腺病毒介导骨形态发生蛋白2基因转染诱导脂肪间充质干细胞向

背景：在众多的骨生长因子中，骨形态发生蛋白2具有显著促进骨缺损修复和骨折愈合的效果。骨修复是一个阶段性过程，不需要转染的目的基因长久表达，因此利用腺病毒载体将目的基因导入脂肪间充质干细胞来促进骨愈合比较理想。目的：观察腺病毒介导的骨形态发生蛋白2基因转染后大鼠脂肪间充质干细胞的分化情况。 设计、时间及地点：细胞学体外对照观察，于2008-01/10在北京大学第三医院中心实验室完成。材料：清洁级3周龄雄性Lewis大鼠24只，由北京大学实验动物学部提供。腺病毒载体Ad-BMP2、Ad-GFP由靳小兵博士构建扩增。方法：胶原酶消化法分离培养大鼠脂肪间充质干细胞，传至第3代分为Ad-BMP2转染组、Ad-GFP转染组和未转染组。各转染组分别向培养液中加入对应的腺病毒液，转染复数MOI=100。主要观察指标：转染后脂肪间充质干细胞碱性磷酸酶活性，von Kossa染色结果，Western blotting法检测骨桥蛋白和骨形态发生蛋白2的表达。结果：与未转染组比较，转染后7，14，21d Ad-GFP转染组细胞碱性磷酸酶活性无明显变化；Ad-BMP2转染组细胞碱性磷酸酶活性明显增高（P < 0.01），且随时间延长碱性磷酸酶活性增加更为显著。Ad-BMP2转染组von Kossa染色出现黑色钙结节，免疫细胞化学染色结果示胞浆内有大量黄染颗粒，骨桥蛋白、骨形态发生蛋白2均呈明显强阳性表达。Western blotting检测结果显示Ad-BMP2转染组脂肪间充质干细胞骨桥蛋白、骨形态发生蛋白2表达水平增高，Ad-GFP转染组中未检测到骨桥蛋白、骨形态发生蛋白2的表达。结论：大鼠脂肪间充质干细胞经腺病毒介导的骨形态发生蛋白2基因转染后，可定向分化为成骨细胞。

Directional differentiation of adipose mesenchymal stem cells into osteoblasts induced by adenovirus-bone morphogenetic protein 2 gene transfection

BBACKGROUND: Among many skeletal growth factors, bone morphogenetic protein-2 can significantly promote the outcomes of bone defect repair and bone fracture healing. Bone repair is a phasic process, and transfection target gene is not needed for a long time. Therefore, target gene is introduced into adipose mesenchymal stem cells using adenovirus vector. This is an ideal method to promote bone healing. OBJECTIVE: To investigate the differentiation of rat adipose mesenchymal stem cells following transfection of adenovirus-bone morphogenetic protein 2 (Ad-BMP2). DESIGN, TIME AND SETTING: The in vitro cytology controlled experiment was performed at the Center Laboratory of Third Hospital, Peking University between January and October 2008. MATERIALS: Totally 24 clean male Lewis rats aged 3 weeks old were supplied by Department of Experimental Animal, Peking University. Adenovirus vector Ad-BMP2 and adenovirus-green fluorescent protein (Ad-GFP) were constructed and amplified by Doctor Jin. METHODS: Rat adipose mesenchymal stem cells were isolated and cultured by the collagenase digestion method. At the third passage, cells were divided into Ad-BMP2 transfection group, Ad-GFP transfection group and non-transfection group. Corresponding adenovirus medium was separately added into the medium in each group, multiplicity of infection=100. MAIN OUTCOME MEASURES: Adipose mesenchymal stem cells following transfection underwent alkaline phosphatase activity detection and von Kossa staining. Expression of osteopontin and BMP-2 was measured by Western blotting. RESULTS: Compared with the non-transfection group, no significant differences in alkaline phosphatase activity were detected in the Ad-GFP transfection group at 7, 14, 21 days transfection. Alkaline phosphatase activity was significantly increased in the Ad-BMP2 transfection group (P < 0.01). With prolongation of time, alkaline phosphatase activity was more significantly increased. Black calcium nodules appeared following von Kossa staining in the Ad-BMP2 transfection group. Immunocytochemical staining results demonstrated that a large number of yellow particles in the cytoplasm. A strong positive reaction to osteopontin and BMP2 was detected. Western blotting results showed that osteopontin and BMP2 expression of adipose mesenchymal stem cells was increased in the Ad-BMP2 transfection group, while osteopontin and BMP2 expression of adipose mesenchymal stem cells was not detected in the Ad-GFP transfection group. CONCLUSION: Rat adipose mesenchymal stem cells can differentiate into osteoblasts following Ad-BMP2 gene transfection.