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目的:研究雷帕霉素(Rapamycin,RAPA)和环孢素A(cyclosporin A,CsA)体内处理对同种移植小鼠急性排斥过程中TLR5及Foxp3表达的影响。方法:建立同种皮肤移植模型,术后给予RAPA或CsA,1次/d,连续14d,同时设生理盐水对照组。每日观察移植物存活状况。术后选取1、7、10、14、21d共5个时间点,提取受体小鼠脾细胞,RT-PCR检测TLR5及Foxp3mRNA表达,探讨不同处理组基因表达与移植物生存的相关性。体外实验利用鞭毛蛋白(flagellin)联合RA-PA和CsA处理正常小鼠T细胞6h,RT-PCR检测TLR5表达。结果:RAPA和CsA可明显延长小鼠同种移植皮片存活期。RAPA可促进脾细胞TLR5mRNA表达升高,停药后的第21天仍明显高于CsA组和对照组(P<0.05);CsA组在第7、10天有显著升高,第21天降低(P<0.05);3组中最高表达的时间点为移植后第10天,此时正值排斥高峰期。RAPA可引起Foxp3mRNA的表达升高(P<0.05),停药后仍维持较高水平;CsA组仅在移植后第7、10天短暂升高,第14天低于对照组(P<0.05)。移植后1、7、10、21d3组的TLR5与Foxp3mRNA变化趋势正相关。体外实验中,RAPA或CsA与flagellin联合使用,可促进TLR5的表达,以前者的作用更为明显。结论:RAPA和CsA在同种移植急性排斥高峰期可引起TLR5和Foxp3表达的协同升高,且RAPA的作用更加明显和持久,鞭毛蛋白体外可以增强这种作用效果。
Objective: To investigate the effects of Rapamycin (RAPA) and cyclosporin A (CsA) treatment on the expression of TLR5 and Foxp3 during acute rejection in allograft mice. Methods: Allogeneic skin graft model was established. RAPA or CsA was given once a day for 14 consecutive days. Meanwhile, normal saline control group was established. Daily observation of graft survival. At 5, 7, 10, 14, and 21 days after operation, the spleen cells of recipients were extracted and the expressions of TLR5 and Foxp3 mRNA were detected by RT-PCR. The correlation between gene expression and graft survival was analyzed. In vitro experiments T cells of normal mice were treated with flagellin combined with RA-PA and CsA for 6h. The expression of TLR5 was detected by RT-PCR. Results: RAPA and CsA could significantly prolong the survival of mice skin allografts. RAPA could promote the expression of TLR5mRNA in splenocytes, and the level of TLR5mRNA in splenocytes was significantly higher than that in CsA group and control group on the21stday (P <0.05); CsA group was significantly increased on the7th and10th days and decreased on the21nd day P <0.05). The time point of the highest expression in the three groups was the 10th day after transplantation, at which time the peak value of exclusion was positive. RAPA caused a significant increase in Foxp3mRNA expression (P <0.05), and maintained a high level after stopping the treatment. CsA only transiently increased on the 7th and 10th days after transplantation, and decreased on the 14th day (P <0.05) . There was a positive correlation between TLR5 and Foxp3mRNA in 1,7,10,21d3 group after transplantation. In vitro experiments, RAPA or CsA combined with flagellin can promote the expression of TLR5, the role of the former is more obvious. CONCLUSION: RAPA and CsA can induce the synergistic increase of TLR5 and Foxp3 expression at the peak of acute rejection in allograft, and the effect of RAPA is more obvious and persistent. Flagellin can enhance this effect in vitro.