血管内皮生长因子165基因转染的骨髓基质细胞异位成骨的初步研究

A primary study on the osteogenic characteristic of MSCs with VEGF165 gene

目的:将血管内皮生长因子165(VEGF165)基因转染的大鼠骨髓基质细胞(MSCs)和β-磷酸三钙复合后,植入原大鼠肌肉中,探索转染外源基因的MSCs异位成骨能力。方法:SD大鼠12只,在全麻及无菌条件下取其左侧股骨的骨髓基质细胞,并用矿化液诱导培养。在脂质体介导下将构建成功的真核表达载体pcDNA3.1-VEGF165转染到诱导培养的MSCs,G-418筛选阳性细胞克隆,将阳性克隆细胞和未转染的MSCs共同接种于β-磷酸三钙上,体外培养7天后,植入原大鼠肌肉内。分别于4周、8周和12周处死动物,观察异位成骨情况。结果:骨髓基质细胞-β-磷酸三钙复合体植入肌肉后,材料周围血管生长较多,随着植入时间的延长,小的骨岛逐渐融合为大的骨块,并有骨髓腔样结构,表现出较好的异位成骨能力。结论:应用VEGF165基因转染MSCs作为种子细胞植入体内后,有利于发挥VEGF165的血管化作用,同时促进组织工程骨的成骨速度,将会成为组织工程骨血管化探索的方向。

Objective To investigate the osteogenic characteristic in vivo of rat bone marrow stroma cells （MSCs） with Vascular endothelial growth factor 165 gene （VEGF165） combined onto β-tricalcium phosphate （β-TCP）. Methods MSCs were obtained from femurs of 12 SD rats under generar anesthesia and sterile condition, and cultured in DMEM supplemented with 10%FBS. MSCs were cultured in a conditional medium in subculture including： dexamethasone, vitamin C, and β-glycerophosphate. At the same time, we constructed the eukaryotic expression vector containing VEGF165 gene. VEGF165 gene was transfected into MSCs by means of LipofectAMINE？2000. The stably gene expressive cells were screened with G-418 for 14 days. The cell mixture were combined onto β-TCP and cultured in vitro for 7 days. The cells-ceramic compound was implanted intramuscularly into the corresponding rat. These rats were executed after 4w, 8w, 12w.The osteogenic characteristic in vivo were investigated by histochemistry. Results As the time passed, much osseous island changed large osseous block. It showed that the cells-ceramic compound implanted intramuscularly could be osteogenesis. Conclusion When VEGF165 gene was transfected into MSCs, there were blood vessel around the osseous island. VEGF165 could improve vessels growth and accelerate osteogenicof tissue engineering bone. The experiment was an interesting research of vascular tissue engineering bone.