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分化(英文)

Differentiation into osteoblasts from marrow mesenchymal stem cells of rabbits transfected by human bone morphogenetic protein-7 gene

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： At present, bone morphogenetie protein （BMP） is the only eytokine characterized as the ability of osteoblast, and it can promote marrow mesenehymal stem cells （MSCs） differentiating into ehondroblast and osteoblast. OBJECTIVE： To investigate the differentiation into osteoblast from MSCs of rabbits transfeeted by human bone morphogenetie protein-7 （hBMP-7） gene with genetic engineering technique. DESIGN： Single sample observation. SETTING： Department of Orthopaedics, the First Hospital Affiliated to Sun Yat-sen University. MATERIALS： pcDNA1.1/AMP-hBMP7 plasmid. METHODS： The experiment was completed at the Surgery Laboratory of the First Hospital Affiliated to Sun Yat-sen University from May 2004 to March 2005. MSCs of rabbits were cultured with whole bone marrow technique, transfeeted with peDNA1.1/AMP-hBMP7 and .peDNA1.1/AMP plasmids respectively in vitro, and left the blank controls. Transcription and expression of transfected genes were detected so as to observe form and growth of cells. Component of calcium content in cytoplasm was measured and osteogenie phenotype was identified with alkaline phosphatase （ALP） staining, calcium salts staining and transmission electron microscopy （TEM）. MAIN OUTCOME MEASURES： ① Form and growth of MSCs of rabbits; ② evaluation of expressive product; ③ results of ALP staining （Ca-Co technique）; ④ results of ehinalizarin staining; ⑤ results of TEM; ⑥ results of scanning electron microscopy （SEM）; ⑦ assay of osteocalein. RESULTS： hBMP-7 gene was contained in MSCs after transfeetion and expressed the relevant mRNA. Cellular form changed a little after expres- sion of amid genes, but growth curve did not changed as compared with that in non-transfected group. Expression of osteoealein was increased as compared with that in non-transfeeted group, and ALP staining and calcium saltg staining of transfected cells were positive. CONCLUSION： Transfeetion of hBMP-7 gene can promote MSCs of rabbits differentiating into osteoblast.