TGF-β1基因修饰的树突状细胞对实验性自身免疫性重症肌无力大鼠外周血单个核细胞CD28／CTLA-4：B7表达的影响

Effect of TGF-β1 gene-modified dendritic cells on expressions of CD28/CTLA-4：B7 in peripheral blood mononuclear cells in rats with experimental autoimmune myasthenia gravis

目的探讨TGF-β1基因修饰的树突状细胞（DC）对实验性自身免疫性重症肌无力（EAMG）大鼠外周血单个核细胞（PBMC）CD28／CTLA-4：B7表达的影响。方法近交系8～10周龄健康雌性Lewis大鼠30只．分为正常组、EAMG组、DC对照组、pcDNA3-TGF-β1-DC组、pcDNA3-DC对照组、生理盐水对照组。除正常组外．其余各组均采用丁氏双鳍电鳐电器官乙酰胆碱受体（AChR）蛋白二次免疫的方法复制EAMG大鼠模型。初次免疫后第5天分别皮下注射2×10^6的DC、pcDNA3-TGF-β1-DC、pcDNA3-DC及等体积的生理盐水，正常组和EAMG组不接受任何治疗。初次免疫后7周分离各组PBMC,应用RT-PCR和流式细胞术方法分别进行CD28、CTLA-4mRNA及B7-1、B7-2蛋白表达水平的检测。结果（1）正常组大鼠PBMCCD28、CTLA-4mRNA的表达水平较低，尤其CTLA-4mRNA极少量表达：EAMG组大鼠CD28、CTLA-4mRNA的表达水平均明显增加；pcDNA3-TGF-β1-DC组CD28mRNA的表达较EAMG组明显降低（氏0．01），而CTLA-4mRNA的表达水平较EAMG组明显增加（P〈0．05）；EAMG组、DC治疗组、pcDNA3-DC对照组和生理盐水对照组之间CD28、CTLA-4mRNA的表达水平无显著性差异（胗0．05）。（2）正常大鼠B7-1、B7．2在PBMC上少量表达；EAMG组B7-1、B7-2两者表达明显增加（P〈0．001）；pcDNA3．TGF-β1-DC治疗组B7-1、B7-2的表达较EAMG组均明显降低（P〈0．011；DC对照组、pcDNA3-DC对照组、生理盐水对照组与EAMG组比较均无明显差异（P〉0．05）。结论EAMG大鼠存在PBMCCD28／CTLA-4：B7协同刺激分子的表达异常，主动调节机体的共刺激通路可能是TGF-β1基因修饰的DC治疗EAMG的机制之一。

Objective To explore the effect of dendritic cells （DC） modified with transforming growth factor β1 （TGF-β1） gene on the expressions of CD28/CTLA-4：B7 costimulatory molecules in peripheral blood mononuclear cells （PBMC） in the Lewis rats with experimental autoimmune myasthenia gravis （EAMG）. Methods Thirty inbreeding line, healthy, female Lewis rats were divided randomly into 6 groups： normal group, EAMG group, DC treatment group, pcDNA3-TGF-β1-DC treatment group, pcDNA3-DC control group and normal saline group. The rats were immunized with the AChR protein extracted from electric organ of Narcine timilei and CFA in the groups except normal group. 2x106 pcDNA3-TGF-β1-DCs/rat were injected subcutaneously into the backs of the rats which had been immunized 5 d earlier with AChR＋CFA. The rats in DC treatment group, pcDNA3-DC control group and normal saline group were injected in parallel with untreated DC, pcDNA3-DC and normal saline, respectively. Seven weeks after the first immunization, the expressions of CD28 mRNA and CTLA-4 mRNA were detected by reverse transcription-polymerase chain reaction （RT-PCR）, and the levels of B7-1 and B7-2 on the surface of PBMC were examined using flow cytometry. Results （1） The low expression of CD28 mRNA and rare expression of CTLA-4 mRNA were found in the normal rats, and both expressions increased markedly in EAMG rats （P〈0.001）. Compared to those in EAMG group, the expression of CD28 mRNA decreased and CTLA-4 mRNA was upregulated after the treatment with pcDNA3-TGF-β1-DC （/9〈0.05）. There was no significant difference in the expressions of CD28 mRNA and CTLA-4 mRNA among the EAMG group, DC treatment group, pcDNA3-DC control group and normal saline group （P〉0.05）. （2） The expressions of CD28, CTLA-4, B7-1 and B7-2 on the surface of PBMC were rare in normal rats, which increased significantly in EAMG rats （P〈0.001）. The levels of CD28, BT-1 and B7-2 in pcDNA3-TGF-β1-DC group were lower than those in EAMG group （P〈0.01）, but the level of CTLA-4 was higher than that in EAMG group （P〈0.05）. They showed no statistically difference among the EAMG group, DC treatment group, pcDNA3-DC control group and normal saline group （P〉0.05）. Conclusions The expressions of CD28/CTLA-4：B7 costimulatory molecules are abnormal in the rats with EAMG. The regulation of CD28/CTLA-4：B7 costimulatory pathways may play a critical role in the mechanism of the treatment with DC transfected with pcDNA3-TGF-β1 in the incipient EAMG rats.