黑睛排斥汤对大鼠高危角膜移植免疫排斥反应的防治研究

背景高危角膜移植患者虽然常规应用免疫抑制性药物，但角膜移植片的排斥率仍很高。近年来传统中药在角膜移植中的作用逐渐引起关注，黑睛排斥汤对大鼠高危角膜移植免疫排斥反应的防治作用需要进一步研究。目的探讨黑睛排斥汤对大鼠高危角膜移植免疫排斥反应的抑制作用。方法16只正常雌性SD大鼠角膜作为供体，32只雌性Wistar大鼠作为受体，用角膜缝线法诱导角膜新生血管（CNV），建立大鼠高危角膜移植模型，然后行封闭群大鼠SD．Wistar组间同种异体全板层角膜移植。采用随机数字表法将大鼠随机分为4个组，包括模型对照组（生理盐水灌服）、CsA组[10mg／（kg·d）CsA灌服]、黑睛排斥汤组[4g／（kg·d）黑睛排斥汤灌服]和联合用药组[10mg／（kg·d）CsA＋4g／（kg·d）黑睛排斥汤灌服]，各组大鼠均于同种异体角膜移植后4d开始灌服相应药物，于术后第4天起裂隙灯显微镜下观察眼前节反应，每隔2天观察1次，连续观察30d。参考Larkin等的标准对角膜}昆浊程度、水肿程度及新生血管程度进行评分，记录移植排斥指数（RI）并观察植片的存活状况，对角膜进行常规组织病理学检查，采用流式细胞分析法对受体角膜局部和全身免疫状态进行检测，对4个组大鼠的各种检测指标进行比较。结果模型对照组大鼠角膜移植术后10d左右发生排斥反应，CsA组和黑睛排斥汤组大鼠均于术后12～13d发生排斥反应，而联合用药组大鼠于术后22d发生排斥反应。与模型对照组比较，CsA组、黑睛排斥汤组和联合用药组大鼠角膜移植后角膜混浊程度评分、角膜水肿评分和新生血管评分均明显下降，差异均有统计学意义（P〈0．05），且联合用药组上述各指标的评分均明显低于CsA组和黑睛排斥汤组，差异均有统计学意义（P〈0．05），而CsA组与黑睛排斥汤组的各种评分差异均无统计学意义（P〉0．05）。模型对照组角膜移植片存活时间平均为（10．38±1．69）d，联合用药组平均为（22．50±3．07）d，差异有统计学意义（t=-9．790，P=0．000）。组织病理学检查表明，术后15d联合用药组大鼠角膜基质中淋巴细胞浸润及新生血管评分少于CsA组和黑睛排斥汤组。流式细胞学检查证实，黑睛排斥汤组、CsA组和联合用药组大鼠角膜移植术后外周血中CD4＋淋巴细胞计数及CD4＋／CD8＋比值均低于模型对照组，联合用药组CD4＋淋巴细胞计数及CD4＋／CD8＋比值均低于CsA组和黑睛排斥汤组，差异均有统计学意义（P〈0．05）。结论黑睛排斥汤对高危角膜移植模型大鼠术后角膜排斥反应有抑制作用，其疗效与0．1％CsA接近，二者联合用药有协同作用。

The prevention and treatment of heijingpaichitang for immune rejection following high-risk corneal transplantation in rats

The rate of corneal graft rejection is still high for high-risk keratoplasty although immune suppression drug is routinely used. The role of traditional Chinese medicine in corneal transplantation is concerned gradually. Heijingpaichitang on the prevention and treatment of rats with high-risk corneal allograft rejection needs further study. Objective This study was to investigate the inhibitory effect of heijingpaichitang on high-risk corneal transplantation immune rejection in rats. Methods Sixteen female SD rats were used as the donors and 32 female Wistar rats were served as recipients. The high-risk corneal trasplantation models were established by corneal suture in 32 Wistar rats, and then homogeneity variant SD-Wistar corneal transplantation was performed. The recipients were randomized into model control group, cyclosporine A （CsA）group, heijingpaichitang group and CsA ＋ heijingpaichitang group. CsA, heijingpaichitang and CsA ＋ heijingpaichitang was orally administered 4 days after operation once per day for 15 days, and normal saline solution was used at the same way in the model control group. Ocular anterior segment reaction was examined under the slit lamp and corneal opacification, edema and neovaseulation were scored based on Larkin＇ s criteria. Rejection index of the corneal graft was recorded and the graft survival time was calculated. The pathological examination of the corneal graft was carried out in all rats, and the inflammatory ceils in the corneas and CD4＋ cells in the periphery blood were assayed using flow cytometry. The use of the animals complied with ARVO Statement. Results Corneal graft rejection occurred in 10 days after operation in the model control group, 12- 13 days in the CsA group and heijingpaichitang group and 22 days in the CsA＋ heijingpaichitang group. Compared with model control group, the scores of the corneal opacification, corneal edema and nenvascularization were significantly lower in the CsA group,heijingpaichitang group and CsA＋heijingpaichitang group （P〈0.05）, and all the scores were declined in the CsA ＋heijingpaiehitang group compared with CsA group and heijingpaichitang group（P〈0.01 ） ,but no significant differences were seen in the scores between the CsA group and heijingpaichitang group（P〉0.05 ）. The mean survival time of grafts was（ 10.38±1. 69）days in the model control group, （ 22.50± 3.07 ） days in the CsA ＋ heijingpaichitang group, with the significant difference （ t = - 9. 790, P =0. 000）. The pathological examination of graft showed that the lymphocytes and new blood vessels were less in the CsA＋heijingpaichitaug group compared with CsA group and heijingpaichitang group 15 days after operation. Flow cytometry verified that the number of lymphocytes in graft,CIM＋cells and CD4＋/CD8＋ in periphery blood were significantly lower in the heijingpaichitang group, CsA group and CsA ＋ heijingpaiehitang group compared with model control group （P〈0. 05）. Conclusions Heijingpaichitang can inhibit immune rejection to certain extent in high-risk corneal transplantation rat and has a similar effect to 0. 1% CsA. Heijingpaichitang and 0. 1% CsA have a synergistic effect.