重组人骨形态发生蛋白-2明胶纳米微球的制备及其对SD大鼠骨髓基质干细胞生物学行为的影响

发布时间：2018-06-09 06:54

本文选题：明胶 + 纳米微球　；参考：《第四军医大学》2015年硕士论文

【摘要】：背景:骨缺损和骨不愈合是临床常见病,如何修复骨缺损和促进骨愈合也是骨组织工程研究的重要内容。支架、细胞和因子是骨组织工程研究的三大要素。近年来,有关骨生长因子的研究日益增多,BMP-2(Bone Morphogenetic Protein-2,骨形态发生蛋白-2)是最重要的骨生长因子之一,其促进骨修复的功能已得到相关研究证实。BMP-2在体内含量很少,基因重组表达技术则实现了rh BMP-2(Recombinant Human Bone Morphogenetic Protein-2,重组人骨形态发生蛋白-2)的批量生产,使其临床应用成为可能,但rh BMP-2的应用存在体内半衰期短、降解快的问题,如果增加其使用量将又会带来一系列的并发症,使其临床应用严重受限。微球缓释技术是解决这个问题的方法之一,rh BMP-2与微球复合,不但能保护因子,还能延长其在体内的作用时间,减少并发症和治疗费用。所以,本研究的目的是为rh BMP-2制备一种纳米载体,使其更广泛地应用于骨科疾病的治疗。目的:1.制备重组人骨形态发生蛋白-2-明胶纳米微球(Recombinant Human Bone Morphogenetic Protein-2-Gelatin nanoparticle,rh BMP-2-GN),检测其理化特性和释药特点。2.检测rh BMP-2-GN对BMSCs(Bone marrow mesenchymal stem cells)的增值和分化效应。3.为rh BMP-2的临床应用提供合适的纳米载体,为下一步动物实验提供必要的依据和更加可靠的解决方案。方法:1.rh BMP-2-GN的制备及检测:(1)制备空白明胶纳米微球。(2)扫描电镜(Scanning Electron Microscope,SEM)观察明胶纳米微球的表面形态,计算其平均粒径,透射电镜(Transmission Electron Microscope,TEM)观察明胶纳米微球的内部结构,粒度分析仪检测其吸水后的平均粒径,计算其溶胀率。(3)将rh BMP-2和GN进行复合制备rh BMP-2-GN,利用酶联免疫吸附法(Enzyme Linked Immuno Sorbent Assay,ELISA)测定加载的rh BMP-2量,计算其包封率和载药量。(4)将rh BMP-2-GN分散到磷酸盐缓冲液(PBS)中,检测其累计释药的百分率。2.rh BMP-2-GN对BMSCs的生物效应:(1)利用CCK-8法检测rh BMP-2-GN对BMSCs早期增殖效应的影响。(2)利用ALP染色法检测rh BMP-2-GN对BMSCs早期分化效应的影响。(3)利用茜素红-S钙结节染色法检测rh BMP-2-GN对BMSCs晚期分化效应的影响。结果:1.rh BMP-2-GN的制备:(1)“二次凝聚法”成功制备空白明胶纳米微球。(2)明胶纳米微球的物理特性:明胶纳米微球表面光滑,球形规整,内部多孔隙、通道,分散均一,干粉状态平均粒径为171.49 nm,吸水膨胀后平均粒径为313.01 nm,溶胀率为1.83。(3)成功制备rh BMP-2-GN,其包封率和载药量分别是98.13±0.131%和58.89±0.079 ng/mg。(4)rh BMP-2-GN的体外释药时间在一个月以上。2.rh BMP-2-GN对BMSCs的生物学作用:(1)rh BMP-2-GN组在第4天和第7天的细胞增值作用均明显优于对照组和明胶纳米微球组(P0.05)。(2)rh BMP-2-GN孔在第4天和第7天的ALP染色均比对照孔和明胶纳米微球孔的染色要深,差异较明显。(3)rh BMP-2-GN孔在第28天出现钙结节的数目能明显多于对照孔和明胶纳米微球孔。结论:1.“二次凝聚法”成功制备空白明胶纳米微球,使rh BMP-2-GN具有较高的包封率和载药量,体外缓释效果良好。2.rh BMP-2-GN通过明胶纳米微球的降解逐步释放rh BMP-2,延长了rh BMP-2的作用时间,明显促进了BMSCs的早期增殖和向成骨细胞分化。
[Abstract]:Background: bone defect and bone nonunion are common clinical diseases. How to repair bone defects and promote bone healing is also an important part of bone tissue engineering research. Scaffolds, cells and factors are three major factors in bone tissue engineering. In recent years, the study of bone growth factors is increasing, BMP-2 (Bone Morphogenetic Protein-2, bone morphogenesis) Protein -2 is one of the most important bone growth factors. Its function of promoting bone repair has been studied to confirm that.BMP-2 is in the body with little content. Recombinant expression technology has realized the mass production of RH BMP-2 (Recombinant Human Bone Morphogenetic Protein-2, recombinant human bone morphogenetic protein -2), so that its clinical application is possible. But the application of RH BMP-2 has the problem of short half-life in the body and fast degradation. If the amount of its use will be increased, a series of complications will be brought about. The microsphere release technique is one of the methods to solve this problem. The combination of RH BMP-2 and microspheres can not only protect the factors, but also prolong the time of action in the body. Reduction of complications and treatment costs. Therefore, the purpose of this study is to prepare a nanoscale for RH BMP-2 and to make it more widely used in the treatment of disease in the Department of