拮抗CD4小分子化合物J2抗小鼠角膜移植排斥机制的初步研究

Initial study on mechanism of a small non-peptidic CD4 inhibitor J2 in prevention corneal allograft rejection

目的探讨拮抗CD4小分子化合物J2抗小鼠角膜移植排斥的机制。方法实验研究。采用随机数字表法将53只Balb／c小鼠分成A、B、c、D及E组，A、C、E组各13只，B、D组各7只，A组为不做任何处理的正常对照，B组为自体角膜原位移植组，C、D、E组为Balb／c—C57BL／6同种移植组，其中B、C组给予不含药物的空白液灌胃，D组给予环孢菌素A（CsA）10mg／kg体重，E组给予J215mg／kg体重。在角膜移植后第1至4周，每周行外周血流式细胞学检查，观察CD3^＋CD4^＋T淋巴细胞、CD3^＋CD8^＋T淋巴细胞的动态变化趋势，在角膜移植后3周行迟发性超敏反应实验，在角膜移植后18d行免疫酶联斑点实验，检测脾脏白细胞介素2（IL-2）、IL-10、γ干扰素（IFN-γ）的变化。实验数据通过单因素方差分析以及重复测量资料的单因素方差分析进行统计学处理。结果流式细胞学检查显示：E组小鼠外周血总CD3^＋T淋巴细胞、CD3^＋CD4^＋T淋巴细胞、CD3^＋CD8^＋T淋巴细胞较B、D组未发生特异性增殖（CD3^＋CD4^＋T淋巴细胞的E与B组间比较，P=0．776；E与D组间比较，P=0．606。CD3^＋CD8^＋T淋巴细胞的E与B组间比较，P=0．941；E与D组间比较，P=0．482）。迟发性超敏反应结果显示：在移植后3周，E组小鼠对供体脾细胞和第3鼠系脾细胞均呈低反应，与A组小鼠比较差异无统计学意义（F=1．00，P=0．422）。免疫酶联斑点试验结果显示：E组分泌IL-2较A组增加（36．0±7．6），分泌IFN-1较A组增加（56．0±42．2），但与B组比较，差异无统计学意义（IL-2比较，P=0．832；IFN-γ比较，P=0．356）。IL-10各组之间差异无统计学意义（F=2．911，P=0．240）。结论J2阻断了CD4与主要组织相容性抗原复合物Ⅱ（MHC-Ⅱ）类分子的抗原提呈过程，特异性抑制了抗原特异性迟发性超敏反应的发生；在J2阻断CIM／MHC-II类分子的抗原提呈过程后，IL-2、IFN-1及IL-10分泌未发生特异性变化。

Objective To discuss the mechanism of J2 on preventing mice＇s corneal rejection. Methods In experimental study, Balb/c mice had been divided into A, B, C, D, E groups randomly. There were 13 mice in each of Group A, C and E, while 7 in both Group B and D. Group A did no management; Group B, autograft control; Group C, D and E, allograft groups （C57BL/6 were used as donors）; Group B and Group C were given placebo; Group D and Group E were treated with orally cyclosporine A （CsA） （10 mg per kilogram of body weight） and J2 （15 mg per kilogram of body weight）, respectively. To observe the variation of lymphocyte subgroup of peripheral blood mononuclear ceils in different groups by flow cytometer analysis every week after corneal transplantation. DTH assay had been done at week 3, cytokines including interleukin-2 （IL-2）, IL-10, interferon-γ （IFN-γ）, secreting level of mouse spleen cells detected by ELLISPOT assay on day 18 after corneal transplantation. To apply single factor analysis of variance, single factor analysis of variance for repeat measured data and so on to analyze the data. Results The results of flow cytometer analysis showed： lymphocyte subgroup of peripheral blood mononuclear cells had not proliferated specifically in Group E, compared to Group B and Group D（ for CD3^＋ CD4^＋ T lymphocyte, E-B,P = 0. 776, E-D,P = 0. 606 ; for CD3^＋ CD8^＋ T lymphocyte, E-B, P = 0. 941, E- D,P =0. 482）. DTH assay showed low reaction to both donor＇s and third strain mouse spleen cells in Group E （F = 1.00,P =0. 422）. The results of ELLISPOT assay indicated： the spot level of IL-2,IFN-γ in Group E raised slightly compared to Group A, but compared to Group B, there was no statistically significant dlfference（for IL-2,P =0. 832;for IFN-γ,,P =0. 356）. The spot level of IL-10 between all groups had no statistically significant difference （ F = 2. 911, P = 0. 240）. Conclusion J2 could block up the process of antigen presentation by inhibiting CD4/major histocompability complex- ]Ⅱ（ MHC-Ⅱ ） moleculeonania, while secreting IL-2, IL-10 and IFN-γ/did not have specificity change.