ADAM28 shRNA干扰载体的构建及对人牙周膜干细胞的抑制效应  

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

作　　者：赵征 邱海燕 傅兰 李杰 ZHAO Zheng;QIU Hai-yan;FU Lan;LI Jie(Department of Geriatric Stomatology,Qingdao Stomatological Hospital.Qingdao 266001,Shandong Province,China)

机构地区：[1]青岛市口腔医院老年口腔病科,山东青岛266001

出　　处：《上海口腔医学》2020年第5期476-481,共6页Shanghai Journal of Stomatology

基　　金：中国博士后科学基金特别资助项目(201003774)。

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。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.

关 键 词：金属蛋白酶解离素28 SHRNA 干扰载体 人牙周膜干细胞 

分 类 号：R781[医药卫生—口腔医学]