【摘要】 目的:采用腺病毒介导人骨形态发生蛋白2基因转染兔脂肪干细胞,观察目的基因在脂肪干细胞中的表达效率及向成骨细胞定向分化的能力。方法:实验于2005-12/2006-09在武汉大学医学院中心实验室和三峡大学医学院病理实验室进行。①实验材料:4月龄健康新西兰大耳白兔2只。质粒pAd-BMP-2由美国哈佛医学院分子骨科中心Oliver博士惠赠;携带β-半乳糖酐酶基因的腺病毒对照载体由李康博士惠赠;大肠杆菌DH5a以及293细胞由本实验室保存。②实验方法:新西兰兔肌注麻醉后,完整取出双侧腹股沟脂肪垫,清除外包膜、明显的结缔组织和小血管,剪碎,离心,体外分离培养脂肪干细胞。表达载体pAd-hBMP-2经293细胞包装重组腺病毒后,取第3代生长良好的脂肪干细胞,按5×105接种于60mm培养皿,将Ad-hBMP-2病毒以感染复数10～20感染细胞。将成功转染骨形态发生蛋白2的脂肪干细胞作为转染组,以转染携带β-半乳糖酐酶基因腺病毒载体的脂肪干细胞作为对照组。两组均置入不含骨形态发生蛋白2的无血清成骨诱导培养基中进行诱导分化。③实验评估:定期观察细胞的形态学变化,MTT法绘制生长曲线,计算增殖时间;成骨诱导培养后测定碱性磷酸酶活性,光倒置显微镜下观察钙化结节形成情况;RT-PCR、免疫组织化学法、Westernblot法检测目的基因人骨形态发生蛋白2和成骨细胞标志性蛋白Ⅰ型胶原、骨钙素的表达。结果:①脂肪干细胞转染前后形态学变化、生长曲线及倍增时间:Ad-hBMP-2基因修饰的脂肪干细胞经诱导培养后形态规则,多角型细胞增多,转染组的细胞生长曲线倍增时间明显短于对照组。②碱性磷酸酶活性及矿化结节形成:转染组脂肪干细胞碱性磷酸酶活性呈增加趋势,成骨诱导培养7,10,14d均显著高于未转染组(P<0.01)。转染组成骨细胞四环素标记显示圆形矿化结节呈金黄色,数量多,结节大。③脂肪干细胞人骨形态发生蛋白2与Ⅰ型胶原、骨钙素的表达:成骨诱导培养7,14d,RT-PCR、免疫组织化学法、Western blot法检测结果显示转染组目的基因人骨形态发生蛋白2、成骨标志性蛋白Ⅰ型胶原、骨钙素的分泌表达均明显强于对照组。结论:经Ad-hBMP-2基因修饰的兔脂肪干细胞在体外能定向分化为成骨细胞,且分化增殖能力强;目的基因骨形态发生蛋白2表达高、持续时间长。更多还原

【Abstract】 AIM: To investigate the expression of human bone morphogenetic protein (hBMP)-2 gene transfected to rabbit adipose-derived stem cell (ADSCs) by adenoviral vector and analyze the cells’ osteoblastic differentiation potential. METHODS: This experiment was performed at the Central Laboratory of Medical College of Wuhan University and Laboratory of Department of Pathology,Medical College of Three Gorges University from December 2005 to September 2006. ①Two 4-month old healthy New Zealand flap-eared rabbits were used and the plasmid vectors of pAd-BMP-2 and pAd-Lac Z were offered by Dr Oliver from Center of Molecule and Orthopaedics of Harvard medical school and Dr Li respectively. The E.coli DH5a and 293 cells were preserved by this lab. ②After the rabbits were anesthetized,double inguina fat pad was excised and outer envelope,connective tissue and small vessels were excluded,sheared and centrifugated and the ADSCs were cultured in vitro. When constructed the pAd-hBMP-2 plasmid vector and recombinated the virus,the 3-passage ADSCs well grown were harvested,cultured in 60 mm culture dish at 5×105. Ad-hBMP-2 virus was transfected for 10-20 MOI. Two groups were the hBMP-2 transfected group and the control group with Lac Z gene. The cells were cultured on complete osteoblastic medium. ③The morphology and localized regions of mineralization of the cells were observed under light inverted microscope. Growth curve was drawn by MTT and proliferation timing was calculated. The expression of alkaline phosphatase (ALP),hBMP-2,CollagenⅠand Osteocalcin were measured by the cytochemisty,reverse transcription polymerase chain reaction (RT-PCR),immunohistochemistry and Western blot,respectively. RESULTS: ①The hBMP-2 transfected ADSCs became the polygon form and its growth curve double time showed shorter than the control group. ②The cytochemisty of ALP of the hBMP-2 transfected group showed significant higher than the control group after transfected for 7 days,10 days and 14 days(P < 0.01). Localized regions of mineralization showed inaurate became more and large. ③The expressions of hBMP-2,CollagenⅠand Osteocalcin were significantly higher than the control group at days 7 and 14 after RT-PCR,Western blot and immunohistochemiscal method. CONCLUSION: The ADSCs transfected by the adenoviral vector mediated hBMP-2 gene have stronger potential to differentiate into osteoblasts. The hBMP-2 gene has showed high and long time expression on the cells.更多还原