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浙江大学学报（医学版）
浙江大学学报（医学版）  2021, Vol. 50 Issue (5): 621-626    DOI: 10.3724/zdxbyxb-2021-0173
原著     
雷帕霉素促进哮喘调节性T细胞分化及功能的机制
金华良,周燕,王利民()
浙江大学医学院附属杭州市第一人民医院呼吸科，浙江 杭州 310006
The mechanism of rapamycin in promoting asthmatic regulatory T cell differentiation and function in vitro
JIN Hualiang,ZHOU Yan,WANG Limin()
Department of Respiratory Medicine, Affiliated Hangzhou First People’s Hospital, Zhejiang University School of Medicine, Hangzhou 310006, China
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摘要：

目的：探讨雷帕霉素对哮喘调节性T细胞体外分化的作用及机制。方法：采用卵蛋白致敏、激发制备哮喘模型。采用超高速流式细胞分选仪收集哮喘小鼠和正常小鼠的脾CD4+CD25– ?T细胞，进一步分为三组，其中模型对照组予转化生长因子β（TGF-β）、白介素（IL）-2，雷帕霉素组予TGF-β、IL-2的同时用雷帕霉素（终浓度为500?nmol/L）干预。采用流式细胞仪检测调节性T细胞水平和CD4+CD25– ?T细胞增殖情况；采用蛋白质印迹法测定哺乳动物雷帕霉素靶蛋白复合体（mTORC）1/2通路相关蛋白S6、Akt磷酸化水平。结果：与模型对照组比较，雷帕霉素组调节性T细胞比例增加，CD4+CD25– ?T细胞增殖水平降低，mTORC1/2信号通路下游S6、Akt磷酸化水平降低（均P<0.05）。结论：雷帕霉素可促进调节性T细胞的分化及功能，其在一定程度上与抑制mTORC1/2信号通路相关。
关键词： 哮喘;  雷帕霉素;  调节性T细胞;  哺乳动物雷帕霉素靶蛋白;  分化;  增殖；体外实验    
Abstract:

Objective:To investigate the mechanism of rapamycin in promoting asthmatic regulatory T cell differentiation in vitro. Methods: Asthma model was prepared by sensitization and challenge of ovalbumin in mice. Spleen CD4+CD25– ?T cells were sorted from the asthmatic mice and normal mice by ultrahigh speed flow cytometer, and divided into three groups. Transforming growth factor-β and interleukin-2, or combined with rapamycin (final concentration of 500?nmol/L) were given in the model group or the rapamycin group. The levels of Treg cells and CD4+CD25– ?T cells were detected by flow cytometry. The phosphorylation level of downstream proteins of S6 and Akt in the mTORC1/2 signaling pathway were examined by Western blotting. Results:Compared with the model group, the differentiation level of Treg cells in the rapamycin group was significantly increased, the proliferation level of CD4+CD25– ?T cells was decreased, and the phosphorylations of the mTORC1/2 substrates, S6 protein and Akt were decreased （all P<0.05）.Conclusion:Rapamycin can promote the differentiation and function of Treg cells via inhibition of the mTORC1/2 signaling pathway.
Key words: Asthma    Rapamycin    Regulatory T cell    Mammalian target of rapamycin    Differentiation    Proliferation; In vitro experiment
收稿日期: 2021-06-23 出版日期: 2021-12-29
:  R562.2  
基金资助: 浙江省自然科学基金（LY19H290011）； 国家自然科学基金（81403247）； 中国博士后科学基金（2016M601982）
通讯作者: 王利民     E-mail: lemonwlm@163.com
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引用本文:

金华良,周燕,王利民. 雷帕霉素促进哮喘调节性T细胞分化及功能的机制[J]. 浙江大学学报（医学版）, 2021, 50(5): 621-626.

JIN Hualiang,ZHOU Yan,WANG Limin. The mechanism of rapamycin in promoting asthmatic regulatory T cell differentiation and function in vitro. J Zhejiang Univ (Med Sci), 2021, 50(5): 621-626.

链接本文:

https://www.zjujournals.com/med/CN/10.3724/zdxbyxb-2021-0173    或    https://www.zjujournals.com/med/CN/Y2021/V50/I5/621

图1  流式细胞术分选正常小鼠和哮喘模型小鼠脾脏来源CD4CD25 T细胞结果
图2  流式细胞术测定各组调节性T细胞分化比例Foxp：叉头状转录因子P亚组.
图3  流式细胞术测定各组调节性T细胞增殖结果CFSE：羧基荧光素二醋酸盐琥珀酰亚胺酯.
图4  各组脾CD4CD25 T细胞S6、Akt磷酸化水平电泳图+–GAPDH：甘油醛-3-磷酸脱氢酶.

组别

n

磷酸化S6/S6

磷酸化Akt/Akt

空白对照组

3

3.062±0.233

0.473±0.024

模型对照组

3

2.358±0.314

0.350±0.008

雷帕霉素组

3

0.929±0.122*

0.179±0.002*
表1  各组CD4CD25 T细胞S6、Akt磷酸化表达水平比较
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