血管内皮生长因子转染后脂肪组织来源干细胞蛋白分泌表达及成骨活性的检测

Detection of protein secretion and expression and osteogenic activity of adipose tissue-derived stem cells after in vitro transfection of vascular endothelial growth factor

目的观察人血管内皮生长因子（VEGF）转染后的人脂肪组织来源干细胞（ADSCs）对VEGF蛋白的分泌和表达情况及转染后细胞的成骨活性变化。方法胶原酶消化脂肪获取人ADSCs，经鉴定后进行培养传代。采用Trizol法获取编码人VEGF成熟肽的基因序列，将获得的目的基因VEGF与含绿色荧光蛋白的双顺反子表达载体连接成重组质粒pSELECT—GFPZeO—VEGF，分别转染第2、3、4、5代ADSCs。荧光显微镜下验证转染结果，酶联免疫（ELISA）检测转染后细胞的VEGF分泌。对第2代转染后的ADSCs于成骨条件下培养，检测上清液中碱性磷酸酶（ALP）和骨钙蛋白（OC）的分泌。结果脂质体介导的VEGF目的基因片段能够成功转染至体外培养的ADSCs；经ELISA定量检测，上清液中VEGFmRNA表达水平在转染组显著增高，对照组无明显变化。成骨条件培养下于转染后第3、5、7天ELISA检测人VEGF转染组对ALP（29．2±2．d）、（38．0±2．3）、（46．7±2．4）U／L（金氏单位）和Oc的分泌量（13．2±1．6）、（18．3±1．6）、（25．3±1．5）μg／L以及实时聚合酶链反应（PCR）检测对ALP（0．1±0．0）、（0．2±0．0）、（0．3±0．0）和OC的分泌量（0．042±0．004）、（0．071±0．008）、（0．127±0．011）均明显高于空载体转染组及空白细胞组（P〈0．01）。免疫印迹法检测人VEGF转染组中ALP／GAPDH和OC／GAPDH灰度比值明显高于空载体转染组和空白细胞组（P〈0．01）。结论经脂质体介导可成功将人VEGF目的基因片段转染至人ADSCs，并能够在体外持续有效地表达具有生物学活性的VEGF mRNA；转染后的ADSCs成骨定向分化能力明显增强。

Objective To examine vascular endothelial growth factor （VEGF） protein secretion and expression and explore the osteogenic activity of adipose tissue-derived stem cells （ADSCs） after transfection of human VEGF. Methods The ADSCs were isolated from human adipose tissue after the digestion of collagenase. After identification by flow cytometry, the cells were cultured and passaged in nutritive medium. Gene sequence encoding human VEGF mature peptide was obtained by Trizol reagent method from human vascular tissue. Target gene VEGF was connected with bicistronic expression vector containing green fluorescent protein to form pSELECT-GFP zeo-VEGF for transfecting 2nd, 3rd, 4th, 5th generation ADSCs mediated by liposome. The transfection results were verified under fluorescence microscope. VEGF protein secretion by transfected cells was detected by enzyme-linked immunosorbent assay （ELISA）. Second- generation transfected ADSCs were cultured under osteogenic conditions. The supernatant levels of alkaline phosphatase （ALP） and osteocalcin （OC） were detected. Results Liposome-mediated VEGF target gene fragment could transfect ADSCs successfully. ELISA quantitative detection showed that VEGF mRNA expression levels in supernatant of the transfected group was significantly higher than the control group. And there were significant differences. After osteogenic culturing, the detections of ELISA, real-time PCR and Western blot showed that the secretion of ALP and OC of VEGF transfected group was significantly higher than that of empty vector transfected and blank cell groups. And there were significant differences （P〈0.01 ）. Conclusion After transfected by liposome-mediated VEGF target gene fragment, human ADSCs can express biologically aetive VEGF mRNA in vitro continuously and effectively. Directional differentiation capacity of transfected ADSCs is signifieantly enhanced.