中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
Chinese Journal of Experimental Ophthalmology
2015年
10期
902-908
,共7页
周琨%高晓唯%蔡岩%李文静%胡裕坤%田丽丽%付燕
週琨%高曉唯%蔡巖%李文靜%鬍裕坤%田麗麗%付燕
주곤%고효유%채암%리문정%호유곤%전려려%부연
角膜移植%植片存活/免疫%免疫耐受%辅助T淋巴细胞/免疫%树突状细胞/免疫%受体,趋化因子受体7/代谢
角膜移植%植片存活/免疫%免疫耐受%輔助T淋巴細胞/免疫%樹突狀細胞/免疫%受體,趨化因子受體7/代謝
각막이식%식편존활/면역%면역내수%보조T림파세포/면역%수돌상세포/면역%수체,추화인자수체7/대사
Corneal transplantation%Graft survival/immunology%Immune tolerance%T-lymphocytes,helper/immunology%Dendritic cells/immunology%Receptors,Chemokine receptor 7/metabolism
背景 角膜移植术后免疫排斥反应是手术失败的主要原因,尤其是高危角膜移植.延长高危角膜移植术植片的存活时间是目前亟待解决的问题.目的 研究趋化因子受体7 (CCR7)基因重组腺病毒(Ad)体外转染供体骨髓来源的未成熟树突状细胞(imDCs)对大鼠高危角膜移植免疫排斥反应的影响,并探讨其可能的机制.方法 取1只雄性Wistar供体大鼠股骨骨髓分离和培养骨髓源imDCs,用携带大鼠CCR7基因的重组Ad转染imDCs,收集1×107个imDCs重悬于500μl含体积分数1％胎牛血清的PBS中.将60只受体SD大鼠以角膜碱烧伤法建立高危角膜移植模型,并用30只Wistar大鼠角膜作为供体进行同种异体角膜移植.采用随机数字表法将SD大鼠分为PBS组、未修饰imDCs组、imDCs+ Ad空载体组和imDCs+ Ad-CCR7组,每组15只,按照分组不同分别于术前7d和术后3d经尾静脉注射相应溶液.术后每日裂隙灯显微镜下观察角膜植片存活情况,术后14d每组任意处死6只大鼠,取角膜植片行常规组织病理学检查,采用逆转录PCR(RT-PCR)法检测辅助性T细胞1(Th1)型细胞因子白细胞介素-2(IL-2)、γ干扰素(IFN-γ)和Th2型细胞因子IL-4、IL-10的mRNA表达水平.结果 PBS组、未修饰imDCs组、imDCs+Ad空载体组和imDCs+Ad-CCR7组角膜植片的平均存活时间分别为(10.44±1.88)、(16.00±2.18)、(15.11±2.03)和(23.67±2.83)d,4个组间总体比较差异有统计学意义(F=53.005,P=0.000);与PBS组比较,未修饰imDCs组、imDCs+ Ad空载体组和imDCs+ Ad-CCR7组角膜植片的平均存活时间均明显延长,差异均有统计学意义(t=5.220、4.385、12.423,均P=0.000);imDCs+Ad-CCR7组植片的平均存活时间较未修饰imDCs组和imDCs+Ad空载体组均明显延长,差异均有统计学意义(t=7.204、8.039,均P=0.000).RT-PCR结果显示,与PBS组相比,未修饰imDCs组、imDCs+Ad空载体组、imDCs+Ad-CCR7组大鼠IFN-γ mRNA和IL-2 mRNA相对表达量均明显降低,而IL-4 mRNA和IL-10 mRNA相对表达量均明显升高,差异均有统计学意义(均P＜0.05);与未修饰imDCs组和imDCs+Ad空载体组比较,imDCs+Ad-CCR7组IFN-γ mRNA和IL-2 mRNA相对表达量明显降低,而IL-4 mRNA和IL-10 mRNA相对表达量明显升高,差异均有统计学意义(均P=0.000).结论 尾静脉注射CCR7基因修饰的imDCs可以明显延长大鼠高危角膜移植术后角膜植片的存活时间,抑制角膜移植免疫排斥反应,可能与CCR7基因修饰的imDCs诱导的Th1/Th2功能偏移有关.
