上海口腔医学

上海口腔医学 ›› 2020, Vol. 29 ›› Issue (5): 476-481.doi: 10.19439/j.sjos.2020.05.006

• 论著 • 上一篇    下一篇

ADAM28 shRNA干扰载体的构建及对人牙周膜干细胞的抑制效应

赵征, 邱海燕, 傅兰, 李杰   

  1. 青岛市口腔医院 老年口腔病科,山东 青岛 266001
  • 收稿日期:2019-05-20 修回日期:2019-09-11 出版日期:2020-10-25 发布日期:2020-11-02
  • 通讯作者: 赵征,E-mail: zhaozheng170@163.com
  • 作者简介:赵征（1973-）,女,口腔医学博士后,副主任医师,副教授
  • 基金资助:
    中国博士后科学基金特别资助项目(201003774)

Construction of ADAM28 shRNA interference vector and its inhibitory effect on human periodontal ligament stem cells

ZHAO Zheng, QIU Hai-yan, FU Lan, LI Jie   

  1. Department of Geriatric Stomatology, Qingdao Stomatological Hospital. Qingdao 266001, Shandong Province, China
  • Received:2019-05-20 Revised:2019-09-11 Online:2020-10-25 Published:2020-11-02

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摘要： 目的：探讨金属蛋白酶解离素28（ADAM28）shRNA干扰载体对人牙周膜干细胞（human periodontal ligament stem cells,HPDLSC）内ADAM28基因表达的抑制作用,为先天性牙根发育不全疾病的基因治疗提供实验依据。方法：设计合成4对特异性shRNA干扰片段,与pGPU6/GFP/Neo 载体连接,构建、鉴定ADAM28 shRNA干扰载体并转染HPDLSC 48 h后,利用实时荧光定量PCR（qRT-PCR）和Western印迹分析检测抑制效率。采用SPSS 21.0软件包对数据进行统计学分析。结果：酶切和测序鉴定证明双链shRNA正确插入表达载体pGPU6/GFP/Neo,重组干扰载体构建成功并高效转染HPDLSC。QRT-PCR和Western 印迹检测显示,pGPU6/GFP/Neo-ADAM28-shRNA1-4均有显著的抑制效率,shRNA1的抑制效率最高;ADAM28-shRNA1-4组与未转染组、阴性对照组比较,差异均有显著性（P< 0.05）。结论：ADAM28 shRNA干扰载体可有效抑制HPDLSC内ADAM28的基因表达。

关键词: 金属蛋白酶解离素28, shRNA, 干扰载体, 人牙周膜干细胞

Abstract: PURPOSE: To investigate the inhibition of ADAM28 shRNA interfering vector on ADAM28 gene expression in human periodontal ligament stem cells(HPDLSC), and provide experimental evidence for gene therapy against congenital hypoplasia of tooth root(CHTR) disease. METHODS: Four pairs of shRNA specific interfering fragments were designed, synthesized, and connected with pGPU6/GFP/Neo vector. ADAM28 shRNA interfering vector was constructed and identified, and transfected into HPDLSC for 48 h, and then the inhibition efficiency was detected by real-time fluorescence quantitative RT-PCR（qRT-PCR）and Western blot. Statistical significance was assessed by SPSS 21.0 software package. RESULTS: Enzyme digestion and sequencing identification demonstrated that the double strands shRNA was correctly inserted into the expression vector pGPU6/GFP/Neo, and the recombinant interfering vector was successfully constructed and highly transfected into HPDLSC. QRT-PCR and Western blot showed that pGPU6/GFP/Neo-ADAM28-shRNA1-4 had significant inhibition efficiency, and shRNA1 had the highest inhibition efficiency. There were significant differences between ADAM28-shRNA1-4 group and non-transfection group, negative control group, respectively(P<0.05). CONCLUSIONS: ADAM28 shRNA interference vector can effectively inhibit ADAM28 gene expression in HPDLSC.

Key words: ADAM28, shRNA, Interference vector, HPDLSC

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