orthopedics. Objective: 1. the preparation of recombinant human bone morphogenetic protein -2- gelatin nanospheres (Recombinant Human Bone Morphogenetic Protein-2-Gelatin nanoparticle, Rh BMP-2-GN), and detection of recombinant human bone morphogenetic protein (Bone Morphogenetic Protein-2-Gelatin nanoparticle, Rh BMP-2-GN) Its physicochemical properties and drug release characteristics.2. detection of the value added and differentiation of RH BMP-2-GN to BMSCs (Bone marrow mesenchymal stem cells).3. provides a suitable nano carrier for the clinical application of RH BMP-2, providing the necessary basis and more reliable solution for the next step animal experiment. Blank gelatin nanospheres. (2) the surface morphology of gelatin nanospheres was observed by scanning electron microscopy (Scanning Electron Microscope, SEM). The average particle size was calculated. The internal structure of gelatin nanospheres was observed by transmission electron microscopy (Transmission Electron Microscope, TEM). The particle size analyzer measured the average particle size after water absorption and calculated its swelling rate. (3) RH BMP-2 and GN were compounded to prepare RH BMP-2-GN, and enzyme linked immunosorbent assay (Enzyme Linked Immuno Sorbent Assay, ELISA) was used to determine the loading capacity of RH, and the encapsulation efficiency and drug loading were calculated. (4) the percentage of cumulative release was detected. (1 The effect of RH BMP-2-GN on the early proliferation of BMSCs was detected by CCK-8. (2) the effect of RH BMP-2-GN on the early differentiation of BMSCs was detected by ALP staining. (3) the effect of RH BMP-2-GN on the late BMSCs differentiation was detected by alizarin red -S calcium nodule staining. Blank gelatin nanospheres. (2) physical properties of gelatin nanospheres: the surface of gelatin nanospheres is smooth, spherical and regular, internal porous, channel, dispersion uniform, the average particle size of dry powder is 171.49 nm, the average particle size is 313.01 nm after water absorption, and the swelling rate is 1.83. (3). The encapsulation efficiency and drug loading are 98., respectively. The biological effect of 13 + 0.131% and 58.89 + 0.079 ng/mg. (4) RH BMP-2-GN in vitro release of.2.rh BMP-2-GN on BMSCs: (1) the cell increment of RH BMP-2-GN group in fourth days and seventh days was significantly better than that of the control group and gelatin nanospheres group (P0.05). (2) RH BMP-2-GN holes were compared to ALP dyeing at fourth days and seventh days. (3) the number of calcium nodules on the twenty-eighth day of RH BMP-2-GN hole was significantly more than that of the control hole and gelatin nanospheres. Conclusion: the 1. "two times coagulation" method successfully prepared blank gelatin nanospheres, which made RH BMP-2-GN have higher encapsulation efficiency and drug loading, and the release effect in vitro. Good.2.rh BMP-2-GN gradually releases RH BMP-2 through the degradation of gelatin nanospheres, prolongs the action time of RH BMP-2 and obviously promotes the early proliferation of BMSCs and differentiation into osteoblasts.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R318.08

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