角膜%角膜移植%共刺激分子/Tim-1%免疫反应,排斥
角膜%角膜移植%共刺激分子/Tim-1%免疫反應,排斥
각막%각막이식%공자격분자/Tim-1%면역반응,배척
Cornea%Keratoplasty%Costimulatory molecule/Tim-1%Immune reaction,reject
背景 角膜移植是目前临床上治疗角膜盲可靠且有效的复明手段,角膜移植术后的免疫排斥反应是角膜移植失败的主要原因. 目的 研究共刺激分子Tim-1在大鼠角膜组织中的表达及其在角膜移植排斥反应发生和发展过程中的作用.方法 40只清洁级成年雌性Wistar大鼠随机分为正常对照组、自体角膜移植组和同种异体角膜移植组.正常对照组大鼠未行任何手术,自体角膜移植组分别以Wistar大鼠作为供体和受体施行穿透角膜移植术,而同种异体角膜移植组以SD大鼠角膜作为供体,Wistar大鼠作为受体行穿透角膜移植术,各组供体角膜植片均为3.5mm,植床直径为3.0 mm.分别于术后7d、14d在裂隙灯显微镜下观察术眼角膜炎症反应情况,按照Larkin的标准进行排斥反应评分,计算排斥反应指数(RI),观察各组角膜植片的平均存活时间和植片存活率.术后7d、14d,3个组各取3只大鼠眼球制备角膜组织切片,分别行组织病理学检查和免疫组织化学检测,观察角膜组织的炎症反应和共刺激分子Tim-1蛋白在角膜组织中的表达;此外分别于术后7d、14d各组取5只大鼠角膜制备组织匀浆,采用实时荧光定量PCR法检测共刺激分子Tim-1 mRNA在大鼠角膜植片中表达量(吸光度,A)的变化.结果 术后7d,自体角膜移植组和同种异体角膜移植组大鼠角膜植片均出现轻度水肿;术后14 d,自体角膜移植组角膜植片水肿消失,植片透明,而同种异体角膜移植组大鼠角膜植片水肿增厚,呈灰白色混浊,可见新生血管形成.自体角膜移植组植片的存活率为100％,同种异体角膜移植组植片存活率为0,平均生存时间为(9.8±1.2)d.组织病理学检查表明,术后7d自体角膜移植组植片基质层可见炎性细胞浸润,但术后14d炎性细胞明显减少;同种异体角膜移植组术后7d植片水肿,基质层可见炎性细胞浸润,术后14 d阳性细胞大量增加,可见新生血管.免疫组织化学法检测表明,正常对照组Wistar大鼠角膜上皮层和内皮层可见少量Tim-1蛋白阳性反应细胞,术后7d自体角膜移植组和同种异体角膜移植组的角膜植片中Tim-1蛋白阳性染色细胞较正常对照组明显增加,术后14d,自体角膜移植组Tim-1蛋白阳性细胞较7d明显减少,而在同种异体角膜移植组中仍然呈高表达.术后7d,正常对照组、自体角膜移植组和同种异体角膜移植组大鼠角膜中Tim-1 mRNA表达量分别为1.24±0.03、5.85±0.08和6.54±0.20,术后14 d自体角膜移植组Tim-1 mRNA表达量下降至1.54±0.10,而同种异体角膜移植组升高至8.62±0.24,各组在不同时间点大鼠角膜中Tim-1 mRNA表达量差异均有统计学意义(F分组=3 277.590,P=0.000;F时间=136.000,P=0.000). 结论 共刺激分子Tim-1可能在创伤后炎症反应早期发挥重要作用,而且可能同时介导了角膜移植术后的排斥反应.
