转染血管内皮生长因子165基因的狗骨髓基质细胞超微结构变化及成骨能力  

Alteration of the ultrastructure of dog marrow stromal cells transfected with vascular endothelial growth factor 165 gene and its osteogenic ability

作　　者：刘日光[1] 杨述华[2] 刘建湘[2] 易诚青[2] 

机构地区：[1]贵阳医学院附属医院骨科,贵州省贵阳市550004 [2]华中科技大学同济医学院协和医院骨科,湖北省武汉市430022

出　　处：《中国临床康复》2006年第33期53-55,i0003,共4页Chinese Journal of Clinical Rehabilitation

基　　金：国家自然科学基金项目(30170945)~~

摘　　要：目的:探讨转染血管内皮生长因子165基因的狗骨髓基质细胞体外生物学特性。方法:实验于2002-05/2005-06在华中科技大学同济医学院协和医院骨科完成。实验分为血管内皮生长因子基因转染组与对照组。从3只成人杂交犬髂骨取骨髓进行骨髓基质细胞分离培养,将重组的血管内皮生长因子165基因用脂质体介导转染狗骨髓基质细胞,用反转录聚合酶链反应方法检测血管内皮生长因子的表达;通过四唑盐、对硝基苯磷酸盐检测细胞增殖与碱性磷酸酶的活性;考马斯亮蓝法检测蛋白质含量;透射电镜观察细胞超微结构。结果:①重组质粒pcDNA3-人血管内皮生长因子165转染骨髓基质细胞后第7,14天,反转录聚合酶链反应方法检测有血管内皮生长因子mRNA表达。②骨髓基质细胞转染基因后,细胞的增殖能力无明显影响。③培养后第6,8,10,12天,转染血管内皮生长因子基因骨髓基质细胞与对照组细胞比较,碱性磷酸酶活性增高,蛋白质合成增多[转染组碱性磷酸酶为(428.09±26.67),(565.11±24.17),(562.94±39.17),(596.45±30.17)nkat,对照组碱性磷酸酶为(363.57±20.67),(536.44±11.42),(537.11±26.83),(548.10±22.34)nkat;转染组细胞蛋白合成为1.41±0.23,1.46±0.24,1.59±0.33,1.74±0.26;对照组细胞蛋白合成为0.82±0.11,0.83±0.09,0.85±0.06,0.91±0.09]。④透射电镜可见转基因细胞内质网增多,线粒体致密,而糖原溶解与脂肪空泡则减少。结论:血管内皮生长因子基因转染可促进骨髓基质细胞的成骨分化。AIM： To investigate the biological properties of cultured dog marrow stromal cells （MSCs） transfected with human vascular endothelial growth factor （VEGF） 165 gene in vitro. METHODS： The experiment was performed at the Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology from May 2002 to June 2005. There were VEGF transfeetion group and control group. MSCs were isolated from 3 human hybridizing dog iliums and cultured. The recombinant pcDrhVEGF 165 plasmid was transfected into the MSCs by lipofectin mediated gene transfer. Expression of VEGF 165 in MSCs was detected by reverse transcription-polymerase chain reaction （RT-PCR）. MSCs proliferation and activity of alkaline phosphatase were detected by MTT method and PNP method, respectively. Protein content was measure with Coomassie light blue. Transmission electron microscope was used for observation of super microstructure in the MSCs. RESULTS： ①The pcD-rhVEGF165 gene mRNA expression was examined with RT-PCR after transfection for 7 and 14 days. ②The proliferation was not affected by transfection of MSCs gene. ③At days 6, 8, 10 and 12 after culture, activity of alkaline phosphatase increased and protein synthesis became more in MSCs after VEGF transfection as compared with the control group [Alkaline phosphatase in VEGF transfection group, was （428.09±26.67）, （565.11 ±24.17）, （562.94±39.17）, （596.45 ±30.17）nkat; Alkaline phosphatase in control group （363.57±20.67）, （536.44±11.42）, （537.11 ±26.83）, （548.10 ±22.34）nkat; protein synthesis in VEGF transfection group was 1.41±0.23,1.46±0.24,1.59±0.33,1.74±0.26; protein synthesis in control group was 0.82±0.11,0.83±0.09,0.85±0.06,0.91 ±0.09]. ④Endoplasmic reticulum enlarged and mitochondria became more fine and close in the transfected MSCs, but glucose body dissolution and liposome bubble were observed little. CONCLUSION： Transfection with VEGF 165 gene can promote the osteogeni

关 键 词：血管内皮生长因子 基因 骨髓 

分 类 号：R551.3[医药卫生—血液循环系统疾病]