发散式冲击波对骨髓间充质干细胞体内成软骨分化影响

发布时间：2018-08-28 15:12

【摘要】：目的:体外冲击波不仅可以通过增加血管内皮生长因子的表达,增加心肌有效灌注和改善左室重塑,从而改善缺血性心脏病患者症状,治疗缺血性心脏病;而且可以使骨细胞将机械信号转化为生化信号后传导给破骨细胞和成骨细胞,引起骨的重建,治疗骨折延迟愈合或不愈合,这提示冲击波具有促进组织再生的作用。然而有关冲击波对骨髓间充质干细胞的成软骨分化影响的研究并不多。本研究利用不同能量的发散式冲击波刺激骨髓间充质干细胞,观察其成软骨效果,探究冲击波在体内对骨髓间充质干细胞成软骨分化的调节作用。方法:从骨髓中分离骨髓间充质干细胞,培养至P3代,通过倒置显微镜观察细胞形态,流式分析细胞表面相关抗原的表达和诱导成骨成脂分化能力鉴定为骨髓间充质干细胞。在探索冲击波调控干细胞成软骨分化时,我们先后采用裸鼠皮下细胞种植模型和新西兰大白兔膝关节软骨损伤两种动物模型。首先将裸鼠分为4组,分别为对照组(藻酸钙种植到裸鼠皮下),干细胞组(包裹干细胞的藻酸钙种植到裸鼠皮下),低能量冲击波组(2.0bar,200次,10Hz的冲击波刺激干细胞后,包裹在藻酸钙中种植到裸鼠皮下),高能量冲击波组(3.0bar, 200次,10Hz的冲击波刺激干细胞后,包裹在藻酸钙中种植到裸鼠皮下)。种植后3个月和12个月,对样本进行组织学检测。然后将新西兰大白兔分为4组,,建立膝关节软骨损伤模型,根据分组将细胞或支架移植到软骨损伤区,分组和细胞处理步骤同裸鼠,移植后3个月,样本进行组织学检测。结果:在裸鼠皮下细胞种植实验中,两个冲击波组样本比其余两组体积更大,Ⅱ胶原和蛋白多糖含量更多,以3. bar压力的冲击波作用效果最佳。在兔膝关节软骨损伤修复实验中,两个冲击波组的软骨损伤区域修复组织更多,Ⅱ胶原和蛋白多糖含量更多,细胞排列更加有序,与周围组织链接更加紧密,以3. bar的冲击波作用效果最佳。结论:1.发散式冲击波在体内具有促进骨髓间充质干细胞向软骨细胞分化的影响。2.冲击波的促进成软骨作用与能量相关,压力为3. 0bar时,冲击波的促进成软骨分化作用优于2. 0bar。
[Abstract]:Objective: extracorporeal shock wave can not only increase the expression of vascular endothelial growth factor, increase myocardial perfusion and improve left ventricular remodeling, so as to improve the symptoms of patients with ischemic heart disease and treat ischemic heart disease. Moreover, bone cells can transform mechanical signals into biochemical signals and transmit them to osteoclasts and osteoblasts, which can cause bone reconstruction and treat fracture healing or nonunion, which indicates that shock wave can promote tissue regeneration. However, there are few studies on the effect of shock wave on the differentiation of bone marrow mesenchymal stem cells. In this study, bone marrow mesenchymal stem cells (BMSCs) were stimulated with divergent shock waves with different energy to observe the effect of chondrogenesis, and to explore the effect of shock wave on the differentiation of bone marrow mesenchymal stem cells (BMSCs) in vivo. Methods: bone marrow mesenchymal stem cells were isolated from bone marrow and cultured to P3 generation. The morphology of mesenchymal stem cells was observed by inverted microscope. The expression of surface related antigen and the ability of inducing osteogenic adipogenic differentiation were identified as bone marrow mesenchymal stem cells by flow cytometry. In order to explore the chondrogenic differentiation of stem cells regulated by shock wave, we used subcutaneous cell implantation model in nude mice and cartilage injury in knee joint in New Zealand white rabbits. At first, the nude mice were divided into four groups: control group (implanted with calcium alginate to the subcutaneous of nude mice), stem cell group (implanted with calcium alginate wrapped in stem cells), low energy shock wave group (2.0 barge 200 times 10 Hz shock wave) after stimulation of stem cells. The high energy shock wave group (3.0 bar. after 200 times 10 Hz shock wave stimulated stem cells, encapsulated in calcium alginate into nude mice subcutaneously). The samples were examined for histology 3 and 12 months after planting. Then New Zealand white rabbits were divided into 4 groups to establish the articular cartilage injury model. The cells or scaffolds were transplanted into the cartilage injury zone according to the groups. The same steps of grouping and cell processing were performed in nude mice. The samples were examined by histology 3 months after transplantation. Results: in the experiment of subcutaneous cell implantation in nude mice, the samples of the two shock wave groups were larger than those of the other two groups, and the contents of collagen and proteoglycan were more than those of the other two groups. The effect of shock wave under the pressure of 3. Bar was the best. In the rabbit knee joint cartilage repair experiment, there were more cartilage repair tissues, more collagen and proteoglycan content, more orderly cell arrangement and more close links with surrounding tissues in the two shock wave groups. The shock wave of 3. Bar is the best. Conclusion 1. Divergent shock wave has the effect of promoting the differentiation of bone marrow mesenchymal stem cells into chondrocytes in vivo. The promotion of chondrogenesis by shock wave is related to energy, and the pressure is 3. In 0bar, the effect of shock wave on cartilage differentiation was better than that of 2. 0bar.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R68

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