解剖学报

解剖学报 ›› 2019, Vol. 50 ›› Issue (2): 166-172.doi: 10.16098/j.issn.0529-1356.2019.02.005

• 细胞和分子生物学 • 上一篇    下一篇

敲减钙离子/钙调蛋白依赖性激酶Ⅳ抑制干扰素-γ诱导C2C12细胞免疫分子的表达

李俊桦1 马永能2 谷瑞彩1 廖华1*   

  1. 1.南方医科大学基础医学院解剖学教研室，广东省医学生物力学重点实验室，广州 510515; 2.四川省绵阳市第三人民医院检验科，四川 绵阳 621002
  • 收稿日期:2018-04-23 修回日期:2018-11-22 出版日期:2019-04-06 发布日期:2019-04-06
  • 通讯作者: 廖华 E-mail:liaohua@fimmu.com
  • 基金资助:
    国家自然科学基金;国家重点研发计划

Knockdown of calcium/calmodulin-dependent protein kinaseⅣ reduce the immune characteristic of C2C12 under stimulation of interferon-γ

LI Jun-hua1 MA Yong-neng2 GU Rui-cai1 LIAO Hua 1*   

  1. Department of Anatomy, Guangdong Key Laboratory of Biomechanics, Southern Medical University, Guangzhou 510515,China; 2. Department of Clinical Laboratory, the Third Hospital of Mianyang,  Sichuan Mianyang 621002,China
  • Received:2018-04-23 Revised:2018-11-22 Online:2019-04-06 Published:2019-04-06
  • Contact: LIAO Hua E-mail:liaohua@fimmu.com

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摘要：

目的 探究敲减钙离子/钙调蛋白依赖性激酶Ⅳ（CaMKⅣ）基因对炎性培养条件下C2C12细胞免疫分子表达的影响。 方法 采用慢病毒基因干扰技术敲减C2C12细胞的CaMKⅣ基因，以2%马血清将C2C12细胞分化成肌管，以干扰素-γ（IFN-γ）刺激C2C12细胞，采用Real-time PCR和Western blotting检测CaMKⅣ的表达，采用Western blotting和免疫荧光检测免疫分子主要组织相容性复合体（MHC） class Ⅰ(H-2Kb)、MHC class Ⅱ(H2-Ea)、Toll样受体3（TLR3）的表达，采用Real-time检测肌细胞分泌型促炎性细胞因子白细胞介素（IL）-1β、IL-6、肿瘤坏死因子（TNF）-α、巨噬细胞炎性蛋白（MIP）-1α和单核细胞趋化因子（MCP）-1的基因表达。 结果 IFN-γ可诱导成肌干细胞以及分化肌管表达或上调表达免疫分子MHC-Ⅰ、MHC-Ⅱ、TLR3，同时上调C2C12细胞促炎性细胞因子IL-1β、IL-6、TNF-α、MIP-1α和MCP-1的基因表达量；敲减CaMKⅣ基因导致上述上调趋势被抑制。 结论 敲减CaMKⅣ基因可有效抑制IFN-γ诱导的免疫分子的表达，可能提示CaMKⅣ参与调节肌细胞的免疫学特性。

关键词: 钙离子/钙调蛋白依赖性激酶Ⅳ, 免疫分子, C2C12细胞, shRNA转染, 实时定量聚合酶链反应, 免疫印迹法, 免疫荧光

Abstract:

Objective Whether calcium/calmodulin-dependent protein kinase Ⅳ (CaMKⅣ) plays a role in regulating immunologic features of muscle cells in inflammatory environment, remains mostly unknown. In this study, we investigated the influence of CaMKⅣ on the immunological characteristics of myoblasts and myotubes received interferon(IFN)-γ stimulation. Methods To investigate the effects of CaMKⅣ on immune characteristics of C2C12 myoblasts and differentiated myotubes, which are firstly knocked down endogenous CaMKⅣ gene and then treated with IFN-γ. Real-time PCR and Western blotting were performed to analyze the expression of CaMKⅣ. Western blotting and immunofluorescence were performed to analyze the expression of major histocompatibility complex(MHC) class-Ⅰ,MHC class-Ⅱ,Toll-like receptor3(TLR3). The expression of interleukin(IL)-1β,IL-6,tumor necrosis factor(TNF)-α,macrophage inflamator protein(MIP)-1α and monocyte chemoattractant protein(MCP)-1 were measured by Real-time PCR. Results Under IFN-γ induced pro-inflammatory milieu, MHC-Ⅰ molecule H-2Kb, MHC-Ⅱ molecule H2-Ea, TLR3 significantly up-regulated in myoblasts and in differentiated myotubes. In striking contrast, CaMKⅣ inhibition in myoblasts and myotubes led to expression suppression of the above molecules. As well, gene levels of pro-inflammatory IL-1β, IL-6, TNF-α, MIP-1α and MCP-1 in C2C12 cells (especially in myotubes) were markedly down-regulated by CaMKⅣ knocking off. Conclusion Knockdown of CaMKⅣ gene can effectively inhibit the expression of IFN-γ-induced immune molecules, suggesting that CaMKⅣ is involved in regulating the immunological properties of muscle cells.

Key words: Calcium/calmodulin-dependent protein kinase Ⅳ, Immune molecules, C2C12 cell, shRNA transfection, Real-time PCR, Western blotting, Immunofluorescence