PI3K-Akt-mTOR信号通路对Foxp3基因表达的影响

Effect of PI3K-Akt-mTOR signal pathway on the expression of Foxp3

目的研究PI3K-Akt-mTOR信号通路对Foxp3基因表达的影响。方法将SPF级昆明系小鼠60只随机分为对照组(A)和实验组(B、C、D),B、C、D三组分别灌胃雷帕霉素0.2、0.4、0.6 mg.d-1,A组每天予以无菌水灌胃,共3周。3周后,无菌条件下心脏采血,EDTA抗凝,分离脾脏,制备单细胞悬液,采用流式细胞仪检测小鼠外周血和脾脏中CD4+CD25+调节性T细胞水平(CD4+CD25+Treg细胞占CD4+T细胞的百分比),RT-PCR检测小鼠脾细胞Foxp3mRNA的表达,免疫组织化学SP法染色观察小鼠脾脏中p-mTOR蛋白表达的变化,并利用计算机图像分析技术测量各实验组及对照组中p-mTOR蛋白表达的平均光密度。结果 Foxp3 mRNA在实验组(B、C、D)小鼠脾脏中表达的程度分别为(0.4853±0.0574、0.7886±0.1085、0.9639±0.2015),明显高于对照组A(0.1345±0.0271)(P<0.05);实验组(B、C、D)小鼠脾脏中p-mTOR蛋白表达的平均光密度分别为(0.2326±0.0431、0.1156±0.0223、0.0556±0.0041),明显低于对照组A(0.4223±0.0534)(P<0.05);两指标经pearson相关分析,小鼠脾脏中Foxp3mRNA的表达与p-mTOR蛋白表达呈负相关。结论抑制PI3K-Akt-mTOR信号通路会促使Foxp3的表达增强;PI3K-Akt-mTOR信号通路的激活程度与Foxp3的表达程度呈负相关。

Aim To investigate the effect of PI3K-Akt-mTOR signal pathway on the expression of Foxp3 in vivo.Methods 60 Kunming-strain mice at the age of 8 weeks were randomly divided into a control group（A） and three experimental groups（B,C,D）.The mice in the groups B,C and D were fed with rapamycin（RAPA） 0.2 mg·d-1,0.4 mg·d-1 and 0.6 mg·d-1 via intragastric administration.The mice in the group A were given with sterile water as the control group.After three weeks,the peripheral blood（with EDTA anticoagulant） was collected under sterile conditions and the splenocytes were seperated to generate a single-cell suspension.The levels of CD4＋CD25＋Treg cells in the peripheral blood and splenocytes of mice were detected by flow cytometer.（The ratio of CD4＋CD25＋ Treg cells to CD4＋CD25＋ Treg cells）.The expression of Foxp3 mRNA in spleen cells of mice were detected with RT-PCR assay.Variation of p-mTOR expression in spleens were observed with immunohistochemical staining（SP method） and the average opticals of protein p-mTOR′s expression between experimental groups and control group were tested with computer image analytical technique.Results The levels of Foxp3 mRNA in spleen cells of mice among experimental groups（B,C,D） were 0.4853±0.0574,0.7886±0.1085 and 0.9639±0.2015 respectively,which were significantly higher than control group A（0.1345±0.0271）（P0.05）;The average opticals of protein p-mTOR′s expression among experimental groups（B,C,D） were 0.2326±0.0431,0.1156±0.0223 and 0.0556±0.0041 respectively,which were lower than control group A（0.4223±0.0534）（P0.05）.It showed that there was a negative correlation between the expression of Foxp3 mRNA and protein p-mTOR which were handled through pearson correlation analysis.Conclusion RAPA can enhance the expression of Foxp3 by inhibiting PI3K-Akt-mTOR signal pathway.There is a negative correlation between the activation of PI3K-Akt-mTOR signal pathway and the expression of Foxp3.