CAJ | 학술논문

目的构建含胰十二指肠同源框-1基因（pancreatic and duodenal homeobox factor 1，PDX1）的重组腺病毒载体，并检测其在人脐带间充质干细胞(human umblic cord msenchymal stem cells，HUCMSCs)的表达情况。方法用BgⅢ/XhoI酶从pUC57-PDX1酶切PDX1，连接到pShuttle-GFP-CMV重组穿梭载体，得到pShuttle-GFP-CMV-PDX1重组穿梭质粒，再将pShuttle-GFP-CMV-PDX1转移到pAdxsi载体上，得到pAdxsi-GFP-PDX1病毒质粒，利用脂质体介导重组腺病毒载体转染293细胞，包装出完整的腺病毒。用重组腺病毒感染HUCMSCs 24 h 后，经荧光显微镜、RT-PCR、免疫荧光、免疫细胞化学、Western Blot等检测PDXI基因及蛋白的表达。结果通过测序、酶切等鉴定PDXI基因正确插入穿梭质粒中，并与病毒骨架质粒重组，重组腺病毒可高效转染HUCMSCs，RT-PCR检测到PDXI mRNA在HUCMSCs中表达，经免疫荧光、免疫细胞化学、Western Blot等证实转染PDXI在HUCMSCs胞核中表达。结论成功构建了PDX1腺病毒载体，并在HUCMSCs中有效表达。
목적 구건함이십이지장동원광-1기인（pancreatic and duodenal homeobox factor 1，PDX1）적중조선병독재체，병검측기재인제대간충질간세포(human umblic cord msenchymal stem cells，HUCMSCs)적표체정황。방법 용BgⅢ/XhoI매종pUC57-PDX1매절PDX1，련접도pShuttle-GFP-CMV중조천사재체，득도pShuttle-GFP-CMV-PDX1중조천사질립，재장pShuttle-GFP-CMV-PDX1전이도pAdxsi재체상，득도pAdxsi-GFP-PDX1병독질립，이용지질체개도중조선병독재체전염293세포，포장출완정적선병독。용중조선병독감염HUCMSCs 24 h 후，경형광현미경、RT-PCR、면역형광、면역세포화학、Western Blot등검측PDXI기인급단백적표체。결과통과측서、매절등감정PDXI기인정학삽입천사질립중，병여병독골가질립중조，중조선병독가고효전염HUCMSCs，RT-PCR검측도PDXI mRNA재HUCMSCs중표체，경면역형광、면역세포화학、Western Blot등증실전염PDXI재HUCMSCs포핵중표체。결론성공구건료PDX1선병독재체，병재HUCMSCs중유효표체。
Objective To construct recombinant adenovirus vector containing human pancreatic and duodenal homeobox factor 1 (PDX1) and detect its expression in human umblical cord mesenchymal stem cells (HUCMSCs). Methods PDX1 obtained by BgⅢ/XhoI enzyme digestion from pUC57-PDX1 was ligated into the recombinant shuttle vector pShuttle-GFP-CMV to obtain the recombinant shuttle plasmid pShuttle-GFP-CMVPDX1. pShuttle-GFP-CMV- PDX1 was shifted to pAdxsi vector to obtain pAdxsi-GFP-PDX1 virus plasmid. The recombinant plasmid was packaged and amplified in 293 cells. The expression of PDX1 gene and protein in HUCMSCs was detected by fluorescence microscopy, RT-PCB, immunofluorescence, immunohistochemistry, and Western Blot. Results PDX1 gene was inserted correctly into shuttle plasmid and the recombinant adenovirus vector was successfully constructed according to the results of sequence and enzyme digestion identification. The adenovirus was effectively transfected into HUCMSCs. RT-PCR verified that PDX1 mRNA was positively expressed in HUCMSCs. Expression of PDX1 protein in the nuclear of HUCMSCs was found by immunofluorescence assay, immunohistochemistry and Western Blot. Conclusion The adenovirus vector containing PDX1 gene is successfully constructed and effectively expressed in HUCMSCs.