TGF-β_1基因在高危角膜移植术后对移植排斥反应的作用

Effects of TGF-β_1 gene on transplanting rejection in high-risk keratoplasty

目的研究高危角膜移植术后，重组腺相关病毒（rAAV）携载的TGF—β1基因（rAAV—TGF—β1）在角膜中的表达及对免疫排斥反应的影响。方法用碱烧伤的方法建立受体角膜新生血管（CNV）化模型。20只Wistar大鼠角膜为供体，40只SD大鼠为受体进行大鼠穿透角膜移植。治疗组术前将20只供体植片置于含rAAV—TGF-β1的DMEM／F12混合培养液中常温培养30min，对照组术前将20只供体植片置于DMEM／F12培养液中常温培养30min，术后裂隙灯下观察植片排斥反应及存活情况。免疫组织化学染色观察2组术后1、2、4周TGF—β1在角膜中的表达。Western blot检测2组术后1、2、4、8周TGF—β1的表达量。结果治疗组角膜平均存活时间为（17．60±2．31）d，对照组为（9．27±1．50）d，治疗组角膜植片混浊及血管化程度明显低于对照组（P〈0．01）。免疫组织化学染色显示治疗组术后1周TGF-v在角膜上皮呈弱阳性表达，术后2～4周角膜上皮及内皮细胞层呈阳性表达并高于对照组。对照组术后1～4周TGF—β1在角膜上皮及基底膜呈弱阳性表达。Western blot检测显示术后1周2组角膜TGF—β1含量差异无统计学意义（P〉0．05），术后2、4、8周治疗组角膜TGF—β1的表达较对照组增高（P〉0．05）。结论rAAV-TGF—β1能减轻高危角膜移植的排斥反应。

Objective Present study was to investigate the expression of TGF-β1 gene transferred by recombinated adenoassociated virus（rAAV） in the implant tablets after high-risk keratoplasty of rats and the effects of TGF-β1 gene on transplant rejection. Methods Corneal neovascularization models were created by putting the filter paper with 1% NaCl solution in the central cornea for 20 seconds in 40 Sprague-Dawley（SD） rats. The penetrating corneal transplatation was performed in the model rats with 20 Wistar rats as donors and models as receiptors. The right eyes of 20 model rats received the grafts cultured in DMEM/ F12 medium with rAAV-TGF-β1in experimental group,and another right eyes of 20 model rats received the grafts cultured in only DMEM/F12 medium as control group. After the operation, survival of grafts was examined under the slit lamp once a day at the first week and afterthere at two-day interval. The grafts rejection was scored according to Plskova criteria. Expression of TGF-β1 in graft was detected in different time points by immunohistochemistry and Western-blotting in 1 week,2,4 and 8 weeks after the operation. The use of the animals followed the Standard of Statement of ARVO. Results The mean graft rejection time was （17.60± 2.31 ） days in experimental group and （9.27 ± 1.50） days in control group, showing a statistically significant difference between them（ t = 6.76, P 〈 0. 01 ）. The degree of vascularizing and the turbidity of graft in treatment group was significantly lower than that the controi group with the statistically significant difference between them （P 〈 0. 01 ）. TGF-β1 showed a weak expression only in corneal epithelium in the initial week and positive expression in whole layer of cornea from 2 weeks through 4 weeks in experimental group. After 2 -4 weeks, TGF-β1 expression in graft was stronger in experimental group compared with control group. In the first week after operation,no significant difference in TGF-β1 expression between two groups（ t = 0. 46, P 〉 0.05 ）. However, the TGF-β1 content in grafts was significantly higher in experimental group than control group from 2 through 8 weeks after operation （P 〈 0.05）. Conclusion The rAAV-TGF-β1 can reduce high-risk corneal transplant rejection.