揹景 角膜移植術後免疫排斥反應是手術失敗的主要原因,尤其是高危角膜移植.延長高危角膜移植術植片的存活時間是目前亟待解決的問題.目的 研究趨化因子受體7 (CCR7)基因重組腺病毒(Ad)體外轉染供體骨髓來源的未成熟樹突狀細胞(imDCs)對大鼠高危角膜移植免疫排斥反應的影響,併探討其可能的機製.方法 取1隻雄性Wistar供體大鼠股骨骨髓分離和培養骨髓源imDCs,用攜帶大鼠CCR7基因的重組Ad轉染imDCs,收集1×107箇imDCs重懸于500μl含體積分數1％胎牛血清的PBS中.將60隻受體SD大鼠以角膜堿燒傷法建立高危角膜移植模型,併用30隻Wistar大鼠角膜作為供體進行同種異體角膜移植.採用隨機數字錶法將SD大鼠分為PBS組、未脩飾imDCs組、imDCs+ Ad空載體組和imDCs+ Ad-CCR7組,每組15隻,按照分組不同分彆于術前7d和術後3d經尾靜脈註射相應溶液.術後每日裂隙燈顯微鏡下觀察角膜植片存活情況,術後14d每組任意處死6隻大鼠,取角膜植片行常規組織病理學檢查,採用逆轉錄PCR(RT-PCR)法檢測輔助性T細胞1(Th1)型細胞因子白細胞介素-2(IL-2)、γ榦擾素(IFN-γ)和Th2型細胞因子IL-4、IL-10的mRNA錶達水平.結果 PBS組、未脩飾imDCs組、imDCs+Ad空載體組和imDCs+Ad-CCR7組角膜植片的平均存活時間分彆為(10.44±1.88)、(16.00±2.18)、(15.11±2.03)和(23.67±2.83)d,4箇組間總體比較差異有統計學意義(F=53.005,P=0.000);與PBS組比較,未脩飾imDCs組、imDCs+ Ad空載體組和imDCs+ Ad-CCR7組角膜植片的平均存活時間均明顯延長,差異均有統計學意義(t=5.220、4.385、12.423,均P=0.000);imDCs+Ad-CCR7組植片的平均存活時間較未脩飾imDCs組和imDCs+Ad空載體組均明顯延長,差異均有統計學意義(t=7.204、8.039,均P=0.000).RT-PCR結果顯示,與PBS組相比,未脩飾imDCs組、imDCs+Ad空載體組、imDCs+Ad-CCR7組大鼠IFN-γ mRNA和IL-2 mRNA相對錶達量均明顯降低,而IL-4 mRNA和IL-10 mRNA相對錶達量均明顯升高,差異均有統計學意義(均P＜0.05);與未脩飾imDCs組和imDCs+Ad空載體組比較,imDCs+Ad-CCR7組IFN-γ mRNA和IL-2 mRNA相對錶達量明顯降低,而IL-4 mRNA和IL-10 mRNA相對錶達量明顯升高,差異均有統計學意義(均P=0.000).結論 尾靜脈註射CCR7基因脩飾的imDCs可以明顯延長大鼠高危角膜移植術後角膜植片的存活時間,抑製角膜移植免疫排斥反應,可能與CCR7基因脩飾的imDCs誘導的Th1/Th2功能偏移有關.
배경 각막이식술후면역배척반응시수술실패적주요원인,우기시고위각막이식.연장고위각막이식술식편적존활시간시목전극대해결적문제.목적 연구추화인자수체7 (CCR7)기인중조선병독(Ad)체외전염공체골수래원적미성숙수돌상세포(imDCs)대대서고위각막이식면역배척반응적영향,병탐토기가능적궤제.방법 취1지웅성Wistar공체대서고골골수분리화배양골수원imDCs,용휴대대서CCR7기인적중조Ad전염imDCs,수집1×107개imDCs중현우500μl함체적분수1％태우혈청적PBS중.장60지수체SD대서이각막감소상법건립고위각막이식모형,병용30지Wistar대서각막작위공체진행동충이체각막이식.채용수궤수자표법장SD대서분위PBS조、미수식imDCs조、imDCs+ Ad공재체조화imDCs+ Ad-CCR7조,매조15지,안조분조불동분별우술전7d화술후3d경미정맥주사상응용액.술후매일렬극등현미경하관찰각막식편존활정황,술후14d매조임의처사6지대서,취각막식편행상규조직병이학검사,채용역전록PCR(RT-PCR)법검측보조성T세포1(Th1)형세포인자백세포개소-2(IL-2)、γ간우소(IFN-γ)화Th2형세포인자IL-4、IL-10적mRNA표체수평.결과 PBS조、미수식imDCs조、imDCs+Ad공재체조화imDCs+Ad-CCR7조각막식편적평균존활시간분별위(10.44±1.88)、(16.00±2.18)、(15.11±2.03)화(23.67±2.83)d,4개조간총체비교차이유통계학의의(F=53.005,P=0.000);여PBS조비교,미수식imDCs조、imDCs+ Ad공재체조화imDCs+ Ad-CCR7조각막식편적평균존활시간균명현연장,차이균유통계학의의(t=5.220、4.385、12.423,균P=0.000);imDCs+Ad-CCR7조식편적평균존활시간교미수식imDCs조화imDCs+Ad공재체조균명현연장,차이균유통계학의의(t=7.204、8.039,균P=0.000).RT-PCR결과현시,여PBS조상비,미수식imDCs조、imDCs+Ad공재체조、imDCs+Ad-CCR7조대서IFN-γ mRNA화IL-2 mRNA상대표체량균명현강저,이IL-4 mRNA화IL-10 mRNA상대표체량균명현승고,차이균유통계학의의(균P＜0.05);여미수식imDCs조화imDCs+Ad공재체조비교,imDCs+Ad-CCR7조IFN-γ mRNA화IL-2 mRNA상대표체량명현강저,이IL-4 mRNA화IL-10 mRNA상대표체량명현승고,차이균유통계학의의(균P=0.000).결론 미정맥주사CCR7기인수식적imDCs가이명현연장대서고위각막이식술후각막식편적존활시간,억제각막이식면역배척반응,가능여CCR7기인수식적imDCs유도적Th1/Th2공능편이유관.
