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| 蛋白激酶C-θ在吗啡诱导Th2细胞分化中的作用 |
| Role of PKC-θ in morphine-induced Th2 effector cells differentiation |
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| DOI：10.12089/jca.2020.06.014 |
| 中文关键词: 蛋白激酶C-θ 吗啡 Th2细胞分化 |
| 英文关键词: Protein kinase C-theta Morphine Th2 cell differentiation |
| 基金项目:江苏省六大人才高峰项目(2016WSW 113)；江苏大学临床医学科技发展基金项目（JLY20180062）；无锡市卫健委青年科研项目（Q201828）;宜兴市科技局社会发展项目（2016 11） |
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| 中文摘要: |
目的 探讨蛋白激酶C-θ（PKC-θ）在吗啡诱导初始T细胞分化模型中的作用及潜在机制。
方法 取小鼠脾脏，制备单细胞悬液，使用分离试剂盒获得初始T细胞。将提纯的初始T细胞分为六组：对照组、吗啡组、纳洛酮（吗啡拮抗剂）组、AEB071（PKC-θ抑制剂）组、吗啡+纳洛酮组和吗啡+AEB071组。按组别加入不同刺激物培养，收取细胞，采用流式细胞仪检测Th1和Th2细胞数量，采用ELISA法检测细胞因子IL-4和IFN-γ浓度，采用Western blot法检测PKC-θ S676、T538两个位点的磷酸化水平，采用实时荧光定量PCR（qPCR）检测GATA3 mRNA表达量，采用凝胶迁移实验（EMSA）检测GATA3转录活性。
结果 与对照组比较，吗啡组Th2细胞数量明显增多，IL-4浓度明显升高，PKC-θ S676、T538两个位点的磷酸化水平明显升高，GATA3 mRNA表达量及转录活性均明显升高（P<0.01）。与吗啡组比较，吗啡+纳洛酮组和吗啡+AEB071组Th2细胞数量明显减少，IL-4浓度明显降低，PKC-θ S676、T538两个位点的磷酸化水平明显降低，GATA3 mRNA表达量及转录活性均明显降低（P<0.01）。六组Th1细胞数量和IFN-γ浓度差异无统计学意义。
结论 吗啡通过μ受体激活PKC-θ调控Th2细胞分化，PKC-θ可能作为逆转吗啡免疫抑制功能的一个干预靶点。 |
| 英文摘要: |
Ojective To investigate the function of protein kinase C theta (PKC-θ) in the model of morphine-induced naive T cells differentiation.
Methods The spleen of the mouse was taken to prepare a single cell suspension, and naive T cells were obtained using a separation kit. The naive T cells were divided into six groups: control group, morphine group, naloxone (morphine antagonist) group, AEB071 (PKC-θ antagonist) group, morphine + naloxone group and morphine + AEB071 group. The purified naive T cells were cultured with different stimulations according to the requirements of different experimental groups. Proportion of T help 1(Th1) and T help 2(Th2) cells were tested by flow cytometry analysis. IL-4 and IFN-γ were measured by ELISA. Phosphorylation of PKC-θ (S676 and T538) was defined by Western blot.The expression and activity of GATA3 was determined by electrophoretic mobility shift assay (EMSA).
Results The content of Th2 cell, the level of IL-4, PKC-θ phosphorylation, transcription factor GATA3 activity and the expression of GATA3 were significantly increased in the morphine group compared with the control group (P < 0.01).Compared with morphine group, treatment with naloxone or AEB071 significantly decreased the content of Th2 cell, the level of IL-4, the level of PKC-θ protein phosphorylation and the expression and activity of GATA3 (P < 0.01). However, the content of Th1 cell and the level of IFN-γ were not significantly changed.
Conclusion Morphine activats PKC-θ via μ receptor to induce the Th2 cell differentiation. It suggests that PKC-θ may serve as an intervention target to reverse morphine-induced immunosuppression. |
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