 7期 636-641 ,共6页

    刘明娜%庄宪丽%高华%李素霞%史伟云

    류명나%장헌려%고화%리소하%사위운

    穿透角膜移植%免疫排斥%虹膜睫状体%血管通透性 穿透角膜移植%免疫排斥%虹膜睫狀體%血管通透性
    천투각막이식%면역배척%홍막첩상체%혈관통투성
    Penetrating keratoplasty%Allograft rejection%Iris and ciliary body%Vascular permeability
    背景 穿透角膜移植术后发生排斥反应是手术失败的主要原因,尤其是存在高危排斥反应条件的患者.以往对角膜移植排斥反应发生机制的研究主要集中于角膜缘和角膜新生血管(CNV)方面,但目前发现,这些因素并不能解释所有角膜植片排斥现象.研究发现,虹膜睫状体组织内存在免疫细胞,但其与角膜植片排斥反应的关系尚不清楚. 目的 研究穿透角膜移植术后虹膜睫状体的血管变化情况. 方法 将8周龄清洁级BALB/c小鼠70只分为同种异体角膜移植组60只和空白对照组10只.角膜移植组以C57BL/6小鼠角膜作为供体,BALB/c小鼠作为受体,行穿透角膜移植术.术后每天裂隙灯显微镜下观察并记录角膜植片混浊情况,并按Hegde的标准进行评分.分别于术后5d、10 d和发生排斥时选取各组小鼠,处死后收集虹膜睫状体标本,分别应用免疫组织化学法和逆转录PCR(RT-PCR)法检测虹膜睫状体中跨膜蛋白occludin、胞质附着蛋白-1(ZO-1)、基质金属蛋白酶-9(MMP-9)、主要组织相容性复合体Ⅱ(MHCⅡ)、趋化因子受体(CCR5、CCR7)及其mRNA在各组小鼠虹膜睫状体组织中的表达,用Image J图像分析软件计算虹膜睫状体组织铺片中上述各因子阳性细胞的数量(个/视野)以及RT-PCR中目的基因的吸光度(A)值. 结果 角膜植片平均存活时间为(17±3)d,术后5d角膜植片评分平均为0.6,术后10d平均评分为0.5,术后18d平均评分为3.3.正常小鼠虹膜睫状体组织中可见大量ZO-1蛋白表达,排斥反应时可见ZO-1蛋白表达明显减少.正常小鼠虹膜睫状体组织中未见MMP-9的表达,角膜移植术后免疫排斥反应时可见大量MMP-9的表达,呈红色荧光.虹膜睫状体组织铺片免疫荧光染色显示,角膜移植术后免疫排斥反应时MHCⅡ+细胞数量较正常小鼠明显增加,分别为(4021.83±495.18)和(1559.67±350.29)个/视野,差异有统计学意义(P=0.000).与正常小鼠相比,角膜移植术后发生免疫排斥反应时ZO-1 mRNA、occludin mRNA表达均降低,A值分别为36.74±3.13 vs.110.11±11.88及57.54±3.41 vs.59.90±3.50,差异均有统计学意义(P＜0.05),而MMP-9 mRNA、CCR5 mRNA、CCR7 mRNA的表达显著增加,A值分别为20.29±1.19 vs.2.77±0.85、35.43±2.56 vs.9.11±0.29、60.83±0.87 vs.0.89±0.95,差异均有统计学意义(P＜0.05).结论 穿透角膜移植术后发生免疫排斥反应时虹膜睫状体中血管通透性明显增加,同时伴有大量抗原递呈细胞的聚集.
    배경 천투각막이식술후발생배척반응시수술실패적주요원인,우기시존재고위배척반응조건적환자.이왕대각막이식배척반응발생궤제적연구주요집중우각막연화각막신생혈관(CNV)방면,단목전발현,저사인소병불능해석소유각막식편배척현상.연구발현,홍막첩상체조직내존재면역세포,단기여각막식편배척반응적관계상불청초. 목적 연구천투각막이식술후홍막첩상체적혈관변화정황. 방법 장8주령청길급BALB/c소서70지분위동충이체각막이식조60지화공백대조조10지.각막이식조이C57BL/6소서각막작위공체,BALB/c소서작위수체,행천투각막이식술.술후매천렬극등현미경하관찰병기록각막식편혼탁정황,병안Hegde적표준진행평분.분별우술후5d、10 d화발생배척시선취각조소서,처사후수집홍막첩상체표본,분별응용면역조직화학법화역전록PCR(RT-PCR)법검측홍막첩상체중과막단백occludin、포질부착단백-1(ZO-1)、기질금속단백매-9(MMP-9)、주요조직상용성복합체Ⅱ(MHCⅡ)、추화인자수체(CCR5、CCR7)급기mRNA재각조소서홍막첩상체조직중적표체,용Image J도상분석연건계산홍막첩상체조직포편중상술각인자양성세포적수량(개/시야)이급RT-PCR중목적기인적흡광도(A)치. 결과 각막식편평균존활시간위(17±3)d,술후5d각막식편평분평균위0.6,술후10d평균평분위0.5,술후18d평균평분위3.3.정상소서홍막첩상체조직중가견대량ZO-1단백표체,배척반응시가견ZO-1단백표체명현감소.정상소서홍막첩상체조직중미견MMP-9적표체,각막이식술후면역배척반응시가견대량MMP-9적표체,정홍색형광.홍막첩상체조직포편면역형광염색현시,각막이식술후면역배척반응시MHCⅡ+세포수량교정상소서명현증가,분별위(4021.83±495.18)화(1559.67±350.29)개/시야,차이유통계학의의(P=0.000).여정상소서상비,각막이식술후발생면역배척반응시ZO-1 mRNA、occludin mRNA표체균강저,A치분별위36.74±3.13 vs.110.11±11.88급57.54±3.41 vs.59.90±3.50,차이균유통계학의의(P＜0.05),이MMP-9 mRNA、CCR5 mRNA、CCR7 mRNA적표체현저증가,A치분별위20.29±1.19 vs.2.77±0.85、35.43±2.56 vs.9.11±0.29、60.83±0.87 vs.0.89±0.95,차이균유통계학의의(P＜0.05).결론 천투각막이식술후발생면역배척반응시홍막첩상체중혈관통투성명현증가,동시반유대량항원체정세포적취집.
