贝伐单抗抑制角膜移植术后新生血管及免疫排斥反应的作用

Effect of bevacizumab on corneal neovascularization and immune rejection after corneal transplantation

目的探讨贝伐单抗对同种异体角膜移植术后免疫排斥反应的影响,评价其对角膜植片存活的疗效及作用机制。设计实验研究。研究对象近交系F344大鼠15只为供体,近交系Lewis大鼠30只为受体。方法受体大鼠右眼行穿透性角膜移植术后随机数字法分为3组:对照组(A组)、贝伐单抗组(B组)、地塞米松组(C组),每组10只。B、C组分别于术后0、3、6、9天结膜下注射40μl贝伐单抗注射液及20μl地塞米松磷酸钠注射液;A组不做处理。术后裂隙灯下观察14天,记录排斥指数(RI,为植片水肿、混浊、新生血管长入评分合计)和新生血管侵袭面积(IA)。术后14天,采用免疫荧光法检测角膜植片中CD4^+、CD8^+免疫细胞的表达。主要指标角膜新生血管面积,免疫排斥时间,免疫细胞的数量。结果术后7天IA分别为:A组(22.50±3.67)mm^2,B组(14.21±2.79)mm^2,C组(15.38±0.84)mm^2;A组明显多于B组及C组(P=0.00和0.01),B组与C组无显著差异(P=0.059)。术后14天IA分别为:A组(27.96±0.50)mm^2,B组(18.76±2.73)mm^2,C组(23.74±2.14)mm^2。A组及C组多于B组(P=0.000)。免疫排斥时间A组8天,B组11天,C组13天。B组及C组角膜植片存活时间均长于A组(P均=0.000)。免疫细胞:CD4^+阳性细胞数:A组(13.2±2.94)个,B组(6.14±1.07)个,C组(3.5±1.78)个;CD8^+阳性细胞数:A组(14.4±2.44)个,B组(4.5±1.51)个,C组(3.38±1.68)个。术后14天,B组、C组角膜植片中CD4^+、CD8^+细胞亦明显少于A组(P均=0.000)。结论结膜下注射贝伐单抗可有效抑制大鼠同种异体穿透性角膜移植术后新生血管的生长,并可能通过此作用延缓移植排斥反应,延长角膜植片存活时间。但贝伐单抗抗移植排斥反应疗效略逊于地塞米松。

Objective To investigate the effects of bevacizumab on prevention of corneal neovascularization in a rat model of penetrating keratoplasty, and to evaluate its effect on the survival of corneal grafts. Design Experimental studies. Participants Fifteen F344 rats were used as donor and 30 Lewis rats as recipients. Methods After penetrating keratoplasty, recipients were randomly divided into three groups： control group（GA）, bevacizumab group （GB） and dexamethasone group （GC）. A single dose of 40 μl bevacizumab was injected subconjunctivally in GB at 0, 3, 6 and 9 days after operation. GC were subconjunctivally injected with 20 μl dexamethasone. GA was not treated. Grafts were examined every three day for two weeks by slit-lamp biomicroscopy, the rejection index （RI, edema, opacity, neovessels） and neovessel invasion area （IA） were calculated. After 14 days, the rats were killed and the expression of CD4^＋ and CD8^＋ cells in corneal grafts was detected by immunofluorescence. Main Outcome Measures Neovessel invasion area, grafts survival time and cell counting of CD4^＋ and CD8^＋. Results Seven days after surgery, IA in GA differed significantly from GB （P=0.00） and GC （P=0.01） , but GB and GC were closed to each other. At Day 7, IA was 22.50±3.67mm^2（GA）, 14.21±2.79mm^2 （GB） and 15.38±0.84mm^2（GC） respectively; at Day 14, IA was 27.96±0.50mm^2（GA）, 18.76±2.73mm^2 （GB） and 23.74±2.14mm^2（GC） respectively. The corneal grafts survival in GB and GC was longer than GA （all P=0.000）. The corneal grafts survival was 8（GA）, 11（GB）,13（GC） days respectively. At Day 14, the CD4^＋, CD8^＋ cells in GA were higher than GB and GC （all P=0.000）. The number of CD4^＋ cells was 13.2±2.94（GA）, 6.14±1.07（GB） and 3.5±1.78（GC） respectively. The number of CD8^＋ cells was 14.4±2.44（GA）, 4.5±1.51（GB） and 3.38±1.68（GC） respectively. Conclusions Subconjunctival administration of bevacizumab may effectively inhibit neovascularization and corneal graft rejection. But bevacizumab is less effective than dexamethasone on inhibiting corneal allograft rejection.