摘要
背景:寻找合适的标记分子作为血管内皮祖细胞谱系追踪观察的标志成为其体内、外诱导分化机制研究中的一项重要课题。目的:体外研究绿色荧光蛋白(green fluorescent protein,GFP)基因转染骨髓源性血管内皮祖细胞的方法,并检测基因转染后细胞的生物学特性。设计、时间及地点:细胞基因工程对照观察,于2007-07/2008-05在苏州大学附属第二医院实验中心完成。材料:3周龄清洁级Wistar大鼠15只用于制备骨髓源性血管内皮祖细胞,绿色荧光蛋白基因及重组腺病毒由苏州大学附属第二医院李晓强教授惠赠。方法:体外分离培养Wistar大鼠血管内皮祖细胞,EGM-2MV培养基培养大鼠骨髓中的单个核细胞。以腺病毒为载体,293A细胞为包装细胞,介导GFP转染血管内皮祖细胞,与未转染的同期细胞作对照。主要观察指标:在荧光显微镜下观察GFP表达效率。酶联免疫吸附法检测培养上清中血管内皮生长因子蛋白水平评价GFP转染血管内皮祖细胞后对细胞功能的影响,MTT法评价GFP转染血管内皮祖细胞后对细胞活性的影响。结果:成功构建了携带绿色荧光蛋白基因的重组腺病毒(Ad-GFP),成功培养了大鼠骨髓源性内皮祖细胞,重组Ad-GFP可高效率转染血管内皮祖细胞,病毒感染组阳性细胞与未感染组细胞血管内皮生长因子蛋白水平表达相当,Ad-GFP转染后对血管内皮祖细胞的增殖没有影响。结论:构建了带有GFP的缺陷型重组Ad-GFP,转染Wistar大鼠血管内皮祖细胞得到了高效表达,且感染细胞的生物学特性和增殖能力未受影响。
BACKGROUND： To seek suitable marked molecule as a mark for blood vessel endothelial progenitor cells （EPCs） has been an important topic for studying differential mechanism in vitro and in vivo. OBJECTIVE： To study an efficient method to transfect green fluorescent protein gene （GFP） into EPCs in vitro and to determine the biological properties of cells following gene transfection. DESIGN, TIME AND SETTING： Gene engineering controlled observation was performed at the Experimental Center, Second Affiliated Hospital, Soochow University between July 2007 and May 2008. MATERIALS： Fifteen three-week-old Wistar rats of clean grade were used to prepare EPCs. GFP gene and recombinant adenovirus were gifted by professor Li Xiao-qiang from the Second Affiliated Hospital of Soochow University. METHODS： Wistar rat bone marrow EPC were separated and purified in vitro. Mononuclear cells isolated from rat bone marrow were incubated in EGM-2MV. GFP were transfected into the EPCs by adenovirus vector after being packed in 293A cells. The nontransfected EPC was used as the controls. MAIN OUTCOME MEASURES： GFP expression efficiency was observed using the fluorescence microscope. Vascular endothelial growth factor protein levels in supernatant were measured by enzyme linked immunosorbent assay to assess GFP-transfected EPCs on cell function. Influence of GFP-transfected EPCs on cell activity was evaluated by MIT. RESULTS： Recombinant adenovirus carrying GFP was successfully constructed, and bone marrow-derived endothelial progenitor cells were cultivated. Recombinant adenovirus carrying GFP could transfect EPCs in a high efficiency. Vascular endothelial growth factor protein levels were equal between positive cells in the infected group and cells in the non-infected group Recombinant adenovirus-GFP transfection had no effect on the proliferation of EPCs. CONCLUSION： The infected EPCs with adenovirus-GFP expressed GFP with much higher efficiency, and maintained the same ability of proliferation and differentiat
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第14期2641-2644,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
江苏省自然科学基金资助项目(BK2007055)~~
