NF-κB抑制剂吡咯二硫氨基甲酸酯(PDTC)对角膜移植大鼠角膜组织的影响

Effect of pyrrolidine dithiocarbamate as an inhibitor of NF-kappa B on corneal tissue in rats after corneal transplantation

目的探讨核转录因子-κB(nuclear factor-κB,NF-κB)抑制剂吡咯二硫氨基甲酸酯(pyrrolidine dithiocarbamate,PDTC)对角膜移植大鼠角膜组织的影响。方法取14只Wistar大鼠为供体鼠,提供双眼角膜; 28只SD大鼠为受体鼠,均以左眼为术眼,右眼设为正常对照,在显微镜下进行同种异体角膜移植术。手术完成后将28只SD大鼠随机分为3组,对照组(10只)以生理盐水滴左眼,实验组(10只)以1 mg·mL^(-1)PDTC滴左眼,均为每天3次,每次1滴,空白组(8只)不做任何处理。术后第1天起,每组分别在术后第5天、第15天、第25天在裂隙灯下观察角膜新生血管的情况并进行评分。对各组角膜植片进行病理学及免疫组织化学染色观察,分析各组大鼠NF-κB及血管内皮生长因子(vascular endothelial growth factor,VEGF)在角膜植片的表达情况。结果对照组、实验组、空白组角膜移植排斥反应发生的时间分别为(10. 80±1. 40) d、(23. 40±2. 30) d、(11. 20±2. 06) d,实验组排斥反应发生的时间较对照组明显延长,差异有统计学意义(P <0. 01)。对照组新生血管一般术后第3～5天出现,并很快进入植片周边,沿缝线处向角膜植片中央粗大生长。实验组新生血管一般术后第5～7天出现,生长速度较慢,血管稀疏。术后不同时间实验组新生血管数、新生血管面积及排斥反应指数均小于对照组,差异均有统计学意义(P <0. 05)。角膜移植术后,实验组大鼠角膜上皮基底细胞内出现较多空泡,角膜基质层内出现间质水肿;炎症细胞浸润,并见新生的毛细血管;缝线及切口周围见大量淋巴细胞浸润,部分植片中央发生散在的炎症细胞浸润、角膜上皮部分脱落。对照组角膜植片水肿及炎症细胞的浸润程度较实验组明显增加,且对照组角膜植片显著增厚,基质结构疏松、紊乱;实验组角膜植片相对基质板层结构排列有序,新生血管数在相同时间内明显减少。免疫组织化学染色显示,NF-κB阳性细胞数,实验组为每400倍视野(4. 236±0. 856)个,较对照组[(18. 451±1. 234)个]明显减少,差异有统计学意义(P <0. 05);空白组大鼠角膜内鲜有阳性表达。VEGF阳性细胞数,实验组为每400倍视野(2. 631±0. 238)个,较对照组[(6. 254±0. 721)个]明显减少,差异有统计学意义(P <0. 05);空白组大鼠角膜内鲜有阳性表达。结论大鼠进行同种异体角膜移植术后,NF-κB抑制剂PDTC可在一定程度上减少角膜新生血管的生成,抑制角膜移植排斥反应的发生。

Objective To investigate the effect of pyrrolidine dithiocarbamate (PDTC),an inhibitor of NF-kappa B,on corneal tissue in rats after corneal transplantation. Methods Fourteen Wistar rats were used as donors and twenty-eight SD rats were used as recipients.The left eyes were used as the operative eye and the right eyes were used as the normal controls.The corneal allograft was performed under the microscope.After the operation,twenty-eight SD rats were randomly divided into three groups.The control group (ten rats) was treated with saline,the experimental group (ten rats) was treated with 1 mg· mL^-1 PDTC for the left eyes three times a day,one drop each time.The blank group (eight rats) did not receive any treatment.Corneal neovascularization was observed under slit lamp on the 5th,15th and 25th day after operation in each group and scored.The expression of NF-kappa B and vascular endothelial growth factors on corneal grafts of rats in each group was analyzed by pathological and immunohistochemical staining. Results The occurrence time of corneal transplantation rejection in control group,experimental group and blank group was (10.80±1.40)d,(23.40±2.30)d,(11.20±2.06)d,respectively.The occurrence time of corneal transplantation rejection in the experimental group was significantly longer than the time in the control group (P<0.01).In the control group,neovascularization usually appeared on the 3rd to 5th day after operation,and soon entered the periphery of the graft and grew coarsely along the suture to the center of the corneal graft.In the experimental group,neovascularization usually appeared on the 5th to 7th day after operation,and the growth rate was slow and the blood vessels were sparse.The number of neovascularization,area of neovascularization and rejection index in the experimental group were less than those in the control group at different times after operation,and the differences was statistically significant (P<0.05).After corneal transplantation,there were many vacuoles in corneal epithelial basal cells,interstitial edema in corneal stroma,infiltration of inflammatory cells and new capillaries,infiltration of lymphocytes around sutures and incisions,scattered infiltration of inflammatory cells in the center of some grafts and partial exfoliation of corneal epithelium in the experimental group.In the control group,corneal graft edema and inflammatory cell infiltration were significantly increased compared with the experimental group,and the corneal graft in the control group was significantly thicker,the matrix structure was loose and disordered;in the experimental group,the corneal graft was arranged orderly relative to the matrix lamellar structure,and the number of new blood vessels were significantly reduced in the same period.Immunohistochemical staining showed that the number of NF-kappa B positive cells in the experimental group was (4.236±0.856)cells per 400 times the field of vision,which was significantly lower than that in the control group [(18.451±1.234)cells],and the difference was statistically significant (P<0.05);and there was little positive expression in the cornea of rats in the blank group.The number of VEGF positive cells in the experimental group was (2.631±0.238)cells per 400 times the field of vision,which was significantly lower than that in the control group [(6.254±0.721)cells],and the difference was statistically significant (P<0.05);there was little positive expression in the cornea of rats in the blank group. Conclusion PDTC,an inhibitor of NF-kappa B,can reduce corneal neovascularization and inhibit corneal transplantation rejection in rats after allograft keratoplasty.