CCR7基因修饰未成熟树突状细胞诱导大鼠高危角膜移植免疫耐受

Induction of CCR7 gene modified immature dendritic cells to immune tolerance in rats high-risk corneal allograft

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＆#215;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.

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 ＆#215; 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.