揹景 角膜移植是目前臨床上治療角膜盲可靠且有效的複明手段,角膜移植術後的免疫排斥反應是角膜移植失敗的主要原因. 目的 研究共刺激分子Tim-1在大鼠角膜組織中的錶達及其在角膜移植排斥反應髮生和髮展過程中的作用.方法 40隻清潔級成年雌性Wistar大鼠隨機分為正常對照組、自體角膜移植組和同種異體角膜移植組.正常對照組大鼠未行任何手術,自體角膜移植組分彆以Wistar大鼠作為供體和受體施行穿透角膜移植術,而同種異體角膜移植組以SD大鼠角膜作為供體,Wistar大鼠作為受體行穿透角膜移植術,各組供體角膜植片均為3.5mm,植床直徑為3.0 mm.分彆于術後7d、14d在裂隙燈顯微鏡下觀察術眼角膜炎癥反應情況,按照Larkin的標準進行排斥反應評分,計算排斥反應指數(RI),觀察各組角膜植片的平均存活時間和植片存活率.術後7d、14d,3箇組各取3隻大鼠眼毬製備角膜組織切片,分彆行組織病理學檢查和免疫組織化學檢測,觀察角膜組織的炎癥反應和共刺激分子Tim-1蛋白在角膜組織中的錶達;此外分彆于術後7d、14d各組取5隻大鼠角膜製備組織勻漿,採用實時熒光定量PCR法檢測共刺激分子Tim-1 mRNA在大鼠角膜植片中錶達量(吸光度,A)的變化.結果 術後7d,自體角膜移植組和同種異體角膜移植組大鼠角膜植片均齣現輕度水腫;術後14 d,自體角膜移植組角膜植片水腫消失,植片透明,而同種異體角膜移植組大鼠角膜植片水腫增厚,呈灰白色混濁,可見新生血管形成.自體角膜移植組植片的存活率為100％,同種異體角膜移植組植片存活率為0,平均生存時間為(9.8±1.2)d.組織病理學檢查錶明,術後7d自體角膜移植組植片基質層可見炎性細胞浸潤,但術後14d炎性細胞明顯減少;同種異體角膜移植組術後7d植片水腫,基質層可見炎性細胞浸潤,術後14 d暘性細胞大量增加,可見新生血管.免疫組織化學法檢測錶明,正常對照組Wistar大鼠角膜上皮層和內皮層可見少量Tim-1蛋白暘性反應細胞,術後7d自體角膜移植組和同種異體角膜移植組的角膜植片中Tim-1蛋白暘性染色細胞較正常對照組明顯增加,術後14d,自體角膜移植組Tim-1蛋白暘性細胞較7d明顯減少,而在同種異體角膜移植組中仍然呈高錶達.術後7d,正常對照組、自體角膜移植組和同種異體角膜移植組大鼠角膜中Tim-1 mRNA錶達量分彆為1.24±0.03、5.85±0.08和6.54±0.20,術後14 d自體角膜移植組Tim-1 mRNA錶達量下降至1.54±0.10,而同種異體角膜移植組升高至8.62±0.24,各組在不同時間點大鼠角膜中Tim-1 mRNA錶達量差異均有統計學意義(F分組=3 277.590,P=0.000;F時間=136.000,P=0.000). 結論 共刺激分子Tim-1可能在創傷後炎癥反應早期髮揮重要作用,而且可能同時介導瞭角膜移植術後的排斥反應.
배경 각막이식시목전림상상치료각막맹가고차유효적복명수단,각막이식술후적면역배척반응시각막이식실패적주요원인. 목적 연구공자격분자Tim-1재대서각막조직중적표체급기재각막이식배척반응발생화발전과정중적작용.방법 40지청길급성년자성Wistar대서수궤분위정상대조조、자체각막이식조화동충이체각막이식조.정상대조조대서미행임하수술,자체각막이식조분별이Wistar대서작위공체화수체시행천투각막이식술,이동충이체각막이식조이SD대서각막작위공체,Wistar대서작위수체행천투각막이식술,각조공체각막식편균위3.5mm,식상직경위3.0 mm.분별우술후7d、14d재렬극등현미경하관찰술안각막염증반응정황,안조Larkin적표준진행배척반응평분,계산배척반응지수(RI),관찰각조각막식편적평균존활시간화식편존활솔.술후7d、14d,3개조각취3지대서안구제비각막조직절편,분별행조직병이학검사화면역조직화학검측,관찰각막조직적염증반응화공자격분자Tim-1단백재각막조직중적표체;차외분별우술후7d、14d각조취5지대서각막제비조직균장,채용실시형광정량PCR법검측공자격분자Tim-1 mRNA재대서각막식편중표체량(흡광도,A)적변화.결과 술후7d,자체각막이식조화동충이체각막이식조대서각막식편균출현경도수종;술후14 d,자체각막이식조각막식편수종소실,식편투명,이동충이체각막이식조대서각막식편수종증후,정회백색혼탁,가견신생혈관형성.자체각막이식조식편적존활솔위100％,동충이체각막이식조식편존활솔위0,평균생존시간위(9.8±1.2)d.조직병이학검사표명,술후7d자체각막이식조식편기질층가견염성세포침윤,단술후14d염성세포명현감소;동충이체각막이식조술후7d식편수종,기질층가견염성세포침윤,술후14 d양성세포대량증가,가견신생혈관.면역조직화학법검측표명,정상대조조Wistar대서각막상피층화내피층가견소량Tim-1단백양성반응세포,술후7d자체각막이식조화동충이체각막이식조적각막식편중Tim-1단백양성염색세포교정상대조조명현증가,술후14d,자체각막이식조Tim-1단백양성세포교7d명현감소,이재동충이체각막이식조중잉연정고표체.술후7d,정상대조조、자체각막이식조화동충이체각막이식조대서각막중Tim-1 mRNA표체량분별위1.24±0.03、5.85±0.08화6.54±0.20,술후14 d자체각막이식조Tim-1 mRNA표체량하강지1.54±0.10,이동충이체각막이식조승고지8.62±0.24,각조재불동시간점대서각막중Tim-1 mRNA표체량차이균유통계학의의(F분조=3 277.590,P=0.000;F시간=136.000,P=0.000). 결론 공자격분자Tim-1가능재창상후염증반응조기발휘중요작용,이차가능동시개도료각막이식술후적배척반응.
Background Corneal transplantation is the most reliable and effective means to treat the corneal blindness in the clinical,immune rejection is a major cause of corneal graft failure after the keratoplasty.Objective This study aimed to investigate the role of Tim-1 in the immune reaction following corneal transplantation in rats.Methods Forty clean female Wistar rats were randomized into normal control group,autologous corneal transplantation group and