兔p38MAPK基因沉默腺病毒载体构建及体外干扰效率的鉴定  

Construction of rabbit p38MAPK gene silencing adenovirus vector and its identification of in-vitro interference efficiency

作　　者：全钢 贾铠宁 于果 许尧祥[2] 岳金[2] 肖文林[2] QUAN Gang;JIA Kaining;YU Guo;XU Yaoxiang;YUE Jin;XIAO Wenlin(Oral and Maxillofacial Surgery,Xinjiang Production And Construction Corps Fourth DivisionHospital,Yining,Xinjiang 835000,China;Department of Stomotology,the Affiliated Hospital ofQingdao University/ School of stomatology,Qingdao University/Key Laboratory of Stomatology,Education Department of Shandong Province,the Affiliated Hospital of Qingdaouniversity.Qingdao,Shandong 266555,China)

机构地区：[1]新疆生产建设兵团第四师医院口腔颌面外科,新疆伊宁835000 [2]青岛大学附属医院口腔科/青岛大学口腔医学院/青岛大学附属医院山东省教育厅口腔重点实验室,山东青岛266555

出　　处：《重庆医学》2019年第9期1467-1471,共5页Chongqing medicine

基　　金：山东省自然科学基金资助项目(ZR2015HM022)

摘　　要：目的构建并筛选p38丝裂原活化蛋白激酶(p38MAPK)基因沉默腺病毒载体,为进一步研究及应用p38MAPK基因沉默进行瘢痕增生基因治疗奠定基础。方法依据兔p38MAPK基因的cDNA序列,设计并合成3对干扰序列,并对腺病毒进行包装。分别将以上3对双链DNA oligo退火后连入pDC316-ZsGreenshRNA载体,经PCR及测序鉴定后,将以上质粒分别与包装质粒混合物共转染HEK293A细胞,包装产生病毒颗粒。各组病毒载体转染兔皮肤成纤维细胞后,运用实时荧光定量PCR(real-time PCR)检测p38MAPK表达水平。结果 PCR和测序证实目的双链DNA oligo片段已被准确克隆到pDC316-ZsGreen-ShRNA载体;realtime PCR检测到各组慢病毒感染兔皮肤成纤维细胞后均可以明显抑制p38MAPK mRNA的表达(P<0.05)。以AD-shRNA-P38MAPK-1组抑制效果最明显。结论成功构建并筛选出针对p38MAPK的最有效RNAi腺病毒表达载体;构建的腺病毒载体可以抑制兔皮肤成纤维细胞p38MAPK的表达。Objective To construct and screen the p38 mitogen-activated protein kinase(p38 MAPK gene silencing adenovirus vector,which lays the foundation for further research and application of p38 MAPK gene silencing for scar hyperplasia gene therapy.Methods According to the cDNA sequence of rabbit p38 MAPK gene,3 pairs of interference sequences were designed and synthesized,and the adenovirus was packaged.The 3 pairs of double-stranded DNA oligo were annealed and ligated into pDC316-ZsGreen-shRNA vector.After PCR and sequencing,the above plasmids were co-transfected into HEK293 Acells with packaging plasmid mixture,and packaged to produce virus particles.After each group of virus vectors transfected rabbit skin fibroblasts,the expression level of p38 MAPK was detected by real-time quantitative PCR(real-timePCR).Results PCR and sequencing confirmed that the target double-stranded DNA oligo fragment was cloned into pDC316-ZsGreen-ShRNA vector.Real-time-PCR detected that the expression of p38 MAPK mRNA was significantly inhibited by lentivirus infection of rabbit skin fibroblasts in each group(P<0.05).The inhibitory effect was most pronounced in the AD-shRNA-P38 MAPK-1 group.Conclusion A p38 MAPK gene silencing adenovirus vector was successfully constructed.The adenovirus vector can inhibit the expression of p38 MAPK in the rabbit skin fibroblasts.

关 键 词：P38丝裂原活化蛋白激酶 基因沉默 腺病毒载体 瘢痕增生 成纤维细胞 

分 类 号：R782.2[医药卫生—口腔医学]