骨性材料复合绿色荧光蛋白标记骨髓间充质干细胞研究体系的建立与应用

Research system of green fluorescent protein labeled bone marrow mesenchymal stem cells growing in bone bio-scaffold

背景:由于骨性生物材料和骨组织本身坚硬致密的物理特性,限制了对生长在其中的靶细胞的示踪研究。目的:针对骨性材料含钙、透光性差的特性,拟建立骨性材料复合绿色荧光蛋白基因标记的骨髓间充质干细胞的研究体系。设计、时间及地点:体外观察实验,于2008-06/2009-01在南方医科大学南方医院骨与软骨再生医学重点实验室完成。材料:β-磷酸三钙多孔骨性生物支架材料(1cm×1cm×0.8cm)购自上海贝奥路生物材料有限公司。3月龄新西兰大白兔用于获取并培养骨髓间充质干细胞。方法:①骨髓间充质干细胞以携带绿色荧光蛋白的慢病毒载体标记后,接种于β-磷酸三钙骨性生物支架材料内部。②以荧光倒置显微镜观测材料中细胞的生长情况,并通过荧光定量聚合酶链反应等检测材料内部细胞的增殖情况。③以自创的半固体脱钙体系对复合细胞的材料脱钙后,制备连续冰冻切片观察;同时,采用不脱钙塑料包埋、扫描电镜等方法观察。主要观察指标:骨性材料中绿色荧光蛋白标记骨髓间充质干细胞的分布、形态及增殖情况。结果:①荧光显微镜下观察到绿色荧光细胞在材料内部生长、增殖。②普通聚合酶链反应可对材料内的绿色荧光细胞进行定性分析,荧光定量聚合酶链反应检测的Ct值和接种的绿色荧光细胞数量呈指数相关,故可以对材料中的绿色荧光细胞进行定量分析。③半固体脱钙可使骨性材料内部坚硬的钙质成分消失,而细胞所在的位置、形态和荧光不变;不脱钙塑料包埋可以原位保留细胞形态和荧光,但仅适用于小块组织;而扫描电镜观察范围有限,细胞无法保留荧光。结论:建立了可在骨性材料内部方便、直观地追踪绿色荧光蛋白标记骨髓间充质干细胞的体系,具有广泛的应用前景。关键词:骨组织工程;绿色荧光蛋白;半固体脱钙;荧光定量聚合酶链反应;

BACKGROUND： It is difficult to study and trace the target cells in bone and bone bio-scaffold, which are very hard, compact and poor lucency. OBJECTIVE： To establish the research system of green fluorescent protein （GFP） labeled bone marrow mesenchymal stem cells growing in the bone bio-scaffold with calcium and poor lucency. DESIGN, TIME AND SETTING： An in vitro experiment was performed at the Key Laboratory of Bone and Cartilage Regenerative Medicine, Nanfang Hospital of Southern Medical University from May 2008 to January 2009. MATERIALS： β-tricalcium phosphate （ β -TCP） porosity bonny bio-material scaffold （1 cm × 1 cm × 0.8 cm） was purchased from Shanghai Bio-lu Bio-materials Company Limited. New-Zealand rabbits, 3 months old, were used to obtain marrow mesenchymal stem cells. METHODS： ① GFP-labeled bone marrow mesenchymal stem cells modified with recombined lentivirus were inoculated in bone bio-scaffold of β-TCP. ② GFP-positive cells in bio-scaffold were observed by fluorescence invert microscope, and their proliferation detected by real-time fluorescent quantitation PCR method. ③ Bio-scaffold with cells were buried in own-made decalcification semi-solid system, and frozen sections were made to observe fluorescence trace. In addition, plastic embedding, scanning electron microscope and paraffin sections were also used. MAIN OUTCOME MEASURES： The distribution, morphology, and proliferation of the GFP-labeled bone marrow mesenchymal stem cells in the bonny bio-material scaffold. RESULTS： ① GFP-labeled cells could be found growing well on internal side of pores of the β -TCP scaffold by fluorescence invert microscope. ② General PCR could qualitatively analyze GFP-positive cells in bone bio-scaffold, while fluorescent quantitation PCR indicated that Ct value of GFP-labeled gene was correlated well to numbers of GFP-positive cells. Therefore, fluorescent quantitation PCR could quantitatively analyze numbers of GFP-positive cells. ③ Hard component in bone bio-scaffold disappeared during semisolid decalcification, but position, morphology, and fluorescence of cells were not changed. Plastic embedding for small section alone could keep morphology and fluorescence in situ, scanning electron microscopy could not keep fluorescence. CONCLUSION： A research system of GFP-labeled bone marrow mesenchymal stem cells is established in bone bio-scaffolds, which would be very useful in many research areas.