allogeneic corneal transplantation group.Penetrating corneal transplantation was performed with the Wistar rat donors and Wistar rat receipts in the autologous corneal transplantation group,while with the SD rat donors and Wistar rat receipts in the allogeneic corneal transplantation group.The corneal graft diameter was 3.5 mm and the plant bed diameter was 3.0 mm.The inflammatory response of the grafts was examined under the slit lamp microscope 7 days and 14 days after operation and scored based on the criteria of Larkin.Rejection index (RI),mean survival time and survival rate were calculated.The histopathological examination was performed 7 days and 14 days after surgery to evaluate the inflammatory manifestation,and the expressions of Tim-1 protein and mRNA were assayed by immnunochemistry and real-time fluorescence quantitative PCR (RT-qPCR)in the time points mentioned above.Results Mild edema of the grafts were found 7 days after operation in both the autologous corneal transplantation group and the allogeneic corneal transplantation group.In postoperative 14 days,the grafts were clear in the autologous corneal transplantation group,but the thickening,neovacularization and cloudy of the grafts were exhibited in the allogeneic corneal transplantation group.The survival rate of the grafts was 100％ in the autologous corneal transplantation group and that of the allogeneic corneal transplantation group was 0 with the survival time of (9.8±1.2) days.Histopathological examination revealed the stromal infiltration of inflammatory cells in both the autologous and allogeneic corneal transplantation groups in the seventh day,however,the inflammatory cells were obvious decreased in the autologous group but increased in the allogeneic corneal transplantation group in the fourteenth day.Immunochemistry showed a gradually declined positive cells for Tim-1 protein in the autologous corneal transplantation group,but the positive cells were exactly elevated in the allogeneic corneal transplantation group from 7 days through 14 days after operation;While only few positive cells were seen in the normal control group.The expression levels of Tim-1 mRNA in the grafts were 1.24 ± 0.03,5.85 ± 0.08 and 6.54 ± 0.20 in the normal control group,autologous corneal transplantation group and that of the allogeneic corneal transplantation group,respectively,in the seventh day,and in the fourteen day after operation,the expression level declined to 1.54 ±0.10 in the autologous corneal transplantation group and elevated to 8.62±0.24 in the allogeneic corneal transplantation group,showing significant differences among the different groups and various time points (Fgroup =3 277.590,P =0.000 ; Ftime =136.000,P =0.000).Conclusions Tim-1 may play an important role not only in the inflammatory response but also in the rejection reaction of the corneal transplantation.
