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

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4×10⁹ 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。

Abstract:

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 4×10⁹ 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 rIL-10 gene in bone marrow mesenchymal stem cells, which provides a basis for gene transplantation therapy of inflammatory bowel disease.