摘要
目的:探讨脂肪组织来源的干细胞(ADSCs)表达外源性基因的可能性。方法:取小香猪腹部皮下脂肪组织,通过I型胶原酶消化、离心等方法分离培养ADSCs,再经原代培养和传代培养。利用成骨诱导剂(10-7mol/L地塞米松、10mmol/Lβ-甘油磷酸钠和50mg/L左旋抗坏血酸)诱导ADSCs向成骨方向分化,观察其生长形态,并通过Gomori改良钙钴法和von Kossa染色对其成骨表型进行鉴定。另设一对照组,培养基中不加入成骨诱导剂。脂质体介导人内皮细胞生长因子(hVEGF)和骨形态发生蛋白-2(hBMP-2)转染ADSCs细胞,观察转染后细胞形态和生长情况,用逆转录-聚合酶链反应(RT-PCR)和免疫细胞化学方法分别检测hVEGF mRNA、BMP-2 mRNA及其蛋白质表达。结果:成骨诱导剂能显著诱导体外培养的ADSCs向成骨细胞分化,诱导19d的ADSCs体积增大,细胞由梭形变为多边形,Gomori改良钙钴法染色显示其胞浆内富含碱性磷酸酶颗粒,诱导15d和19d,碱性磷酸酶阳性细胞分别为(25.0±7.5)%和(49.7±6.9)%。von Kossa染色表明聚集的细胞团中央有钙化结节形成。RT-PCR检测证实,经质粒转染的ADSCs中hVEGF和hBMP-2基因表达明显增强,对照组呈弱表达。免疫组织化学检测证实转染ADSCs内有棕色的阳性颗粒出现,未转染细胞则呈现阴性。结论:ADSCs能被成功诱导为成骨细胞,hVEGF和hBMP-2基因能成功地转入ADSCs并表达,这为促进骨组织工程的血管化和成骨活性奠定了基础。
Objective ： To investigate the possibility of exogenous gene expression in transfection adipose tissuederived stromal cells （ADSCs） . Methods： ADSCs were obstained from the swine adipose tissue and were cultured in DMEM/F12 with fetal bovine serum. DMEM/F12 medium was supplemented with 10% fetal bovine serum, 50 mg/L freshly prepared ascorbic acid, 10 mmol/L β-sodium glycerophosphated and 10^-8 mol/ L dexamethasone for osteoblast-inducing culture. The osteoblast phenotype was identified with Gomori and Von Kossa staining. The expression plasmid of human vascular endothelial growth factor （hVEGF） and human bone morphogenetic protein- 2 （hBMP-2） genes were transfected into ADSCs. The expression of hVEGF and hBMP-2 in the transfected cells was detected by reverse transcription-polymerase chain reaction （RT-PCR） and immunohistochemistry. The ADSCs cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum only served as controls. Results： ADSCs grew as adherent cells and appeared fibroblast-liked in the culture, and they could stably proliferate and passage. The cells became polygon-shaped and were characterized by large size with a relatively large nucleus and rich in cytoplasm after being induced. There was a large number of alkaline phosphatase （ALP） particles as shown by Gomori stain. After 15-day and 19-day induction, positive cells for ALP were （25.0±7. 5）% and （49.7± 6.9） %, respectively. Von Kossa staining demonstrated formation of calcium nodules in the extracellular matrix. The hVEGF and hBMP-2 genes were successfully transferred into ADSCs and successfully expressed in ADSCs. RT-PCR showed that the hVEGF and hBMP-2 gene expressions were significantly up-regulated in transfected cells, but weakly expressed in the cells of the control group. Immunohistochemistry showed brown particles in ADSCs transfeeted by the two genes, but they could not be found in non-transfeeted cells. Conclusion： ADSCs can be successfully induced to differentiatc into osteoblasts in vitro. The swine ADSCs with transference of the human VEGF and BMP-2 genes, can express the two genes and proteins. It might be used to increase the osteogenic and vascularization capability of ADSCs in the study of bone tissue engineering.
出处
《感染．炎症．修复》
2008年第2期77-81,F0003,共6页
Infection Inflammation Repair
基金
国家重点基础研究发展规划项目(2005CB522603)