    Background Allograft rejection is a main cause of failure of penetrating keratoplasty,especially in the patient with high risk of rejection condition.Previous study on allograft rejection mechanism focused on limbal and corneal neovascularization,but these factors did not explain all the phenomena of allograft rejection.Research found that immune cells appeared in iris and ciliary body when rejection occurred,but the relationship between these immune cells and allograft rejection is unclear Objective This study was to evaluate the relationship between diversity of vascular permeability in the iris and ciliary body and allograft rejection after penetrating keratoplasty.Methods Seventy clean eight-week-old BALB/c mice were divided into allogeneic corneal transplantation group (60 mice) and blank control group (10 mice).Allogeneic corneal transplantation was performed with the same age of C57BL/6 mice as donor and BALB/c mice as the recipients.The grafts were examined under the slit lamp microscope and scored based on the criteria of Hegde.The mice were sacrificed and iris and ciliary tissue were obtained 5,10 days and rejection after surgery.Immunohistochemistry and reverse transcription PCR (RT-PCR) was used respectively to detect the expression diversities of occludin,zonula occludens protein-1 (ZO-1),matrix metalloproteinase-9(MMP-9),major histocompatibility complex-Ⅱ (MHC-Ⅱ),and CCR5,CCR7 and their mRNA in iris and ciliary body.Image-J image analysis software was used to calculate the quantity of positive cells on iris wholemount,and absorbance of target genes (A values).The use and care of the experimental animals complied the ARVO Resolution on the Use of Animals in Research.Results The mean survival time of corneal gratts was (17±3) days after operation.The mean score was 0.6 in 5 days and 0.5 in 10 days,and 3.3 in 18 days after operation.Expression of ZO-1 reduced significantly,and that of MMP-9 increased obviously at the time of rejection.MHC Ⅱ + cells were scattered in iris and ciliary body in normal mice,and the number of the positive cells (cells/field) was increased after operation with a peak value when rejection occurred.A significant difference was seen between normal mice and rejection mice (1559.67±350.29 vs.4021.83±495.18) (P=0.000).The expressions of occludin mRNA and ZO-1 mRNA in the iris and ciliary body decreased obviously in the rejection mice.Compared with normal mice,theA value of ZO-1 and occluding were 36.74±3.13 vs.110.11±11.88 and 57.54±3.41 vs.59.90±3.50respectively,with significant differences between them (all P＜0.05).The expressions of MMP-9 mRNA,CCR5 mRNA and CCR7 mRNA in the iris and ciliary body increased gradually with the time lapse after operation and peaked when the rejection appeared.The A value of MMP-9 mRNA,CCR5 mRNA and CCR7 mRNA were significantly higher than those of normal mice (20.29±1.19 vs.2.77±0.85 for MMP-9 mRNA; 35.43±2.56 vs.9.11±0.29 for CCR5 mRNA,and 60.83±0.87 vs.0.89 ±0.95 for CCR7 mRNA) respectively (all P＜0.05).Conclusions The permeability of vascules in the iris and ciliary body increase during the allograft rejection after penetrating keratoplasty.Increased antigen presenting cells were also detected.
    원문보기 원문다운로드 사이트로이동
저자의 최근 논문