糖基化pIRES-BMP2-VEGF165/MSCs靶向归巢治疗骨缺损的作用及机制研究

The repair of bone defect is complicated, involving synergistic effects of kinds of cells and cytokines.In clinic, the treatment of large bone defects is a conundrum .Our previous researches have found MSCs tranfected with BMP2 and VEGF165 have stronger osteogenic ability, and MSCs with glycosylation modification show obvious ability of targeting migration . Combined with the latest progress, our group present a new strategy:Application of genetic engineering technology，multipotential stem cells will be transformed into that，which have active osteogenesis ability and targeting。It should promote the repair of bone defect.In this study, we using BMP2 and VEGF165 plasmid transfecte MSCs, turn them into pIRES-BMP2-VEGF165/MSCs, which can express BMP-2 and VEGF165,and active osteogenesis ability. Then, changing such pIRES-BMP2-VEGF165/ MSCs’ surface CD44 molecule glycogen phenotype into HCELL, which could combine to the E-selectin receptor in vascular endothelial cell surface.Then, observing the effects of treating bone defect, and evaluating its ability of targeting and osteogenesis. This strategy will provide a new principle and new technique for treating the bone defect and has a wide application prospect.

骨缺损修复是一个复杂的过程，需要多种细胞和细胞因子的协同作用，其治疗更是骨科临床的一大难题。课题组研究发现BMP2、VEGF165双基因转染MSCs具有更强的成骨能力，糖基化改造的MSCs具有明显靶向迁移能力。结合最新进展，项目组提出：应用基因工程技术将MSCs改造成有活跃成骨能力和靶向性的干细胞，则可能促进骨缺损修复。本课题拟用BMP2、VEGF165质粒转染MSCs，将其改造成pIRES-BMP2-VEGF165/MSCs，使其表达BMP-2和VEGF165两种成骨相关的细胞因子，增强MSCs成骨能力；再用糖基化技术改变pIRES-BMP2-VEGF165/MSCs表面CD44分子糖原表型，使其能够和血管内皮细胞表面E-选择素结合，获得靶向归巢的能力,并在动物实验中观察其治疗骨缺损的效果，评价其靶向归巢及成骨能力,为临床应用该策略治疗骨缺损提供新原理和新技术，具有广泛应用前景。

创伤、感染及肿瘤导致的较大骨缺损仍是骨科临床治疗的一大难题。针对上述问题，如能将多能干细胞改造成具有靶向骨缺损部位的干细胞，则可能促进骨缺损修复。本课题分离出多向分化潜能的骨髓间充质干细胞（MSCs），用糖基化技术改变MSCs分子表面糖原表型，使其能够和损伤是表达于血管内皮细胞表面的E、P选择素结合，获得靶向迁移至病损部位的能力。然后将均转染有绿色荧光蛋白（GFP）的FuT7-MSCs及非糖基化MSCs 注射至骨缺损小鼠体内观察其靶向定植和成骨能力。研究结果显示经糖基化处理后的MSCs可高表达唾液酸化路易斯寡糖(sLeX)结构，使细胞能高效地与E、P选择素结合，在骨缺损小鼠体内明显地定向归巢至骨缺损部位。同时，糖基化处理后并不抑制MSCs细胞的增殖、成骨分化能力。在小鼠的骨缺损模型中，通过尾静脉注射后7d、14d进行小动物CT成像检测，观察到FuT7-MSCs注射组的骨缺损修复情况明显好于对照组，组织学病理检查亦提示较好的骨缺损修复效果。整个实验提示糖基化处理的MSCs能明显靶向募集于骨缺损部位，并促进骨缺损修复，实现了该课题的假设。该研究为临床应用该策略治疗骨缺损提供了新理论基础，经进一步研究，极可能具有广泛的应用前景。