摘要
目的构建同时表达绿色荧光蛋白(green fluorescent protein,GFP)报告基因和人类Nel1型蛋白[homo sapiens NEL-like1,NELL1)]基因的重组腺病毒载体pAdxsi-GFP-NELL1,并转染大鼠BMSCs,观察其表达情况,为进一步研究NELL1蛋白的成骨作用提供理论基础。方法设计特异性引物从NELL1质粒中扩增NELL1,将测序正确的片段用XhoI/BglII酶切处理,定向插入至pShuttle-GFP-CMV(-)TEMP重组穿梭载体,然后将验证正确的重组穿梭质粒中的插入片段转移至pAdxsi载体构建重组腺病毒载体质粒,用PacI限制性内切酶线性化后转染HEK293细胞,扩增纯化重组腺病毒,半数组织培养感染量法测定重组腺病毒滴度。培养大鼠BMSCs,采用流式细胞仪鉴定表面标志物,并行成骨、成脂诱导鉴定。用构建的pAdxsi-GFP-NELL1及空病毒pAdxsi-GFP(作为对照)转染鉴定正确的BMSCs,RT-PCR检测NELL1的表达,免疫荧光检测GFP基因及NELL1的表达情况,细胞计数试剂盒(cell counting kit-8,CCK-8)法检测对细胞增殖的影响。结果成功构建同时表达NELL1和GFP基因的重组腺病毒载体(pAdxsi-GFP-NELL1),纯化后获得滴度达1×1011pfu/mL的重组腺病毒。成功分离获得大鼠BMSCs并传代、纯化,经流式细胞仪鉴定表面标志物及成骨、成脂诱导鉴定为BMSCs。pAdxsi-GFP-NELL1转染BMSCs后,RT-PCR检测示NELL1mRNA阳性表达,荧光显微镜观察示细胞爬片GFP阳性表达,NELL1抗体免疫荧光观察示NELL1蛋白阳性表达。CCK-8法鉴定显示转染后对BMSCs生长无明显影响。结论构建并纯化后的重组腺病毒载体(pAdxsi-GFP-NELL1)可高效转染大鼠BMSCs,并稳定表达NELL1和GFP两种基因,为进一步研究新的成骨基因NELL1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-GFP-NELL1 that co-expressing green fluorescent protein （GFP） and homo sapiens NEL-like 1 （NELL1） protein （a protein strongly expressed in neural tissue encoding epidermal growth factor like domain）,to observe its expression by transfecting the recombinant adenovirus into rat bone marrow mesenchymal stem cells （BMSCs） so as to lay a foundation for further study on osteogenesis of NELL1 protein. Methods From pcDNA3.1-NELL1,NELL1 gene sequence was obtained,then NELL1 gene was subcloned into pShuttle-GFP-CMV （-）TEMP vector which was subsequently digested with enzyme and insterted into pAdxsi vector to package the recombinant adenovirus vector （pAdxsi-GFP-NELL1）. After verified by enzyme cutting and gel electrophoresis,pAdxsi-GFP-NELL1 was amplified in HEK293 cells and purif ied by CsCl2 gradient purification,titrated using 50% tissue culture infective dose （TCID50） assay. The rat BMSCs were cultured and identifi ed byflow cytometry and directional induction,then were infected with adenoviruses （pAdxsi-GFP-NELL1 and pAdxsi-GFP）. NELL1 expression was verified by RT-PCR and immunofluorescence; GFP gene expression was verified by the intensity of green fluorescence underfluorescence microscope. Cell counting kit-8 （CCK-8） was used for investigate the influence of vectors on the proliferation of rat BMSCs. Results Recombinant adenoviral vector pAdxsi-GFP-NELL1,which encodes a fusion protein of human NELL1,was successfully constructed and amplified with titer of 1 × 1011 pfu/mL. The primary BMSCs were cultured and identified by flow cytometric analysis,osteogenic and adipogenic induction,then were used for adenoviral transfection efficiency and cell toxicity tests. An multiplicity of infection of 200 pfu/cell produced optimal effects in transfer effi ciency without excessive cell death in vitro. Three days after transfectionwith 200 pfu/cell pAdxsi-GFP-NELL1 or pAdxsi-GFP,over 60% BMSCs showed green fluorescent by fluorescence microscopy. Imunofluorescence with NELL1 antibody also revealed high level expression of human NELL1 protein in red fluorescent in these GFP expressing cells. RT-PCR analysis confirmed that the exogenous expression of NELL1 upon transfection with pAdxsi-GFP-NELL1 at 200 pfu/cell,whereas NELL1 remained undetectable in Ad-GFP-transfected rat BMSCs. The proliferative property of primary rat BMSCs after adenoviral NELL1 transfection was assayed by CCK-8 in growth medium. Growth curve demonstrated no significant difference among BMSCs transfected with pAdxsi-GFP-NELL1,pAdxsi-GFP,and no treatment control at 7 days （P 0.05）. Conclusion Recombinant adenovirus vector pAdxsi-GFP-NELL1 can steady expressing both GFP and NELL1 protein after being transfected into rat BMSCs. It provides a useful tool to trace the expression of NELL1 and investigate its function in vitro and in vivo.
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2010年第5期606-612,共7页
Chinese Journal of Reparative and Reconstructive Surgery
基金
国家自然科学基金资助项目(30872621)
国家科技支撑计划资助项目(2006BAI16B02)~~
关键词
BMSCS
重组腺病毒载体
人类Nel1型蛋白
绿色荧光蛋白
基因转染
大鼠
Bone marrow mesenchymal stem cells Recombinant adenovirus vector Homo sapiens NEL-like 1 protein Green fluorescent protein Gene transfection Rat
