MicroRNA-214抑制剂调节成骨、破骨过程防治早期股骨头骨坏死塌陷

发布时间：2018-05-06 01:12

本文选题：股骨头骨坏死 + 动脉灌注　；参考：《中国人民解放军医学院》2017年博士论文

【摘要】：一、目的:拟在研究人股骨头骨坏死标本骨结构的基础上,探索坏死股骨头内miRNA-214表达与局部成、破骨细胞活性之间的关系及潜在机制,制备腺相关病毒搭载miRNA-214抑制剂,观察药物调节成骨、破骨活性防治大鼠股骨头骨坏死塌陷的效果。二、方法:①2015年3月至2016年5月期间,收集我院16例股骨头骨坏死标本。标本进行Micro-CT扫描后,根据骨质密度的不同,将每个标本分为不同区域,分别采用病理学检测、实时荧光定量PCR、WesternBlot等方法对骨微观结构及成骨、破骨细胞活性进行分析。PCR检测股骨头骨坏死标本骨组织,观察不同区域成骨、破骨相关基因表达情况及miRNA-214表达情况。②制备以腺相关病毒为载体的miRNA-214抑制剂药物,评价其体外调节成骨、破骨的能力。③建立大鼠股骨头骨坏死模型,观察AAV-anti-miRNA-214药物的治疗效果。三、结果:①标本坏死区域miRNA-214表达显著增高,破骨活性显著增高,成骨活性显著降低。而硬化区成骨活性显著增高,破骨活性显著降低。同时,miRNA-214在股骨头骨坏死标本坏死区域显著增高;②成功制备AAV-anti-miRNA-214,发现其能够明显抑制破骨细胞的功能,促进成骨细胞的功能,同时可以显著下调miRNA-214的表达,TRAP染色结果显示AAV-anti-miRNA-214可以显著抑制骨髓间充质干细胞向破骨细胞分化的能力。MC3T3-E1 细胞加入AAV病毒包裹的anti-miRNA-214 , 发现AAV-anti-miRNA-214能够明显促进成骨细胞的功能,ALP染色的结果显示成骨细胞的功能增强;③AAV-anti-miRNA-214可以促进成骨活性,抑制破骨活性,防治大鼠股骨头骨坏死塌陷。四、结论:AAV- anti-miR-214可以促进成骨活性,抑制破骨活性,其双向调节作用可有效防治大鼠早期股骨头骨坏死塌陷。
[Abstract]:Objective: to explore the relationship between the expression of miRNA-214 and local osteoclast and osteoclast activity in the necrotic femoral head on the basis of studying the bone structure of the osteonecrosis specimen of human femoral head, and to prepare the adeno-associated virus carrying miRNA-214 inhibitor. To observe the effect of drug regulating osteogenesis and osteoclast activity preventing and treating osteonecrosis of femoral head in rats. Methods: from March 2015 to May 2016, 16 cases of osteonecrosis of femoral head were collected. After Micro-CT scanning, each specimen was divided into different regions according to the different bone density. The microstructure and osteogenesis of bone were detected by pathological examination and real-time fluorescence quantitative PCR Western blot. Osteoclast activity was analyzed. PCR was used to detect bone tissue of osteonecrosis specimens of femoral head. Osteogenesis, osteoclast-associated gene expression and miRNA-214 expression were observed in different regions. 2 miRNA-214 inhibitor drugs were prepared using adeno-associated virus as vector. To evaluate the ability of osteoregulation and osteoclast in vitro to establish osteonecrosis model of femoral head in rats and to observe the therapeutic effect of AAV-anti-miRNA-214 drugs. Results: the expression of miRNA-214, osteoclast activity and osteogenesis activity in the necrotic region were significantly increased and osteogenic activity was significantly decreased. However, osteogenic activity in sclerotic area was significantly increased and osteoclast activity was significantly decreased. At the same time, AAV-anti-miRNA-214 was successfully prepared by miRNA-214 in the necrotic area of osteonecrosis of femoral head. It was found that AAV-anti-miRNA-214 could significantly inhibit the function of osteoclasts and promote the function of osteoblasts. At the same time, the expression of miRNA-214 was significantly down-regulated by trap staining. The results showed that AAV-anti-miRNA-214 could significantly inhibit the ability of bone marrow mesenchymal stem cells to differentiate into osteoclasts. MC3T3-E1 cells were added to anti-miRNA-214 wrapped with AAV virus. It was found that AAV-anti-miRNA-214 could significantly promote osteoblasts. The results of ALP staining showed that the function of osteoblasts was enhanced. 3AAV-anti-miRNA-214 can promote osteogenic activity, inhibit osteoclast activity and prevent osteonecrosis of femoral head in rats. Conclusion: AAV- anti-miR-214 can promote osteogenic activity and inhibit osteoclast activity, and its bidirectional regulation can effectively prevent and cure early osteonecrosis of femoral head in rats.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R681.8

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