褪黑素缓释对人间充质干细胞成骨发生作用研究

发布时间：2018-03-27 13:05

本文选题：褪黑素　切入点：PLGA　出处：《中山大学》2012年硕士论文

【摘要】：褪黑素主要由松果体合成与分泌，近年来众多实验证明，褪黑素对于骨的重建过程具有重要作用，一方面能促进骨的发生，另一方面能防止骨的退化，这两方面的作用主要通过受体依赖的或非受体依赖的方式进行。在体外，纳米级浓度的褪黑素促进了多种前成骨细胞系以及人骨髓间充质干细胞的进一步分化。褪黑素作为一种常用药物，其给药方式主要包括，腹腔注射、口服和静脉注射。即便这些方式不至造成微环境不适宜的浓度，也可能产生某些副作用。在骨治疗的应用中，局部给药也存在一些缺陷，主要包括给药剂量太高，给药时间太短等。乳酸羟基乙酸共聚物（PLGA），由于具有良好的生物可降解性及生物相容性而作为药物承载工具，被广泛应用于骨组织材料工程研究和临床试验，具有亲水基团，体内分解后产物为二氧化碳和水。在制备中通过改变乳酸和羟基乙酸的比例调整降解的理化性质，以适应各种药物不同的释放速率。本研究目的是将褪黑素包埋封装于乳酸羟基乙酸共聚物微球中，构建一种褪黑素持续性缓释系统PLGA-MEL-MS，并评估该缓释系统所释放的褪黑素对人骨髓间充质干细胞体外成骨发生诱导的作用效果。本研究中，采用单相乳液溶剂蒸发技术制备成PLGA载褪黑素微球（PLGA-MEL-MS）。扫描电子显微镜结果表明，包埋褪黑素后的PLGA微球与空载PLGA微球的外观形态特征基本一致。体外药物释放试验显示，PLGA-MEL-MS具有二重药物释放方式：在起始突释效率下，头三天药物释放量约达到40%；然后经过相对缓慢稳定的持续释放阶段，在第25天，约85%的药物释放到微球外。细胞增殖实验结果指出，PLGA-MEL-MS对于人骨髓间充质干细胞的增殖没有显著作用。PLGA-MEL-MS具有良好的药物缓释效果，，对于细胞体外增殖无明显毒性副作用。另外，本研究进行了骨髓间充质干细胞的成骨分化诱导，将构建的PLGA-MEL-MS添加到成骨诱导培养体系中，在基因水平和蛋白水平上检测成骨分化的效果。试验的结果显示，PLGA-MEL-MS相对于对照组明显提高了碱性磷酸酶（ALP）的mRNA表达水平并且促进了ALP的活性。同时，其他几个成骨细胞分化的标志基因Runx2、骨钙素（Osteocalcin）及骨桥蛋白（Osteopontin）的表达水平也呈现显著上调趋势。茜素红染色定量分析结果也表明，PLGA-MEL-MS相对于对照的空载PLGA微球，显著地增加了间充质干细胞分化后所分泌的钙沉淀。因此，本研究中所构建的PLGA载褪黑素微球药物释放系统能控制褪黑素的释放，并得到了在成骨分化所需时间内，褪黑素浓度相对稳定的微环境，促进了体外诱导人骨髓间充质干细胞成骨分化的过程。在今后深入的临床试验中，此褪黑素微球缓释系统可作为骨生长促进剂，为应用于体内的骨损伤愈合康复提供了有意义的研究基础。
[Abstract]:Melatonin is mainly synthesized and secreted by pineal body. In recent years, many experiments have proved that melatonin plays an important role in the process of bone reconstruction. On the one hand, it can promote the formation of bone, on the other hand, it can prevent the degeneration of bone. These two effects are mainly done in receptor-dependent or non-receptor-dependent ways. In vitro, Melatonin promoted the further differentiation of many proosteoblasts and human bone marrow mesenchymal stem cells. Melatonin, as a common drug, was administered mainly by intraperitoneal injection. Oral and intravenous administration. Even if these methods do not cause inappropriate concentrations in the microenvironment, they may have some side effects. Because of its good biodegradability and biocompatibility, it has been widely used in bone tissue material engineering and clinical trials, and has hydrophilic group, because of its good biodegradability and biocompatibility. After decomposition in vivo, the products were carbon dioxide and water. The physicochemical properties of degradation were adjusted by changing the ratio of lactic acid and glycolic acid to adapt to the different release rates of various drugs. The aim of this study was to encapsulate melatonin in the microspheres of lactate glycolic acid copolymers. A sustained release system of melatonin (PLGA-MEL-MSS) was constructed, and the effect of melatonin released by the system on osteogenesis of human bone marrow mesenchymal stem cells in vitro was evaluated. In this study, PLGA loaded melatonin microspheres (PLGA-MEL-MSN) were prepared by single-phase emulsion solvent evaporation technique. The morphological features of the PLGA microspheres after melatonin implantation were basically the same as those of the PLGA microspheres without loading. In vitro drug release tests showed that PLGA-MEL-MS had a dual drug release mode: under the initial release efficiency. In the first three days, the amount of drug released was about 40; then, after a relatively slow and steady period of sustained release, on the 25th day, The results of cell proliferation test showed that PLGA-MEL-MS had no significant effect on the proliferation of human bone marrow mesenchymal stem cells. PLGA-MEL-MS had a good drug release effect and had no obvious toxic side effect on the proliferation of human bone marrow mesenchymal stem cells in vitro. In addition, the osteogenic differentiation of bone marrow mesenchymal stem cells was induced, and the constructed PLGA-MEL-MS was added to the osteogenic culture system. The results showed that PLGA-MEL-MS significantly increased the expression of mRNA and the activity of ALP compared with the control group. The expression of several other markers of osteoblast differentiation, Runx2, osteocalcinin and osteopontin, also showed an up-regulation trend. The quantitative analysis of alizarin red staining also showed that PLGA-MEL-MS was significantly higher than the control PLGA microspheres. The calcium precipitate secreted by mesenchymal stem cells after differentiation was significantly increased. Therefore, the drug release system of PLGA loaded melatonin microspheres in this study can control the release of melatonin and obtain a relatively stable microenvironment of melatonin concentration within the time required for osteogenic differentiation. It promotes the process of osteogenic differentiation of human bone marrow mesenchymal stem cells in vitro. In further clinical trials, the melatonin microspheres can be used as bone growth promoters. It provides a meaningful research basis for healing and healing of bone injury in vivo.
【学位授予单位】：中山大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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