天津医药

天津医药 ›› 2019, Vol. 47 ›› Issue (6): 561-565.doi: 10.11958/20181907

• 专题 肛肠 •    下一篇

靶向沉默TrkB基因对结肠癌细胞SW620 失巢凋亡水平的影响

祁麟1 ， 齐春胜1 ， 肖波2△   

  1. 1中国医学科学院北京协和医学院血液病医院血液学研究所 （邮编300020）； 2天津医科大学基础医学院免疫学系
  • 收稿日期:2018-11-30 修回日期:2019-04-24 出版日期:2019-06-15 发布日期:2019-06-15
  • 通讯作者: 肖波 E-mail:tjydxb@163.com
  • 基金资助:
    淋巴细胞特异性转录因子Aiolos 在肺癌远端转移中的功能研究

The effect of targeted silencing of TrkB gene on apoptotic level of anoikis in colon cancer cell SW620

QI Lin1#br# , QI Chun-sheng1#br# , XIAO Bo2△   

  1. 1 Institute of Hematology ＆Hospital of Blood Diseases, Chinese Academy of Medical Sciences ＆Peking Union Medical College, Tianjin 300020, China; 2 Department of Immunology, Tianjin Medical University
  • Received:2018-11-30 Revised:2019-04-24 Published:2019-06-15 Online:2019-06-15

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摘要： 摘要： 目的 在结肠癌细胞SW620中靶向沉默神经营养因子酪氨酸激酶受体B （TrkB） 基因以探讨其对肿瘤细胞的失巢凋亡水平的影响。方法 构建TrkB慢病毒干扰载体， 使用TrkB-shRNA慢病毒感染SW620细胞， 利用实时定量PCR和Western blot法分别检测TrkB基因在mRNA和蛋白水平表达量的变化； 酶联免疫吸附试验 （ELISA） 检测下调TrkB基因表达后贴壁和悬浮生长状态下细胞失巢凋亡率的变化。结果 成功构建TrkB-shRNA慢病毒干扰载体，获得SW620-iTrkB稳定感染细胞系。与SW620-iLuc对照组相比， 转染SW620-iTrkB细胞中TrkB基因的mRNA和蛋白相对表达水平均降低 （P＜0.05）。不同生长状态和不同转染质粒转染均对SW620-iTrkB细胞的失巢凋亡水平有影响， 存在交互效应 （P＜0.05）。与贴壁状态相比， 悬浮状态下SW620-iLuc组和SW620-iTrkB组细胞凋亡率均明显上升 （P＜0.05）； 在悬浮状态下， SW620-iTrkB组细胞凋亡率明显高于SW620-iLuc组 （P＜0.05）。结论 慢病毒载体介导的TrkB-shRNA能在结肠癌细胞SW620中产生特异性的基因沉默效应， 显著增加细胞的失巢凋亡水平。

关键词: 受体蛋白质酪氨酸激酶类, 失巢凋亡, 基因沉默, 肿瘤转移, shRNA

Abstract: Abstract: Objective To investigate the targeted silencing of neurotrophic factor tyrosine kinase receptor B (TrkB) gene in SW620 colon cancer cell line on the apoptotic level of anoikis. Methods TrkB-shRNA lentivirus interference vector was constructed to infect SW620 cells. Real-time PCR and Western blot assay were used to detect the expression levels of TrkB gene at levels of RNA and protein respectively. ELISA assay was performed to investigate the apoptotic level of anoikis after down-regulation of TrkB gene expression in adhering and suspension cells. Results TrkB-shRNA lentivirus interference vector was successfully constructed to obtain SW620-iTrkB stable infected cell line. Compared with the SW620-iLuc control group, the mRNA and protein relative expression levels of TrkB gene were significantly decreased in SW620-iTrkB cells (P＜0.05). It was found that different growth states and different transfection plasmid transfections showed effects on the apoptotic levels of anoikis in SW620-iTrkB cells, and there were interaction effects between them (P＜ 0.05). Compared with the adherent cells, the apoptosis rates were significantly increased in the suspension cells of SW620- iLuc group and SW620-iTrkB group (P＜0.05). In the suspension group, the apoptosis rate was significantly higher in SW620-iTrkB group than that of SW620-iLuc group (P＜0.05). Conclusion Lentiviral vector-mediated TrkB-shRNA can produce specific gene silencing effect in colon cancer SW620 cells, which significantly increases the apoptotic level of anoikis.

Key words: receptor protein-tyrosine kinases, anoikis, gene silencing, neoplasm metastasis, shRNA