摘要
目的构建同时表达绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因和人胰岛素(insulin,INS)基因的重组腺病毒载体pAdxsi-EGFP-INS,并感染人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUCMSCs),观察其表达及诱导hUCMSCs成脂分化的作用。方法用EcoRI/XhoI将胰岛素基因从原始质粒上切下,定向插入至pShuttle-CMV-EGFP穿梭质粒,将验证正确的pShuttle-CMV-EGFP-INS中的EGFP-INS片段转移至pAdxsi载体上,构建pAdxsi-EGFP-INS重组腺病毒载体,用PacI限制性内切酶线性化后转染人胚肾细胞系HEK293细胞,扩增纯化重组腺病毒,测定病毒滴度。原代培养hUCMSCs,分别用pAdxsi-EGFP-INS(感染组)及pAdxsi-EGFP(对照组)腺病毒感染hUCMSCs,观察荧光蛋白的表达情况,成脂诱导剂诱导分化,油红O染色观察胰岛素对hUCMSCs成脂的诱导作用。结果成功构建pAdxsi-EGFP-INS重组腺病毒载体,包装、纯化后获得滴度达4×1011pfu/ml的重组腺病毒。分离获得hUCMSCs并传代、纯化,pAdxsi-EGFP-INS感染hUCMSCs后,EGFP-INS在胞浆及胞核表达;成脂诱导培养18 d后,感染组细胞呈现出含有大量小脂滴的脂肪样细胞,而对照组细胞含有少量较大的脂滴,感染组含脂滴的脂肪样细胞数量多于对照组。结论获得的pAdxsi-EGFP-INS重组腺病毒可高效感染hUCMSCs,为进一步研究胰岛素在干细胞成脂诱导分化中的作用,追踪其在体内、体外表达情况提供了新工具。
Objective To construct a recombinant adenovirus vector pAdxsi-EGFP-INS with enhanced green flu- orescent protein （EGFP） and insulin （INS） and to observe its expression by transfecting the recombinant adenovirus into human umbilical cord mesenchymal stem cells （bUCMSCs） and the effect of induced hUCMSCs lipoid differentiation. Methods Insulin gene was obtained with EcoRI/XhoI from primitive vector and subcloned into pShuttle-CMV-EGFP plasmid. The EGFP-INS fragment from pShuttle-CMV-EGFP-INS plasmid was subsequently transferred into pAdxsi vector to obtain pAdxsi-EGFP-INS after being verified by enzyme cutting and gel electrophoresis, pAdxsi-EGFP-INS plasmid was linearized by Pacl and transfected into HEK293 cells. The adenovirus recombinant was purified and titrated, and virus ti- ter was detected. The primary culture of hUCMSCs was infected with pAdxsi-EGFP-INS virus （infection group） and pAdxsi-EGFP virus （control group） respectively. The fluoresein expression was observed with immunofluorescence. The hUCMSCs were induced and differentiated into adipocytes by culturing them in adipogenic differentiation medium. The in- duction of insulin in hUCMSCs adipogenic ceils were observed with oil red 0 stain. Results Recombinant of adenovirus vector pAdxsi-EGFP-INS was successfully constructed, and amplified with titer of 4 x 101~ pfu/ml after packaging and deputation. The hUCMSCs were transfected with the recombinant adenovirus pAdxsi-EGFP-INS after generation and dep-uration, and the diffuse EGFP-INS fluorescence was observed in the cytoplasm and nucleus. After adipocyte differentia- tion was induced for 18 d, there were plenty of small lipid droplets of adipogenic ceils in infection group ; there were a few big lipid droplets of adipogenic cells in control group, and the cell number in infection group was less than that in control group. Conclusion The adenovirus recombinant pAdxsi-EGFP-INS can effectively transfect hUCMSCs, which can pro- vide a new method to further study the effect of insulin on
出处
《解放军医药杂志》
CAS
2013年第10期6-10,共5页
Medical & Pharmaceutical Journal of Chinese People’s Liberation Army
基金
国家自然科学基金资助项目(30872689)
全军"十二五"医学科学技术研究重大项目(06MA079)
甘肃省自然科学基金资助项目(3ZS06-A25-099)
关键词
胰岛素基因
绿色荧光蛋白质类
腺病毒
间质干细胞
脐带
Insulin gene
Green fluorescent protein
Adenovirus
Mesenchymal stem cell
Umbilical cord
