种植体表面RNAi功能化修饰以及形貌分子机制的系列研究

发布时间：2018-06-08 02:19

  本文选题：钛种植体 + 表面修饰　； 参考：《第四军医大学》2017年博士论文

【摘要】：【背景】钛种植体是目前应用于口腔牙齿缺失较为理想的修复材料。虽然大量的临床病例证实该技术成功率较高,然而仍存在由于骨结合不良造成的失败案例。因此,如何提高种植体骨结合能力仍是研究的热点。利用生物分子进行种植体的表面活化具有生物活性强、生物相容性好等优势,是目前较为常用的手段。其中,利用RNAi技术对种植体表面活化具有作用靶向、稳定、持久等优点,是生物活化中的研究热点。种植体的表面物理形貌优化也具有很强的应用前景,而对于形貌的信号转导机制尚不明确。我们课题组在前期进行了大量的关于微米坑结构和纳米管结构影响成骨分化相关的分子信号通路进行了初步阐释,然而尚有诸多问题未得到深入研究。因此,深入探索种植体表面形貌的信号转导机制也是研究的重点方向。为此,本研究共分为两个部分,第一部分是采用相关物理化学手段对钛种植体表面进行RNAi功能化修饰,主要包括简单吸附、分子自组装和阴极电沉积等方法;第二部分是研究钛种植体表面形貌的信号转导,主要从亚细胞结构变化和细胞应激反应角度进行阐释。本研究旨在寻找更为优良的种植体表面修饰技术,进而提高种植体骨结合;另一方面,为了更好的阐释生物材料表面微观结构对细胞行为影响的本质,从而为指导生物材料的表面结构设计提供理论支持。【目的】探索钛种植体表面RNAi功能化修饰的可行性和修饰方法;探索si RNA转运与细胞自噬的关系;阐明钛种植体表面形貌对细胞器的影响;阐明钛种植体表面形貌对细胞自噬的影响。【方法】第一部分:钛种植体表面RNAi功能化修饰1.采用微弧氧化、阳极氧化等方法在钛种植体表面构建微米级孔洞和纳米管形貌;使用的转运载体主要为壳聚糖;mi RNA包括mi R-148b和阴性对照;si RNA包括si GFP和si Ckip-1等;采用直接混合的方式形成不同的转染复合物。2.利用简单吸附过程,将壳聚糖/mi RNA转染复合物涂布在微弧氧化钛表面,真空冷冻干燥;分子自组装过程利用带正电的壳聚糖/si RNA复合物和带负电的透明质酸交替孵育沉积在光滑钛表面;阴极电沉积方法将纳米管化的钛种植体连接到负极,使用壳聚糖的盐酸溶液与si RNA形成复合物作为电解液,石墨做正极,低电压下进行加载。3.使用扫描电镜、荧光显微镜、激光共聚焦、原子力显微镜等手段进行材料表面表征;利用荧光强度定量检测涂层加载效率和释放规律;细胞转染后激光共聚焦观察转染效率;流式细胞仪和实时定量PCR检测基因沉默效率;在材料表面进行成骨分化实验,检测ALP活性、胶原分泌以及矿化状况,评估种植体表面促进成骨分化的能力。4.分别使用脂质体2000和壳聚糖与si RNA形成lipoplex和polyplex复合物转染H1299细胞,MDC染色和western blot检测自噬水平变化;在自噬调节药物存在情况下转染细胞,流式细胞仪检测细胞摄取效率和基因沉默效率;免疫荧光染色观察细胞内si RNA和自噬小体的分布。第二部分:钛种植体表面形貌的信号转导1.采用酸蚀+阳极氧化、碱热处理、过氧化氢腐蚀等方法在钛种植体表面构建不同的形貌。2.将成骨细胞系MG63培养在不同形貌材料表面,采用细胞器特异性荧光探针对细胞进行染色,激光共聚焦观察细胞器的分布状况;流式细胞仪检测荧光强度;实时定量PCR和western blot检测细胞器特异性标识表达;透射电镜观察内质网形态;实时定量PCR检测PERK通路关键分子表达。3.将成骨细胞系MC3T3-E1培养在不同形貌材料表面,采用MDC方法对细胞进行染色,检测自噬活性;透射电镜观察细胞内自噬泡的形成和结构特征;western blot检测自噬相关蛋白表达变化;罗丹明标记鬼笔环肽进行细胞骨架染色;扫描电镜观察细胞伪足;钙离子探针染色胞浆钙离子浓度;在种植体表面进行成骨诱导分化,干预自噬活性,检测ALP活性和矿化形成。【结果】第一部分:钛种植体表面RNAi功能化修饰1.微弧氧化和阳极氧化分别能够在纯钛种植体表面形成微米级孔洞结构和纳米管阵列。2.利用简单吸附过程能够实现壳聚糖/mi RNA复合物在微弧氧化表面的均匀加载,复合物能够深入微米孔洞内部;扫描电镜下涂层分布均匀;早期涂层中mi RNA释放迅速,随后释放缓慢;可以实现大鼠原代培养的骨髓间充质干细胞的转染;使用mi R-148b后能够显著促进其成骨分化;细胞活性维持在80%以上。3.分子自组装技术能够在钛表面形成多层结构包裹壳聚糖/si RNA复合物;原子力显微镜显示表面粗糙度发生交替波动;扫描电镜显示壳聚糖与透明质酸形成嵌合结构;si RNA的加载量随着层数的增加而增加;释放速率缓慢;使用si GFP加载后可持续的沉默H1299细胞中GFP的表达,细胞相容性良好;使用si Ckip-1后能够显著促进MG63的成骨分化。4.利用阴极电沉积可迅速的将壳聚糖/si RNA复合物加载到纳米管表面;荧光显微镜和扫描电镜显示随着电流密度的增加,涂层分布逐渐覆盖纳米管口;在短时间内si RNA加载量与加载电压和加载时间成正比;在不同p H中si RNA释放模式不同,在酸性环境中较快,中性和碱性环境中较慢;加载si GFP后能够实现靶基因持续性沉默和细胞高效转染;对细胞粘附和活力无显著影响;使用si Ckip-1后能够显著促进细胞成骨分化。