摘要
目的：淋巴细胞具有独立的胆碱能系统,包括乙酰胆碱(Ach)、胆碱乙酰基转移酶(ChAT)、乙酰胆碱酯酶(AChE)、M型乙酰胆碱受体(mAChR)和N型乙酰胆碱受体(nAChR).小鼠胸腺中未成熟的T淋巴细胞、大鼠脾脏CD3+T淋巴细胞和多种T细胞株均表达α7烟碱型乙酰胆碱受体(a7nAChR),且此受体在胆碱能抗炎通路中也发挥重要作用。前期研究证实小鼠CD4+CD25+调节性T细胞(Treg)表面表达a7nAChR,且激活该受体可以增强CD4+CD25+Treg的免疫抑制功能。本实验对CD4+CD25+Treg中a7nAChR与JAK3-STAT5信号通路之间的关系进行了进一步的研究。
方法：1.采用免疫磁珠法分选正常C57BL/6J小鼠脾脏CD4+CD25+Treg,流式细胞术鉴定CD4+CD25+Treg的纯度,台盼蓝染色鉴定其活性；2.a7nAChR激动剂烟碱、受体特异性拮抗剂α-银环蛇毒预处理Treg后,与CD4+CD25-T细胞共培养,使用MTT法检测CD4+CD25-T细胞的增殖变化情况；3.通过激光共聚焦、Western blot技术检测CD4+CD25+Treg细胞表面a7nAChR下游相关信号通路蛋白JAK3及STAT5的表达情况；4.同样方法采用烟碱及α-银环蛇毒预处理CD4+CD25+Treg后,通过Western blot法检测细胞中JAK3及STAT5蛋白的表达变化情况。
结果：1.免疫磁珠法分选得到的CD4+CD25+Treg细胞纯度在90%以上,台盼蓝染色显示细胞活性大于97%。2.MTT检测显示10μmol/L烟碱刺激可增强Treg细胞对CD4+CD25-T细胞的抑制效应,1μmol/Lα-银环蛇毒单独作用、烟碱与α-银环蛇毒联合作用Treg细胞后,与对照组(未处理Treg细胞)相比Treg细胞的免疫抑制功能无明显变化。3.激光共聚焦荧光检测分析显示,CD4+CD25+Treg胞浆中存在JAK3、STAT5蛋白及其磷酸化形式p-JAK3、p-STAT5。同一视野下,CD4+CD25+Treg在495nm激光激发下,细胞表面的CD25-PE发出橙色荧光,胞浆中FITC二抗间接标记的JAK3、p- JAK3、STAT5和p-STAT5发出绿色荧光,软件叠加后图片显示为橘黄色荧光。Western blot技术进一步在蛋白水平检测CD4+CD25+Treg内存在JAK3、p- JAK3、STAT5和p-STAT5表达,其分子量分别为106kD、106kD、92kD和94kD。4.烟碱处理后,CD4+CD25+Treg上a7nAChR下游信号通路p-JAK3及p-STAT5蛋白表达增高,α-银环蛇毒单独作用、烟碱与α-银环蛇毒联合作用Treg细胞后,上述两蛋白水平无显著改变。
结论：1.激活a7nAChR,可以增强CD4+CD25+Treg细胞对CD4+CD25-T细胞的免疫抑制效应。2.CD4+CD25+Treg细胞存在JAK3. p- JAK3. STAT5和P-STAT5表达。3.烟碱可以增强p- JAK3和P-STAT5表达,a7nAChR特异性拮抗剂可以逆转该效应。
Objectives:Lymphocytes have their own independent cholinergic system, which includes acetylcholine (ACh), choline acetylase (ChAT), acetylcholinesterase (AChE), muscarinic ACh receptor (mAChR) and nicotinic ACh receptor (nAChR). The a7 nicotinic acetylcholine receptor (a7nAChR) that plays an important role in the cholinergic anti-inflammtory pathway is expressed by the immature T lymphocytes in mouse thymus gland, CD3 positive T cell in rat spleen and multiple T cell strains. Former study has confirmed that mouse CD4+CD25+ regulatory T cell (Treg) expressed a7nAChR on its surface, and activation of this receptor could enhance the suppressive function of Treg on other kinds of immune cells. The present study investigated the association between a7nAChR and the JAK3-STAT5 signaling pathway in mouse CD4+CD25+ Treg.
Methods:1. CD4+CD25+ regulatory T cells from the spleen of C57BL/6J mice were separated by means of MACS microbeads and then identified for purity and activity using flow cytometry and trypan blue staining; 2. Pretreating these cells with nicotine (agonist of a7nAChR) and a-bungarotoxin (receptor's specific antagonist), then we co-cultured them with CD4+CD25- T cells whose proliferative situation was then assessed by MTT assay; 3. Treg cells were tested for the expression of signaling pathway protein JAK3 and STAT5 through confocal microscopy and western blotting; 4. Similarly pretreating CD4+CD25+ Treg cells with nicotine and a-bungarotoxin, we evaluated the expression changes of JAK3 and STAT5 protein with western blot test.
Results:1. The purity of the separated CD4+CD25+ Treg cells was above 90% and more than 97% of which were alive; 2. MTT assay showed that Treg cells which were treated with 10μmol/L nicotine had higher suppressive effect on CD4+CD25- T cells' proliferation compared with control group (untreated Treg cells), and Tregs which were treated with 1μmol/L a-bungarotoxin alone or with nicotine and a-bungarotoxin co-administration had no such effect; 3. Confocal microscopy assay showed that there were JAK3, STAT5, p-JAK3 and p-STAT5 proteins expressed in the cytoplasm of CD4+CD25+ Treg. Furthermore, Western blot test got protein strips of JAK3, STAT5, p-JAK3 and p-STAT5 whose molecular weight were 106kD,106kD,92kD and 94kD respectively in Treg cell's gross protein extracts; 4. Pretreated with nicotine, CD4+CD25+ Treg cells expressed more p-JAK3 and p-STAT5 proteins than untreated ones, but a-bungarotoxin alone or nicotine and a-bungarotoxin co-administration treatment had no such effect mentioned above.
Conclusions:1. The results suggest that activation of a7nAChR may enhance the immunosuppressive function of CD4+CD25+ Treg cells; 2. It is found that JAK3, p-JAK3, STAT5 and p-STAT5 proteins be expressed in CD4+CD25+ Treg cells.3. Nicotine may enhance the expression of p- JAK3 and p-STAT5 in Treg cells, however, such effect can be reversed by the specific antagonist of a7nAChR a-bungarotoxin.
引文
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