摘要
目的研究重组腺相关病毒介导的转化生长因子-β1(recombinant adeno-associat-ed virus mediated transforming growth factor-β1,rAAV-TGF-β1)对大鼠高危角膜移植免疫排斥反应的抑制作用以及对白细胞介素-10(interleukin-10,IL-10)表达的影响。方法构建rAAV-TGF-β1真核表达质粒载体。40只角膜新生血管化的SD大鼠行穿透性角膜移植术。实验组(20只):角膜植片预浸泡在rAAV-TGF-β1(10×1012v.g.L-1)的达尔伯克必需基本培养液F12混合培养液中37℃孵育30min;对照组(20只):角膜植片预浸泡在0.1mol.L-1磷酸盐缓冲液中37℃孵育30min。裂隙灯观察TGF-β1对大鼠角膜植片存活时间、排斥反应指数的影响。术后第1周、第2周、第4周、第2个月对角膜植片行组织病理学检查,免疫组织化学方法检测角膜组织中IL-10的表达。结果对照组角膜植片平均存活时间为(10.40±2.45)d,实验组为(18.70±1.51)d,比对照组显著延长,差异具有显著统计学意义(P<0.01);术后第2周实验组角膜植片排斥反应指数(3.48±0.86)明显低于对照组(7.65±0.42),差异具有显著统计学意义(P<0.01)。IL-10在术后主要表达于角膜的上皮层与内皮层。术后各时期实验组IL-10的表达明显高于对照组,差异具有统计学意义(P<0.05)。结论rAAV-TGF-β1能显著延长角膜植片存活时间,抑制大鼠高危角膜移植免疫排斥反应,其机制与提高角膜组织中IL-10的表达有关。重组腺相关病毒是眼科基因治疗的有效载体。
Objective To study the immunosuppressive action of recombinant adeno-associated virus mediated transforming growth factor-β1 ( rAAV-TGF-β1 ) on the rat models of high-risk penetrating keratoplasty rejection and the effect of rAAV-TGF-β1 on the expression of interleukin-10 (IL-10). Methods The eukaryotic plasmid vector containing rAAV-TGF-β1 was constructed. Forty SD rats were operated with penetrating keratoplasty, and randomly divided into 2 groups, experimental group and control group ,20 animals in each group. Corneal neovascularization(CNV) were induced by alkaline cauterization of the central cornea. In experimental group, the donor cornea graft was incubated in 10 ×10^12 v. g · L^-1 rAAV-TGF-β1 dulbecco' s minimum essential medium mixed culture medium for 30 minutes at 37℃ ,and in control group,the donor graft was treated in phosphate;buffered solution for 30 minutes at 37℃ before surgery. Mean survival time and rejection index were clinically observed and compared with slit light microscope. The allografts were detected by histopathology, and expression of IL-10 was evaluated by immunhistochemistry at the 1st week,2nd week,4th week and 2nd month postoperatively. Resultu The mean survival time of control group were ( 10.40 ±2.45 ) d, were ( 18.70 ±1.51 ) d in experimental group, which was statistically significant compared with that of control group(P 〈 0.01 ). Rejection index (RI) of rAAV-TGF-β1 group was markedly lower compared with that of control group(P 〈0.01 ). The expression of IL-10 were increased at corneal epithelium and endothelium in experimental group compared with those in control group (P 〈 0. 05 ). Conclusions Treatment with rAAV-TGF-β1 can significantly prolong the survival time of corneal allograft and suppress the immunological rejection after high-risk penetrating keratoplasty, which may be associated with upregulating the expression of IL-10 in corneas. Recombinant adeno-associated virus is the effective vector for ophthalmologi
出处
《眼科新进展》
CAS
北大核心
2009年第1期12-17,共6页
Recent Advances in Ophthalmology
基金
国家自然科学基金资助(编号:30672290)~~