论文部分内容阅读
背景:骨形态发生蛋白质是目前发现惟一的一类具有成骨能力的细胞因子,它能刺激基质干细胞向成软骨细胞和成骨细胞方向分化。目的:应用基因工程方法,观察人骨形态发生蛋白质7基因转染兔骨髓基质干细胞向成骨细胞的定向分化。设计:单一样本观察。单位:中山大学附属第一医院骨科。材料:pcDNA1.1/AMP-hBMP7质粒。方法:实验于2004-05/2005-03在中山大学附属第一医院外科实验室完成。全骨髓培养法培养兔骨髓基质干细胞,在体外分别转染pcD-NA1.1/AMP-hBMP7和pcDNA1.1/AMP并留置空白对照。检测转染基因的转录和表达,观察细胞形态和生长情况,通过对感染细胞的碱性磷酸酶染色、钙盐染色、电镜观察胞质中钙质成分以及检测骨钙素鉴定其成骨分化表型。主要观察指标:①兔骨髓基质干细胞的形态及生长。②表达产物的鉴定。③碱性磷酸酶染色(钙钴法)结果。④茜素红染色结果。⑤透射电镜观察结果。⑥扫描电镜观察结果。⑦骨钙素的测定结果。结果:人骨形态发生蛋白质7基因存在于转染后的骨髓基质干细胞中并表达相应的mRNA。目的基因表达后的细胞形态略有变化,但生长曲线与未转染组无明显变化。骨钙素表达较未转染组明显增强。转染组细胞的碱性磷酸酶染色和钙盐染色均为阳性。结论:人骨形态发生蛋白质7基因转染可促进兔骨髓基质干细胞向成骨细胞表型分化。
BACKGROUND: Bone morphogenetic protein (BMP) is the only type of osteogenic cell factor that has been found to stimulate the differentiation of stromal stem cells into chondrogenic cells and osteoblasts. OBJECTIVE: To observe the directional differentiation of human bone marrow stromal stem cells into osteoblasts transfected with BMP-7 gene by gene engineering. Design: Single sample observation. Unit: First Affiliated Hospital of Sun Yat-sen orthopedic. Materials: pcDNA1.1 / AMP-hBMP7 plasmid. Methods: The experiment was performed in the Surgical Laboratory of the First Affiliated Hospital of Sun Yat-sen University from May 2004 to March 2005. Bone marrow stromal stem cells were cultured by whole-bone culture method, pcD-NA1.1 / AMP-hBMP7 and pcDNA1.1 / AMP were transfected in vitro and blank control was set aside. The transcription and expression of transfection gene were detected and the cell morphology and growth were observed. The osteogenic differentiation of osteoblastic differentiation was evaluated by alkaline phosphatase staining, calcium staining of infected cells, calcium content in cytoplasm under electron microscope and osteocalcin type. MAIN OUTCOME MEASURES: ① Morphology and growth of rabbit bone marrow stromal stem cells. ② expression product identification. ③ alkaline phosphatase staining (calcium and cobalt method) results. ④ alizarin red staining results. ⑤ TEM observation results. ⑥ scanning electron microscopy observations. ⑦ osteocalcin determination results. RESULTS: Human BMP-7 gene was found in transfected bone marrow stromal stem cells and expressed the corresponding mRNA. After the target gene expression, the morphology of the cells changed slightly, but the growth curve did not change significantly with the untransfected group. Osteocalcin expression than the non-transfected group was significantly enhanced. Transfection group cells were positive for alkaline phosphatase staining and calcium salt staining. CONCLUSION: Human bone morphogenetic protein 7 gene transfection can promote the differentiation of rabbit bone marrow stromal stem cells into osteoblasts.