天津医药

天津医药 ›› 2019, Vol. 47 ›› Issue (4): 376-381.doi: 10.11958/20181630

• 细胞与分子生物学 • 上一篇    下一篇

氧化苦参碱下调ZNF580以抑制LTC-14细胞分泌细胞外基质的分子机制研究

陈凯 1，张青 2△，刘百庆 1，陈伟 1，周芳 2，张兰 2，夏时海 1，许威 1   

  1. 1中国人民武装警察部队特色医学中心，天津市肝脏胰腺纤维化与分子诊疗重点实验室（邮编300162）；2天津市西青医院
  • 收稿日期:2018-10-26 修回日期:2019-02-22 出版日期:2019-04-15 发布日期:2019-05-27
  • 通讯作者: 张青 E-mail:finezhangqing@163.com
  • 作者简介:陈凯（1983），男，硕士，技师，主要从事胰腺疾病的基础研究
  • 基金资助:
    天津市西青医院科研基金;天津市应用基础与前沿技术研究计划青年项目;国家自然科学基金

Oxymatrine suppressed extracellular matrix expression in LTC-14 cells by down-regulating ZNF580 expression

CHEN Kai1，ZHANG Qing2△, LIU Bai-qing1, CHEN Wei1, ZHOU Fang2, ZHANG Lan2, XIA Shi-hai1, XU Wei1   

  1. 1 Tianjin Key Laboratory of Hepatopancreatic Fibrosis and Molecular Diagnosis & Treatment, Characteristic Medical Center of PAP, Tianjin 300162, China; 2 Tianjin Xiqing Hospital
  • Received:2018-10-26 Revised:2019-02-22 Published:2019-04-15 Online:2019-05-27
  • Contact: Qing ZHANG E-mail:finezhangqing@163.com

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摘要： 目的 探讨氧化苦参碱（OM）对转化生长因子-β1（TGF-β1）诱导的大鼠胰腺星状细胞 LTC-14中细胞外 基质（ECM）分泌的影响及其分子机制。方法 取生长良好的 LTC-14细胞株，分为对照组（正常培养的 LTC-14细 胞），TGF-β1组（10 μg/L TGF-β1刺激 12 h），TGF-β1+OM组（1 g/L OM预处理 30 min，再加入 10 μg/L TGF-β1刺激 12 h），TGF-β1 + SiZNF850组（LTC-14细胞中瞬时转染 ShRNA-ZNF580质粒，24 h后加入 10 μg/L TGF-β1刺激 12 h）。实时定量 PCR、Western blot 检测细胞内 Smad2、Smad3、ZNF580、α-肌动蛋白（α-SMA）、基质金属蛋白酶-2 （MMP-2）、基质金属蛋白酶组织抑制剂1（TIMP1）的mRNA和蛋白表达情况，酶联免疫吸附试验（ELISA）检测ECM中 胶原蛋白-Ⅰ（Col-Ⅰ）、Col-Ⅲ、纤维连接蛋白（FN）、肿瘤坏死因子-α（TNF-α）和白细胞介素-1β（IL-1β）的分泌情 况。结果 TGF-β1诱导的 LTC-14细胞中 Smad2、Smad3、ZNF580和 α-SMA的 mRNA和蛋白表达水平均升高（P＜ 0.05），MMP-2/TIMP1比值下降（P＜0.05），且 Col-Ⅰ、Col-Ⅲ、FN、TNF-α和 IL-1β的分泌水平升高（P＜0.05）；而用 OM预处理或沉默 ZNF580基因后，LTC-14细胞中 TGF-β1/Smads通路 Smad2、Smad3、α-SMA和 ZNF580的 mRNA和 蛋白表达水平均下调（P＜0.05），MMP-2/TIMP1表达比值升高（P＜0.05），细胞外基质 Col-Ⅰ、Col-Ⅲ、FN、TNF-α和 IL-1β分泌水平均降低（P＜0.05）。结论 OM通过抑制胰腺星状细胞LTC-14中TGF-β1/Smads通路激活，下调ZNF580， 阻滞胰腺星状细胞的活化，使ECM分泌减少、降解增多，减轻胰腺纤维化。ZNF580可能是OM作用的分子靶点。

关键词: 氧化苦参碱, 细胞外基质, 转化生长因子β1, ZNF580, 胰腺星状细胞

Abstract: Objective To probe the molecular mechanism and effect of oxymatrine (OM) on the secretion of extracellular matrix (ECM) in rat pancreatic stellate cell line LTC-14 induced by TGF-β1. Methods LTC-14 cells were divided into control group (LTC-14 cells in normal culture), TGF-β1 group (LTC-14 cells stimulated by 10 μg/L TGF-β1 for 12 h), TGF-β1+OM group (LTC-14 cells pretreated with 1 g/L OM for 30 min before TGF-β1 stimulation) and TGF-β1+ SiZNF850 group (LTC-14 cells were transiently transfected with ShRNA-ZNF580 plasmids for 24 h before TGF - β1 stimulation). After cultivation, cell supernatant was collected and total RNA and total protein were extracted. The expressions of Smad2, Smad3, ZNF580, α - SMA, MMP2 and TIMP1 were detected by Real-time PCR and Western blot assay. The secretion of ECM components, Col-I, Col-III, FN, TNF-α and IL-1β were detected by ELISA. Results The mRNA and protein expressions of Smad2, Smad3, ZNF580 and α-SMA were increased (P＜0.05), the expression ratio of MMP2/TIMP1 was down-regulated (P＜0.05), and the secretion of ECM components was increased in LTC-14 cells induced by TGF-β1 (P＜0.05). However, after OM intervention or ZNF580 gene knockdown, the mRNA and protein expressions of Smad2 and Smad3 were suppressed (P＜0.05), the mRNA and protein expressions of ZNF580 decreased, the expression ratio of MMP-2/ TIMP1 was up-regulated (P＜0.05), and the secretion of ECM components was reduced (P＜0.05). Conclusion Oxymatrine can reduce ECM secretion, increase ECM degradation and alleviate pancreatic fibrosis by inhibiting TGF- β1/ Smads/ZNF580 signaling pathway and blocking the activation of pancreatic stellate cells. ZNF580 may be a molecular target of OM in reducing pancreatic fibrosis.

Key words: oxymatrine, extracellular matrix, transforming growth factor beta 1, ZNF580, pancreatic stellate cell