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钙联蛋白在心肌成纤维细胞活化过程中的作用研究

田格尔 赵明月 周骏腾 权月 吴文超 刘小菁

田格尔, 赵明月, 周骏腾, 权月, 吴文超, 刘小菁. 钙联蛋白在心肌成纤维细胞活化过程中的作用研究[J]. 仁和测试, 2020, 37(3): 450-459. doi: 10.7507/1001-5515.202001052
引用本文: 田格尔, 赵明月, 周骏腾, 权月, 吴文超, 刘小菁. 钙联蛋白在心肌成纤维细胞活化过程中的作用研究[J]. 仁和测试, 2020, 37(3): 450-459. doi: 10.7507/1001-5515.202001052
Geer TIAN, Mingyue ZHAO, Junteng ZHOU, Yue QUAN, Wenchao WU, Xiaojing LIU. The potential role of calnexin in the activation of cardiac fibroblasts[J]. Rhhz Test, 2020, 37(3): 450-459. doi: 10.7507/1001-5515.202001052
Citation: Geer TIAN, Mingyue ZHAO, Junteng ZHOU, Yue QUAN, Wenchao WU, Xiaojing LIU. The potential role of calnexin in the activation of cardiac fibroblasts[J]. Rhhz Test, 2020, 37(3): 450-459. doi: 10.7507/1001-5515.202001052

钙联蛋白在心肌成纤维细胞活化过程中的作用研究

doi: 10.7507/1001-5515.202001052
基金项目: 国家自然科学基金(11672197)
详细信息
    通讯作者:

    刘小菁,Email:liuxq@scu.edu.cn

The potential role of calnexin in the activation of cardiac fibroblasts

Funds: The National Natural Science Foundation of China
More Information
  • 摘要: 钙联蛋白(Calnexin)是内质网上的凝集素样分子伴侣蛋白,也是线粒体结合内质网膜上的重要蛋白,在心脏疾病中发挥着重要作用,但其在心肌成纤维细胞活化中的作用和机制并未阐明。本研究通过小鼠胸主动脉缩窄(TAC)模型来观察体内心肌纤维化情况,并利用心脏超声,苏木精-伊红、Masson 和天狼星红染色,以及心脏纤维化标志物表达水平,鉴定模型是否成功。体外实验利用转化生长因子 TGFβ1 刺激建立心肌成纤维细胞活化模型,通过定量 PCR、Western blot、EdU 渗入法及划痕实验等方法检测其活化、细胞增殖、迁移的变化。利用腺病毒过表达和沉默 Calnexin,来观察 Calnexin 在体外心肌成纤维细胞活化中的作用。结果显示,Calnexin 在 TAC 模型和体外心肌成纤维细胞活化过程中均表达降低。过表达 Calnexin 能缓解心肌成纤维细胞活化;相反,沉默 Calnexin 导致其活化加重。进一步探究发现,内质网应激在心肌成纤维细胞活化过程中被激活,过表达 Calnexin 后内质网应激缓解,相反,敲低 Calnexin 后内质网应激加重,与活化表型变化一致。结果表明,过表达 Calnexin 可能通过缓解内质网应激从而减轻心肌成纤维细胞的活化程度。
  • 图  1  Calnexin 在小鼠 TAC 模型中的表达降低

    a.心脏重量与体重比值;b.小鼠 TAC 术后超声心动图检测图像;c-g. 超声心动图检测心功能指标;h. 心脏组织病理切片;i. 纤维化指标 mRNA 相对表达量;j. Calnexin 的 mRNA 相对表达量;k. Calnexin 的蛋白相对表达量;l. Calnexin 在 TAC 术后不同时间的 mRNA 相对表达量。n = 6,与 sham 组相比,* P < 0.05,** P < 0.01,*** P < 0.001,**** P < 0.000 1

    Figure  1.  The expression of calnexin was decreased in TAC model of mice

    a. heart weight/body weight; b. representative images of an echocardiographic assessment of mice after TAC; c-g. the cardiac function indicators by echocardiogram; h. the stained sections of hearts; i. the mRNA levels of COL-1 and α-SMA; j. the mRNA level of calnexin; k. the protein level of calnexin; l. the mRNA levels of calnexin with time gradient. n = 6, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.000 1, compared with sham group

    图  2  Calnexin 在心肌成纤维细胞活化过程中的表达降低

    a. EdU 渗入法检测心肌成纤维细胞的增殖能力;b. 划痕实验检测心肌成纤维细胞的迁移能力;c. 原代心肌成纤维细胞荧光染色;d. 心肌成纤维细胞活化标志物 mRNA 的相对表达量;e. Calnexin 的蛋白相对表达量。n = 6,与对照组相比,* P < 0.05,** P < 0.01,*** P < 0.001,**** P < 0.000 1

