發(fā)布時間：2018-06-09 06:54

  本文選題：明膠 + 納米微球��； 參考：《第四軍醫(yī)大學(xué)》2015年碩士論文

【摘要】：背景:骨缺損和骨不愈合是臨床常見病,如何修復(fù)骨缺損和促進骨愈合也是骨組織工程研究的重要內(nèi)容。支架、細(xì)胞和因子是骨組織工程研究的三大要素。近年來,有關(guān)骨生長因子的研究日益增多,BMP-2(Bone Morphogenetic Protein-2,骨形態(tài)發(fā)生蛋白-2)是最重要的骨生長因子之一,其促進骨修復(fù)的功能已得到相關(guān)研究證實。BMP-2在體內(nèi)含量很少,基因重組表達(dá)技術(shù)則實現(xiàn)了rh BMP-2(Recombinant Human Bone Morphogenetic Protein-2,重組人骨形態(tài)發(fā)生蛋白-2)的批量生產(chǎn),使其臨床應(yīng)用成為可能,但rh BMP-2的應(yīng)用存在體內(nèi)半衰期短、降解快的問題,如果增加其使用量將又會帶來一系列的并發(fā)癥,使其臨床應(yīng)用嚴(yán)重受限。微球緩釋技術(shù)是解決這個問題的方法之一,rh BMP-2與微球復(fù)合,不但能保護因子,還能延長其在體內(nèi)的作用時間,減少并發(fā)癥和治療費用。所以,本研究的目的是為rh BMP-2制備一種納米載體,使其更廣泛地應(yīng)用于骨科疾病的治療。目的:1.制備重組人骨形態(tài)發(fā)生蛋白-2-明膠納米微球(Recombinant Human Bone Morphogenetic Protein-2-Gelatin nanoparticle,rh BMP-2-GN),檢測其理化特性和釋藥特點。2.檢測rh BMP-2-GN對BMSCs(Bone marrow mesenchymal stem cells)的增值和分化效應(yīng)。3.為rh BMP-2的臨床應(yīng)用提供合適的納米載體,為下一步動物實驗提供必要的依據(jù)和更加可靠的解決方案。方法:1.rh BMP-2-GN的制備及檢測:(1)制備空白明膠納米微球。(2)掃描電鏡(Scanning Electron Microscope,SEM)觀察明膠納米微球的表面形態(tài),計算其平均粒徑,透射電鏡(Transmission Electron Microscope,TEM)觀察明膠納米微球的內(nèi)部結(jié)構(gòu),粒度分析儀檢測其吸水后的平均粒徑,計算其溶脹率。(3)將rh BMP-2和GN進行復(fù)合制備rh BMP-2-GN,利用酶聯(lián)免疫吸附法(Enzyme Linked Immuno Sorbent Assay,ELISA)測定加載的rh BMP-2量,計算其包封率和載藥量。(4)將rh BMP-2-GN分散到磷酸鹽緩沖液(PBS)中,檢測其累計釋藥的百分率。2.rh BMP-2-GN對BMSCs的生物效應(yīng):(1)利用CCK-8法檢測rh BMP-2-GN對BMSCs早期增殖效應(yīng)的影響。(2)利用ALP染色法檢測rh BMP-2-GN對BMSCs早期分化效應(yīng)的影響。(3)利用茜素紅-S鈣結(jié)節(jié)染色法檢測rh BMP-2-GN對BMSCs晚期分化效應(yīng)的影響。結(jié)果:1.rh BMP-2-GN的制備:(1)“二次凝聚法”成功制備空白明膠納米微球。(2)明膠納米微球的物理特性:明膠納米微球表面光滑,球形規(guī)整,內(nèi)部多孔隙、通道,分散均一,干粉狀態(tài)平均粒徑為171.49 nm,吸水膨脹后平均粒徑為313.01 nm,溶脹率為1.83。(3)成功制備rh BMP-2-GN,其包封率和載藥量分別是98.13±0.131%和58.89±0.079 ng/mg。(4)rh BMP-2-GN的體外釋藥時間在一個月以上。2.rh BMP-2-GN對BMSCs的生物學(xué)作用:(1)rh BMP-2-GN組在第4天和第7天的細(xì)胞增值作用均明顯優(yōu)于對照組和明膠納米微球組(P0.05)。(2)rh BMP-2-GN孔在第4天和第7天的ALP染色均比對照孔和明膠納米微球孔的染色要深,差異較明顯。(3)rh BMP-2-GN孔在第28天出現(xiàn)鈣結(jié)節(jié)的數(shù)目能明顯多于對照孔和明膠納米微球孔。結(jié)論:1.“二次凝聚法”成功制備空白明膠納米微球,使rh BMP-2-GN具有較高的包封率和載藥量,體外緩釋效果良好。2.rh BMP-2-GN通過明膠納米微球的降解逐步釋放rh BMP-2,延長了rh BMP-2的作用時間,明顯促進了BMSCs的早期增殖和向成骨細(xì)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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R318.08

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