Background Graft rejection is a primary cause of corneal transplantation failure,especially in high-risk keratoplasty.How to extend the survival time of graft is a problem to be solved.Objective This study was to investigate the influence of immune tolerance on high-risk rat keratoplasty induced by donor bone marrowderived immature dendritic cells (imDCs) transfected by chemokine receptor 7 (CCR7) recombinant adenovirus (Ad).Methods Bone marrow-derived imDCs were isolated and cultured from femur marrow of one male Wistar donor rat.The cells were transfected using recombinant Ad vector with rat CCR7 gene and resuspended in 500 μl PBS containing 1％ fetal bovine serum with the cells 1 × 107.High-risk corneal transplantaion models were established using monolateral corneal alkali-burn method in 60 SD rat recipients, and then allograft keratoplasty was performed with the 30 Wistar rats as donors.The models were randomized into the PBS group,imDCs group,imDCs with blank Ad vector group and imDCs with Ad-CCR7 group following the corresponding solution injection via caudal vein on preoperative day 7 and postoperative day 3 respectively.The survival time of graft was evaluated under the slit lamp microscope once per day.On the 14th day after operation, corneal sections were prepared from 6 eyes of each group for the pathological examination,and the relative expression levels of T helper cell 1 (Th1)-related factors,interleukin-2 (IL-2) mRNA and interferon-γ(IFN-γ) mRNA,as well as Th2-related factors, IL-4 mRNA and IL-10 mRNA, were detected by reverse transcription PCR (RT-PCR).The use and care of animals complied with the ARVO statement Results The mean survival time of grafts was (10.44±1.88) , (16.00±2.18) , (15.11±2.03) and(23.67±2.83) days in the PBS group,imDCs group,imDCs with blank Ad group and imDCs with Ad-CCR7 group, respectively, with a significant difference among the 4 groups (F =53.005, P =0.000).Compared with the PBS group, the mean survival time of grafts was considerably extended in the imDCs group,imDCs with blank Ad group and imDCs with Ad-CCR7 group (t=5.220,4.385,12.423 ,all at P=0.000) ,and a remarkble prolongation of graft survival duration was seen in the imDCs with Ad-CCR7 group in comparison with the imDCs group and the imDCs with blank Ad group (t =7.204,8.039,both at P=0.000).The relative expression levels of IFN-γ mRNA and IL-2 mRNA in the grafts were significantly lower,but the relative expression levels of IL-4 mRNA and IL-10 mRNA were significantly higher in the imDCs group,imDCs with blank Ad group and the imDCs with Ad-CCR7 group than those in the PBS group (all at P＜0.05).Compared with the imDCs group and the imDCs with blank Ad group, the relative expression levels of IFN-γ mRNA and IL-2 mRNA in the grafts were remarkably delined,but the relative expression levels of IL-4 mRNA and IL-10 mRNA were remarkably elevated in the imDCs with Ad-CCR7 group (all at P =0.000).Conclusions Application of imDCs transfected with CCR7 recombinant Ad via vena caudalis can prolong the survival time of grafts after keratoplasty of SD rats probably by affecting the balance of Th1/Th2 cytokines.
