鼠IL-10基因腺病毒载体的构建及在骨髓间充质干细胞中的表达  

Construction of rat interleukin-10 adenoviral vector and its expression in bone marrow mesenchymal stem cells

作　　者：张文婷[1] 唐娟 赵红梅[1] 游洁玉[1] ZHANG Wen-Ting;TANG Juan;ZHAO Hong-Mei;YOU Jie- Yu(Department of Gastroenterology,Hunan Children's Hospital,Changsha 410007,China)

机构地区：[1]湖南省儿童医院消化营养科,湖南长沙410007 [2]湖南中医药大学,湖南长沙410007

出　　处：《中国当代儿科杂志》2019年第7期708-712,共5页Chinese Journal of Contemporary Pediatrics

基　　金：湖南省卫生厅(B2012-102)

摘　　要：目的构建携带鼠IL-10(rIL-10)基因重组腺病毒载体,并探讨其能否在鼠骨髓间充质干细胞(MSCs)中稳定表达.方法 rIL-10引物经PCR扩增rIL-10 cDNA序列,将回收到的656 bp rIL-10 DNA片段克隆入pcDNA3.1载体中,构建pcDNA3.1-IL-10,将其与腺病毒载体共转染HEK293细胞,进行细胞内同源重组,经测序及聚合酶链反应(PCR)鉴定重组是否成功;反复冻融裂解HEK293细胞,将获得的含rIL-10基因的病毒液感染MSCs,Western blot法检测rIL-10在MSCs中的表达.结果经测序及PCR验证,rIL-10基因腺病毒载体构建成功,病毒滴度达4×10^9 PFU/mL.含rIL-10基因的病毒液体外感染MSCs后,可检测到IL-10的表达.结论构建重组腺病毒能够介导rIL-10基因在MSCs中的稳定表达,为下一步基因移植治疗炎症性肠病提供基础.Objective To construct the recombinant adenoviral vector carrying the rat interleukin-10 (rIL-10) gene, and to investigate whether it is stably expressed in bone marrow mesenchymal stem cells. Methods The rIL-10 gene was amplified by PCR from template rIL-10 cDNA, and the recovered 656 bp rIL-10 DNA fragment was cloned into pcDNA3.1 to construct pcDNA3.1-IL-10. Then HEK293 cells were transfected with pcDNA3.1-IL-10 and adenoviral vector for homologous recombination, and sequencing and PCR were used to evaluate whether recombination was successful. HEK293 cells were lysed by repeated freeze-thaw cycles, and bone marrow mesenchymal stem cells were infected with the virus solution containing the rIL-10 gene. Western blot was used to measure the expression of rIL-10 in bone marrow mesenchymal stem cells. Results Sequencing and PCR verified that the rIL-10 adenoviral vector was successfully constructed, with a virus titer of 4×10^9 PFU/mL. The expression of IL-10 was detected after bone marrow mesenchymal stem cells were infected by the virus solution containing the rIL-10 gene. Conclusions The constructed rIL-10 recombinant adenovirus can mediate the stable expression of rILgene in bone marrow mesenchymal stem cells, which provides a basis for gene transplantation therapy of inflammatory bowel disease.

关 键 词：炎症性肠病 IL-10基因 腺病毒载体 骨髓间充质干细胞 大鼠 

分 类 号：R725.7[医药卫生—儿科]