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目的:探讨应用植入转染血小板源性生长因子(Platelet-derived growth factor,PDGF)基因的骨髓间充质干细胞的灭活同种异体跟腱重建兔前交叉韧带(anterior cruciate ligament,ACL)的效果。方法:取新西兰白兔自体骨髓间充质干细胞(mesenchymal stem cells,MSCs),培养后感染稳定表达PDGF-BB逆转录病毒,种植于经γ射线照射灭活的同种异体跟腱上,进行前交叉韧带重建。实验分对照组(移植物为单纯灭活跟腱)、细胞组(移植物为种植MSCs的灭活的跟腱)和基因组(移植物为转染基因的MSCs种植于灭活的跟腱)三组。术后3周、6周、12周,分别进行组织学及免疫组织化学染色,观察关节腔内移植物的变化过程及力学拉伸试验观察韧带的生物力学改变。结果:术后3周移植物表面已经覆盖大量从受体来的炎性细胞,对照组细胞量明显少于细胞组和基因组。细胞由移植物表面逐渐进入深部,基因组中随大量细胞进入的同时还伴有血管的增生,形成典型的肉芽组织。而对照组和细胞组血管增生不明显。手术后6周,对照组、细胞组和基因组都显现细胞到达移植物深部,甲苯胺蓝染色显示细胞组和基因组移植物的异染阳性,但对照组为阴性。手术后12周,对照组显现移植物内有大量的成纤维细胞,甲苯胺蓝染色轻度异染;而细胞组和基因组细胞量减少,显现类软骨性状,细胞排列与正常前交叉韧带接近。胶原分型的免疫组化染色三组均显示Ⅰ型胶原为主的移植物在植入后经过Ⅲ型胶原替代,后由Ⅲ型胶原为主逐步过渡到重新以Ⅰ型胶原为主。这个转换过程基因组最快,对照组最慢。手术后3、6和12周移植物的刚度和最大载荷与移植前相比均有明显下降,以3周下降最明显。术后6周,移植物刚度和最大载荷开始上升,细胞组和基因组上升明显,与对照组有显著性差异。结论:转染PDGF-BB基因的自体骨髓间充质干细胞有促进重建韧带血管化、加速韧带成熟的作用。
OBJECTIVE: To investigate the effect of an in vivo anterior cruciate ligament (ACL) reconstruction of allogenic Achilles tendon implanted with bone marrow mesenchymal stem cells transfected with platelet-derived growth factor (PDGF) gene effect. METHODS: New Zealand white rabbit autologous bone marrow mesenchymal stem cells (MSCs) were cultured and infected with PDGF-BB retroviruses. The retroviruses were implanted in allograft Achilles tendon inactivated by γ-ray irradiation. Cruciate ligament reconstruction. The experimental groups were divided into three groups: the Achilles tendon (inactivation) and the cell group (the Achilles tendon inactivation of MSCs), and the genome (MSCs transplanted with transgene were planted in inactivated Achilles tendon) group. After 3 weeks, 6 weeks and 12 weeks after operation, histological and immunohistochemical staining were performed respectively to observe the change process of the graft in the joint cavity and the mechanical tensile test to observe the biomechanical changes of the ligaments. Results: After 3 weeks, the graft surface covered a large number of inflammatory cells from the recipient. The amount of cells in the control group was significantly less than that in the cell group and the genome. Cells from the graft surface gradually into the deep, with a large number of cells into the genome at the same time also accompanied by vascular proliferation, the formation of typical granulation tissue. The control group and cell group vascular proliferation was not obvious. At 6 weeks after surgery, cells in the control group, cell group and genome were found to have reached the deep part of the graft. Toluidine blue stain showed a heterogeneous positive staining in the cell group and the genomic graft, but the control group was negative. Twelve weeks after the operation, the control group showed a large number of fibroblasts in the graft with toluidine blue stain slightly heterochromatic; while the cell group and genomic cell number decreased, showing cartilaginous traits, cell arrangement and normal anterior cruciate ligament close. Collagen type immunohistochemical staining of the three groups showed that type I collagen-based grafts were implanted after the type III collagen replacement, after the gradual transition from type III collagen to re-type I collagen-based. This conversion process is the fastest in the genome and slowest in the control group. At 3, 6 and 12 weeks after operation, the stiffness and the maximum load of the grafts decreased significantly compared with those before transplantation, and the most significant reduction occurred at 3 weeks. After 6 weeks, the graft stiffness and the maximum load began to rise, the cell group and the genome increased significantly, with the significant difference between the control group. CONCLUSION: Autologous bone marrow mesenchymal stem cells transfected with PDGF-BB gene can promote the vascularization of the ligament and accelerate the ligament maturation.