转染血管内皮生长因子基因的人成骨细胞增殖及其生物学功能

Proliferation and biological function of human osteoblasts transfected with vascular endothelial growth factor gene

背景:传统的自体或异体骨移植治疗骨缺损都存在难以克服的缺点,基因治疗可能是一种新途径。目的:观察转染外源性血管内皮生长因子基因的人成骨细胞体外生物学特性。设计、时间及地点:对比观察,细胞学实验,实验于2005-05/2006-05在辽宁医学院科学实验中心完成。材料:人髂骨骨块取自需髂骨植骨的颈椎病患者的植骨块修洁剩余部分的骨质,患者知情同意。质粒pCDI/VEGF121为北京大学人类疾病治疗中心马大龙教授惠赠;感受态大肠杆菌DH5a为辽宁医学院刘丹平教授惠赠。方法:体外分离和培养人成骨细胞。实验分为血管内皮生长因子基因转染组与对照组。利用阳离子脂质体将pCDI-VEGF121真核表达质粒导入体外培养的人成骨细胞。主要观察指标:传代后1,3,5,7d观察血管内皮生长因子在人成骨细胞内的表达,及其对人成骨细胞增殖活力和细胞分泌骨钙素和碱性磷酸酶能力的影响。结果:pCDI-VEGF121真核表达质粒转染人成骨细胞后第3,7天,以反转录聚合酶链反应方法检测到其中有血管内皮生长因子mRNA表达。转染组的细胞数在第1,3,5,7天均高于对照组(P<0.05或P<0.01)。培养3d时转染组成骨细胞的碱性磷酸酶阳性率大于对照组(P<0.01);转染组的骨钙素含量高于对照组(P<0.05或<0.01)。结论:血管内皮生长因子基因转染可以促进体外培养人成骨细胞的增殖能力和相应的生物学功能。

BACKGROUND： Traditional methods of repairing bone defect such as autograft and allograft have some disadvantages that are hard to deal with, gene treatment may be a new approach. OBJECTIVE： To investigate the biological properties of cultured human osteoblasts transfected with vascular endothelial growth factor （VEGF） gene. DESIGN, TIME AND SETTING： A controlled experiment based on cytology was carried out in the Scientific Experiment Centre of Liaoning Medical College from May 2005 to May 2006. MATERIALS： Human lilac born block was harvested from a cervical spondylosis patient who required lilac bone graft with his informed consent of this patient. Plasmid pCDI/VEGF121 was given as gift from Professor Ma, Peking Unviersity Human Disease Genomics Research Center. Competent Escherichia coil was given as gift from Professor Liu, Liaoning Medical College. METHODS： Human osteoblasts were isolated and cultured in vitro. There were a VEGF transfection group and a control group in the experiement. Using cation liposome, the pCDINEGF121 eukaryotic expression plasmis was induced into human osteoblasts. MAIN OUTCOME MEASURES： At 1, 3, 5, 7 days following passage culture, the expression of VEGF in human osteoblasts was detected. Its effects on the cell proliferation, the secretion of osteocalcin and alkaline phosphatase were investigated. RESULTS： After the plasmid pCDI-VEGF121 was transferred into human osteoblasts 3 and 7 days, VEGF mRNA expression was detectable by RT-CPR method. The cell number of transfection group was larger than that of control group （P 〈 0.05 or P 〈 0.01 ）. When the cells were cultured for 3 days, the positive rate of alkaline phosphatase in the transfection group was increased compared with control group （P 〈 0.01）; the secretion of osteocalcin in the transfection group was higher than that of control group （P〈 0.05 orP〈 0.01）. CONCLUSION： VEGF gene transfection can improve the proliferation and biological function of human osteoblasts cultured in vitro.