上海交通大学学报（医学版）

上海交通大学学报（医学版） ›› 2019, Vol. 39 ›› Issue (12): 1375-.doi: 10.3969/j.issn.1674-8115.2019.12.006

• 论著·基础研究 • 上一篇    下一篇

沉默结缔组织生长因子基因对肝星状细胞生长和细胞周期的影响

张荣华1，董岸莺2，柏干苹1，万 萍1，蒋 毅1，吴 红1   

  1. 1. 陆军军医大学西南医院中医与风湿免疫科，重庆400038；2. 库尔勒解放军951医院心肾呼吸科，库尔勒841000
  • 出版日期:2019-12-28 发布日期:2020-02-06
  • 通讯作者: 张荣华（1962—），女，教授，主任医师，博士；电子信箱：zhrhlggg@163.com。
  • 作者简介:张荣华（1962—），女，教授，主任医师，博士；电子信箱：zhrhlggg@163.com。
  • 基金资助:
    国家自然科学基金（81273918）；基础研究军民融合项目(SWH2017JCZD-5)；军队中医药科研专项课题（2010ZYZ231）；重庆市中医药科技项目（2012-2-63）

Effects of connective tissue growth factor gene-silencing on cell growth and cell cycle of hepatic stellate cells

ZHANG Rong-hua, DONG An-ying, BAI Gan-ping, WAN Ping, JIANG Yi, WU Hong   

  1. 1. Department of Traditional Chinese Medicine and Rheumatology, Southwest Hospital, the Army Medical University of Chinese PLA, Chongqing 400038, China 2. Department of Heart, Kidney and Respiration, No.951 Hospital of PLA, Korla 841000, China
  • Online:2019-12-28 Published:2020-02-06
  • Supported by:
    National Natural Science Foundation of China, 81273918; Basic Research Military-Civil Integration Project, SWH2017JCZD-5; Specific Project of Traditional Chinese Medicine of Chinese PLA, 2010ZYZ231; Project of Traditional Chinese Medicine Science and Technology of Chongqing, 2012-2-63

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摘要： 目的·探讨沉默结缔组织生长因子（connective tissue growth factor，Ctgf）基因对大鼠肝星状细胞HSCT6的生长、细胞周期及其转化生长因子-β1（transforming growth factor-β1，TGF-β1）、Smad3和Smad7表达的影响。方法·采用RNA干扰技术构建Ctgf基因的pCDH/Ctgf-shRNA重组慢病毒载体。该重组载体经病毒包装后获得高感染力的pCDH/Ctgf-shRNA病毒颗粒用于感染HSCT6细胞。荧光显微镜观测被感染HSCT6细胞的绿色荧光蛋白（green fluorescent protein，GFP）的表达情况；CCK-8试剂盒检测被感染HSCT6细胞的生长变化；流式细胞术检测被感染HSCT6细胞的周期变化。Real-time PCR和Western blotting分别检测pCDH/Ctgf-shRNA病毒对HSCT6细胞的Ctgf基因的沉默效应及对TGF-β1、Smad3和Smad7表达的影响。结果·成功构建Ctgf基因的重组病毒载体pCDH/Ctgf-shRNA。荧光显微镜观测表明，被感染重组病毒的HSCT6细胞显著表达GFP。CCK-8检测结果证实，与对照细胞相比，沉默Ctgf基因的HSCT6细胞生长明显受抑，72 h开始两者差异具有统计学意义（PCtgf基因的HSCT6细胞可被阻滞于S期。Real-time PCR和Western blotting检测表明，pCDH/Ctgf-shRNA病毒能有效沉默HSCT6细胞的Ctgf基因，下调TGF-β1、Smad3基因及其蛋白的表达，上调Smad7基因及其蛋白的表达；与对照比较，差异皆有统计学意义（均P结论·沉默Ctgf基因能抑制HSCT6细胞生长，使其TGF-β1、Smad3的表达下调，同时上调其Smad7的表达；这种生长受抑可能与其TGF-β1/Smads（Smad3/Smad7）信号通路受阻紧密相关。

关键词: 结缔组织生长因子, 肝星状细胞, 转化生长因子-&, beta, 1, Smad3, Smad7

Abstract:

Objective · To investigate the effects of silencing connective tissue growth factor (Ctgf) gene on the growth, cell cycle and the of TGF-β1, Smad3 and Smad7 of rat hepatic stellate cell line HSCT6. Methods · The recombinant lentivirus vector pCDH/Ctgf-shRNA of Ctgf gene was constructedRNA interference. The recombinant vector was packaged to obtain highly infectious pCDH/Ctgf-shRNA lentivirus particles for HSCT6 infection. The of green fluorescent protein (GFP) in the transfected HSCT6 cells was observed under fluorescence microscope. The effects of Ctgf-shRNA lentivirus on the growth of HSCT6 cells were testedCCK-8. The effects of Ctgf-shRNA lentivirus on the cell cycle of HSCT6 cells were analyzedflow cytometry (FCM). The effects of Ctgf-shRNA lentivirus on the of mRNA of Ctgf, Tgf-β1, Smad3 and Smad7, and their proteins in HSCT6 cells were detectedreal-time PCR and Western blotting, respectively. Results · The lentiviral vector pCDH/Ctgf-shRNA has been constructed successfully. The HSCT6 cells transfectedCtgf-shRNA lentivirus significantly expressed GFP under fluorescence microscope. The results of CCK-8 confirmed that the growth of HSCT6 cells transfectedCtgf-shRNA lentivirus was slower than that of controls and the differences were statistically significant after being cultured for 72 h (P0.05). The results of FCM revealed that the growth of HSCT6 cells transfectedCtgf-shRNA lentivirus was blocked in the S phase of cell cycle. The results of real-time PCR and Western blotting showed that the Ctgf-shRNA lentivirus effectively silenced Ctgf gene, down-regulated the of genes and encoding proteins of TGF-β1, and Smad3 of HSCT6 and up-regulated the of genes and encoding proteins of Smad7 of HSCT6 cells. The differences between transfected cells and controls were statistically significant (P0.05). Conclusion · Silencing Ctgf gene can effectively inhibit the growth of HSCT6 cells, down-regulate the of TGF-β1 and Smad3 and up-regulate the of Smad7. The inhibition of the growth of HSCT6 cells may be closely related to interference of the TGF-β1/Smads (Smad3 and Smad7) signaling pathway.

Key words: connective tissue growth factor (CTGF), hepatic stellate cell, transforming growth factor-β1 (TGF-β1), Smad3, Smad7