中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2020, Vol. 15 ›› Issue (1): 169-177.doi: 10.4103/1673-5374.264463

• 原著:周围神经损伤修复保护与再生 • 上一篇    下一篇

Claudin-15过表达抑制体外培养许旺细胞的增殖并促其凋亡

  

  • 出版日期:2020-01-15 发布日期:2020-05-22
  • 基金资助:
    国家自然科学基金(81671220)；中国吉林省科技发展计划(20160101077JC)；中国吉林省学校联合建设专项项目(SXGJQY2017-13)

Claudin-15 overexpression inhibits proliferation and promotes apoptosis of Schwann cells in vitro

Jian-Nan Li1, Zhan Zhang1, Guang-Zhi Wu1, Deng-Bing Yao2, Shu-Sen Cui1   

  1. 1 China-Japan Union Hospital of Jilin University, Changchun, Jilin Province, China
    2 School of Life Sciences, Jiangsu Key Laboratory of Neuroregeneration, Co-innovation Center of Neuroregeneration, Nantong University, Nantong, Jiangsu Province, China
  • Online:2020-01-15 Published:2020-05-22
  • Contact: Shu-Sen Cui, PhD, MD,sscui916@126.com.
  • Supported by:
    This study was supported by the National Natural Science Foundation of China, No. 81671220 (to SSC); the Scientific and Technological Development Program of Jilin Province of China, No. 20160101077JC (to SSC); the School Joint Construction Special Project of Jilin Province of China, No. SXGJQY2017-13 (to SSC).

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摘要：

课题组以往研究结果表明，Claudin-15在坐骨神经损伤模型大鼠远端神经的许旺细胞中表达上调，但Claudin-15在许旺细胞中具体发挥什么作用以及作用机制尚不清楚。为此，实验观察Claudin-15对体外培养许旺细胞增殖和凋亡的影响，以明确Claudin-15在许旺细胞中的作用及机制。实验体外培养了大鼠原代许旺细胞，并对许旺细胞进行Claudin-15 siRNA转染(siRNA-1组，Claudin-15基因敲除)，并设置单纯阴性对照序列转染的NC组作对照，对许旺细胞进行pGV230-Claudin-15质粒转染(pGV230-Claudin-15组，Claudin-15基因过表达)，并设置单纯pGV230转染的pGV230组作对照；通过EdU实验检测Claudin-15对许旺细胞增殖的影响，通过流式细胞仪分析Claudin-15对许旺细胞凋亡的影响，通过Transwell小室分析Claudin-15对细胞迁移的影响，通过Western blot及实时PCR检测许旺细胞相关蛋白及mRNA的表达。结果显示：(1)与NC组相比，siRNA-1组细胞增殖率升高，AKT的蛋白磷酸化水平下降；细胞凋亡率及c-Jun蛋白磷酸化水平，PKCα，Bcl-2，Bax的mRNA表达量下降；神经营养因子bFGF，NT3的mRNA表达量升高，细胞迁移率相近。(2)与pGV230相比，pGV230-Claudin-15组细胞增殖率下降；细胞凋亡率及c-Jun的蛋白磷酸化升高，c-Fos的蛋白表达量升高，PKCα、Bax的mRNA表达量下降；神经营养因子bFGF、NT3的mRNA表达量下降。(3)上述实验结果证实，Claudin-15过表达可在体外抑制许旺细胞增殖，促进许旺细胞凋亡，沉默Claudin-15表达可逆转此现象并产生神经保护作用。实验于2016年3月5日经吉林大学实验动物伦理委员会批准(批准号：2016-nsfc001)。

orcid: 0000-0003-2840-8166 (Shu-Sen Cui)

关键词: 周围神经损伤, 许旺细胞, Claudin-15, 细胞增殖, 细胞凋亡, c-Jun, Bax, PKCα, 瓦勒溃变, 神经再生

Abstract: Our previous experiments have discovered that Claudin-15 was up-regulated in Schwann cells of the distal nerve stumps of rat models of sciatic nerve injury. However, how Claudin-15 affects Schwann cell function is still unknown. This study aimed to identify the effects of Claudin-15 on proliferation and apoptosis of Schwann cells cultured in vitro and explore the underlying mechanisms. Primary Schwann cells were obtained from rats. Claudin-15 in Schwann cells was knocked down using siRNA (siRNA-1 group) compared with the negative control siRNA transfection group (negative control group). Claudin-15 in Schwann cells was overexpressed using pGV230-Claudin-15 plasmid (pGV230-Claudin-15 group). The pGV230 transfection group (pGV230 group) acted as the control of the pGV230-Claudin-15 group. Cell proliferation was analyzed with EdU assay. Cell apoptosis was analyzed with flow cytometric analysis. Cell migration was analyzed with Transwell inserts. The mRNA and protein expressions were analyzed with quantitative polymerase chain reaction assay and western blot assay. The results showed that compared with the negative control group, cell proliferation rate was up-regulated; p-AKT/AKT ratio, apoptotic rate, p-c-Jun/c-Jun ratio, mRNA expression of protein kinase C alpha, Bcl-2 and Bax were down-regulated; and mRNA expression of neurotrophins basic fibroblast growth factor and neurotrophin-3 were increased in the siRNA-1 group. No significant difference was found in cell migration between the negative control and siRNA-1 groups. Compared with the pGV230 group, the cell proliferation rate was down-regulated; apoptotic rate, p-c-Jun/c-Jun ratio and c-Fos protein expression increased; mRNA expression of protein kinase C alpha and Bax decreased; and mRNA expressions of neurotrophins basic fibroblast growth factor and neurotrophin-3 were up-regulated in the pGV230-Claudin-15 group. The above results demonstrated that overexpression of Claudin-15 inhibited Schwann cell proliferation and promoted Schwann cell apoptosis in vitro. Silencing of Claudin-15 had the reverse effect and provided neuroprotective effect. This study was approved by the Experimental Animal Ethics Committee of Jilin University of China (approval No. 2016-nsfc001) on March 5, 2016.

Key words: apoptosis, Bax, cell proliferation, c-Jun, Claudin-15, nerve regeneration, peripheral nerve injury, protein kinase C alpha, Schwann
cells, Wallerian degeneration