    Figure  2.  The expression of calnexin was decreased in activated cardiac fibroblasts

    a. the proliferation rate of cells by EdU staining; b. the migration capability of cardiac fibroblasts by wound healing assay; c. immunofluorescence staining of α-SMA in cardiac fibroblasts; d. the mRNA levels of fibrotic markers; e. the protein level of calnexin. n = 6, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.000 1, compared with control group

    图  3  过表达 Calnexin 缓解 TGFβ1 诱导的心肌成纤维细胞活化

    a. 腺病毒转导效率检测;b. 过表达 Calnexin 后心肌成纤维细胞活化指标 mRNA 水平变化;c. EdU 渗入法检测心肌成纤维细胞的增殖能力;d. 划痕实验检测心肌成纤维细胞的迁移能力;e. 心肌成纤维细胞荧光染色;f. 心肌成纤维细胞活化标志物 mRNA 的相对表达量。n = 6,* P < 0.05,** P < 0.01,*** P < 0.001,**** P < 0.000 1

    Figure  3.  Calnexin overexpression alleviated the activation of cardiac fibroblasts induced by TGFβ1

    a. the transduction effect of adenovirus; b. the mRNA levels of fibrosis markers; c. the proliferation rate of cells by EdU staining; d. the migration capability of cardiac fibroblasts by wound healing assay; e. immunofluorescence staining of α-SMA in cardiac fibroblasts; f. the mRNA levels of CTGF, α-SMA and TGF-β. n = 6, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.000 1

    图  4  敲低 Calnexin 加重 TGFβ1 诱导的心肌成纤维细胞活化

    a. 腺病毒转导效率检测;b. 沉默 Calnexin 后心肌成纤维细胞活化指标 mRNA 水平变化;c. EdU 渗入法检测心肌成纤维细胞的增殖能力;d. 划痕实验检测心肌成纤维细胞的迁移能力;e. 心肌成纤维细胞荧光染色;f. 心肌成纤维细胞活化标志物蛋白相对表达量。n = 6,* P < 0.05,** P < 0.01,*** P < 0.001,**** P < 0.000 1

    Figure  4.  Calnexin knockdown aggravated the activation of cardiac fibroblasts induced by TGFβ1

    a. the transduction effect of adenovirus; b. the mRNA levels of fibrosis markers; c. the proliferation rate of cells by EdU staining; d. the migration capability of cardiac fibroblasts by wound healing assay; e. immunofluorescence staining of α-SMA in cardiac fibroblasts; f. the protein levels of CTGF, α-SMA and TGF-β. n = 6, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.000 1

    图  5  Calnexin 影响 ER stress 来调节 TGFβ1 诱导的心肌成纤维细胞活化

    a. TGFβ1 诱导的心肌成纤维细胞活化激活 ER stress;b. 病毒敲低 Calnexin 后,心肌成纤维细胞中 ER stress 通路蛋白的表达水平;c. 病毒过表达 Calnexin 后,心肌成纤维细胞中 ER stress 通路蛋白的表达水平。* P < 0.05,** P < 0.01,*** P < 0.001

    Figure  5.  Calnexin regulates ER stress in TGFβ1-induced cardiac fibroblasts activation

    a. the protein expression of BIP, p-IRE1α, ATF4, and c-ATF6 with TGFβ1 treatment in cardiac fibroblasts; b. the protein levels of BIP, p-IRE1α, ATF4, and c-ATF6 with sh-adenovirus transduction in cardiac fibroblasts; c. the protein levels of BIP, P-IRE1α, ATF4, and c-ATF6 with overexpression-adenovirus transfection in cardiac fibroblasts. * P < 0.05, ** P < 0.01, *** P < 0.001

    表  1  qPCR 反应使用的引物序列

    Table  1.   The sequences of primers in qPCR

    基因 引物序列
    Calnexin F: ATGGAAGGGAAGTGGTTACTGT
    R: GCTTTGTAGGTGACCTTTGGAG
    α-SMA F: ACAACTGGTATTGTGCTGGACT
    R: TCAGCAGTAGTCACGAAGGAAT
    TGF-β F: TGAGTGGCTGTCTTTTGACG
    R: ACTGAAGCGAAAGCCCTGTA
    COL-1 F: ACGTCCTGGTGAAGTTGGTC
    R: TCCAGCAATACCCTGAGGTC
    CTGF F: CTACCGACTGGAAGACACATTT
    R: TTCCTCTAGGTCAGCTTCACAG
    β-actin F: ACTATCGGCAATGAGCGGTTC
    R: ATGCCACAGGATTCCATACCC
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  • 收稿日期:  2020-01-20
  • 修回日期:  2020-04-17
  • 刊出日期:  2020-03-17

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