小分子化合物J2抑制小鼠角膜移植术后排斥反应的研究

Prevention of mouse corneal allograft rejection by micromolecular compound J2

目的探讨新型药物J2抗同种异基因小鼠角膜移植术后排斥反应的作用及其机制。方法实验研究。采用随机分组设计的研究方法。建立以C57BL／6小鼠为供体，BALB／c小鼠为受体的角膜移植实验模型，其中供体38只，受体100只，随机分为A、B、C、D四组，每组25只。A组为BALB／c小鼠自体原位角膜移植，B、C及D组均采用C57BL／6-BALB／c同种异体角膜移植，术后采用腹腔注射给药，自手术当日开始，A组、B组给予安慰剂，C组给予CsA10mg／kg体重，D组给予J215mg／kg体重，每天1次，连续给药12d。观察各组植片生存指数变化，苏木素-伊红染色及冰冻切片免疫组织化学观察角膜植片病理改变；分别在术后1、2、3及4周行逆转录聚合酶链反应（RT—PCR）检测角膜植片中细胞因子的表达。各组间植片存活时间差异采用One—Way ANOVO分析进行比较，两组之间比较采用SNK法。结果B组角膜植片平均存活时间为（18．88±4．19）d，D组发生排斥时间明显延迟为（33．62±6．80）d，两组比较差异有统计学意义（q=8．33，P=0．00），而D组与C组比较差异无统计学意义（q=0．85，P=0．60）。组织学检查证实，术后21d，B组见大量细胞浸润，而D组与C组植片未见明显的细胞浸润。免疫组织化学检测显示安慰剂组角膜植片大量CD4^＋和CD8^＋T淋巴细胞浸润，J2组与CsA组植片仅有少量的CD4^＋和CD8^＋T淋巴细胞浸润。RT—PCR结果显示，在正常小鼠角膜未见IL-10和IFN-γ基因表达；异基因移植组从第1周开始表达各种基因，第3周表达明显增强；而J2组与CsA组从第1周开始表达，第2、3周表达减弱，第4周表达强于前3周。结论J2通过拮抗CD4与主要组织相容性抗原（MHC）Ⅱ分子结合抑制CD4^＋T淋巴细胞激活，从而具有抗小鼠角膜移植排斥作用。

Objective To investigate the effects of J2 on the prevention of corneal allograft rejection Randomized control design was applied. One hundred C57BL/6 and thirty eight BALB/c mice were used as donors and recipients, rspetively, to establish a corneal allograft model. These mice were randomly divided into A, B, C and D groups. There were 25 mice in each group. Group A, autograft control; Group B, allograft control （ placebo was used in this group ） ; Group C, allograft and treated with intraperitoneal ciclosporin A （CsA, 10 mg · kg^-1 · d^-1 ） and Group D, allograft and treated with intraperitoneal J2 （15 mg · kg^-1 · d^-1 ）. All medications were applied on the day of transplantation and were continuously administrated for 12 days. Graft survival index, paraphin sections with HE staining and immunohistochemical staining of frozen sections were observed in each group 1, 2, 3 and 4 weeks after operation. IL-2 and IL-10 expression of cornea was detected by RT-PCR. One-Way ANOVO analysis was used to compare transplant survival time of the corneal allograft of each group. Results The average transplant survival time in the corneal allograft controls was （18.88 ±4. 19） d. Treatment with J2 resulted in a significant increase of grafts＇ survival time （ 33.62 ± 6. 80 ） d （ q = 0. 85, P 〈 0.01 ）. There was no significant difference between J2 and CsA groups. Histological examination showed that numerous lymphocytes infiltrated into the grafts 21 days after the operation in the control groups. Mild lymphocytes infiltration was found in both CsA and J2 groups. Immunohistochemical studies showed that large amount of CD4^＋ and CD8^＋ T-lymphocytes infiltrated into the grafts of the placebo group at 21 days. J2 and CsA groups only showed a small number of CD4^＋ and CD8^＋ T-lymphocytes in the grafts. RT-PCR showed that few IL-2 and IL-10 genes were expressed in the autologous transplantation group in the first week, and the expression of these two cytokines showed no significant increase during following three weeks. Both genes were expressed from the first week in the allogeneic transplantation group and significantly increased in the third week. Both genes were expressed from the first week in CsA and J2 group, expression was reduced in the second and third weeks. Expression of these cytokines in the fourth week was stronger than those in previous three weeks. Conclusion J2 can inhibit corneal rejection in allograft mice model and its effect is similar to that of CsA.