N-(3,4-二甲氧基肉桂酰)邻氨基苯甲酸介导小鼠皮肤移植免疫负向调节的作用及机制研究

Study on effect and mechanism of N-(3,4-dimethoxycinnamonyl)anthranilic acid mediated negative immune regulation in mice skin transplantation

目的研究N-(3,4-二甲氧基肉桂酰)邻氨基苯甲酸[N-(3,4-dimethoxycinnamonyl)anthranilic acid,3,4-DAA]在小鼠皮肤移植中的免疫负调节作用,并探索其发挥作用的机制。方法 体外实验中,取同种异基因小鼠皮肤移植7d后的BALB/C小鼠脾脏制得淋巴细胞单细胞悬液,一半用四甲基偶氮唑蓝比色法(methyl thiazolyl tetrazolium,MTT)检测小鼠脾脏淋巴细胞生长抑制率;另一半分为3组,分别加入RPMI1640培养液10μl(c1组)、3,4-DAA100μmol/L(c2组)、加入3,4-DAA200μmol/L(c3组),培养72h,进行病理学检查。同时用蛋白免疫印迹法检测吲哚胺2,3-二氧化酶(indoleamine2,3-dioxygenase,IDO)表达水平、用反相高压液相质谱法测定IDO活性。体内实验中,应用不同浓度3,4-DAA或合并使用西罗莫司进行药物干预,皮肤移植后10d取移植皮片行病理检查,用蛋白免疫印迹法检测脾脏淋巴细胞单细胞悬液中IDO的表达水平,用反相高压液相质谱法测定移植小鼠血清的IDO活性。结果 体外实验中,MTT检测结果示3,4-DAA对小鼠脾脏淋巴细胞增殖有抑制作用,呈现明显的剂量效应关系(P<0.05～P<0.01);在倒置显微镜下观察,与c1组比较,c3组、c2组皮肤移植小鼠的脾脏淋巴细胞悬液中的淋巴细胞生长明显受抑制,且c3组的淋巴细胞生长抑制较之c2组更显著,显示3,4-DAA有抑制脾脏淋巴细胞生长的作用。体内实验中,给予3,4-DAA处理组小鼠移植皮肤免疫损伤程度明显减轻。体外实验与体内实验结果示3,4-DAA处理组IDO表达水平、IDO活性较空白对照组均明显提高(P<0.05～P<0.01)。结论 3,4-DAA能够抑制小鼠皮肤移植排斥反应,显著减轻移植物排斥损伤,其作用机制是通过增强IDO活性实现的。

Objective To investigate the effect of N-（3,4-dimethoxycinnamonyl）anthranilic acid（3,4-DAA）in mice skin transplantation and explore the mechanisms.Methods In vitro experiments,T lymphocytes monoplast suspension was isolated from BALB/C mice splenocytes on day 7 after allogene mice skin transplantation.Half of the suspension was used to measure the splenic T lymphocyte proliferation inhibition rate using methyl thiazolyl tetrazolium（MTT）measurement.The other half of suspension was divided into 3 groups：c1 group（added RPMI 1640 solution 10 μl）,c2 group（added 3,4-DAA 100 μmol/L）and c3 group（added 3,4-DAA 200 μmol/L）.All the samples were cultured for 72 h before pathematology examination.Western blot was also applied to analyze the expression of indoleamine 2,3-dioxygenase（IDO）.High performance liquid chromatography（HPLC）was used to determine the IDO activity.In vivo experiment,diff-erent concentrations of 3,4-DAA were used as drug interposition and sometimes combined with sirolimus.The skin grafts were collected for pathematology examination on day 10 after transplantation.The expression of IDO in the splenic T lymphocytes monoplast suspension was measured by Western blot.And the IDO activity in the serum of transplanted mice was measured by HPLC.Results In vitro experiments,the MTT results showed that 3,4-DAA significantly decreased the T lymphocyte proliferation and had significant dosage effect（P0.05-P0.01）.Under the inverted microscope,the lymphocyte proliferation was significantly restrained in the splenic T lymphocytes monoplast suspension of skin transplanted mice in c3 and c2 group,compared with c1 group.And the restraint was more significant in c3 group compared with c2 group,which showed that 3,4-DAA could restrain the lymphocyte proliferation in the spleen.In vivo experiment,the immunologic injury condition of skin graft significantly relieved in the 3,4-DAA treated group.Both in vitro and in vivo experiments showed that IDO expression and IDO activity significantly increased in 3,4-DAA treated group compared with the control group（P0.05-P0.01）.Conclusion 3,4-DAA can suppress the rejection of mice skin transplantation and relieve the damage of allograft rejection through enhancing the IDO activity.