5.使用lipoplex和polyplex转染H1299细胞后能够诱发m TOR-independent自噬反应;不同自噬调节药物对细胞摄取效率无显著影响;rapamycin和TG显著促进si RNA沉默效率,Li Br和3-MA显著减弱si RNA沉默效率;不同自噬调节药物对细胞内游离si RNA的比例产生不同影响,与沉默效率相对应。第二部分:钛种植体表面形貌的信号转导1.采用酸蚀+阳极氧化、碱热处理、过氧化氢腐蚀等方法分别能在钛种植体表面构建出微米坑-纳米管、纳米线、纳米孔洞形貌。2.微纳米形貌表面MG63细胞器分布和数量未受到显著影响;与光滑钛相比,细胞器特异性标识表达均在第1天时下降,第3天后恢复正常水平;透射电镜显示内质网腔扩张;PERK通路关键分子表达上调。3.纳米管形貌诱发MC3T3-E1暂时性、可逆性的自噬反应;形貌诱发的自噬反应类型属于m TOR-independent类型;细胞骨架分布在纳米管表面铺展明显,细胞边缘丝状伪足丰富,胞浆内钙离子浓度上升;纳米线、纳米孔洞均可诱发类似的自噬反应;纳米管结构同时也能够在Hela和H1299两种肿瘤细胞系中诱发自噬反应;应用自噬潮抑制剂后种植体表面成骨细胞ALP活性显著下调;细胞在与纳米管形貌早期接触后分离,纳米管促进成骨分化的能力仍能够保持。【结论】1.钛种植体表面RNAi功能化修饰具有可行性,利用简单吸附、分子自组装和阴极电沉积的方法均可以在钛种植体表面加载RNAi转染复合物,促进细胞的成骨分化能力。2.钛种植体表面微纳米形貌对细胞器的数量和分布未产生显著影响,但在早期抑制了细胞器特异性标识表达,诱发内质网应激反应。3.钛种植体表面形貌能够激活m TOR-independent自噬反应,具有暂时性和可逆性的特点,对细胞成骨分化具有重要的调控意义。
[Abstract]:[background] titanium implant is an ideal repair material for dental tooth loss. Although a large number of clinical cases have confirmed the high success rate of this technique, however, there are still cases of failure due to poor bone binding. Therefore, how to improve the bone binding ability of the implant is still a hot spot. The surface activation of the body has the advantages of strong biological activity and good biocompatibility, which is the most commonly used means at present. Among them, the application of RNAi technology to the surface activation of the implant has the advantages of targeting, stabilizing and lasting. It is a hot topic in the study of biological activation. The mechanism of signal transduction of morphology is not clear. A large number of molecular signaling pathways related to the effect of micron pits structure and nanotube structure on osteogenesis are preliminarily explained in our group. However, many problems have not been studied in depth. Therefore, the signal transduction mechanism of the surface morphology of the implant is also explored. Therefore, this study is divided into two parts. The first part is the use of related physical and chemical methods to modify the surface of titanium implants by RNAi, including simple adsorption, molecular self-assembly and cathodic electrodeposition, and the second part is the signal transduction of the surface morphology of titanium implants, mainly from subthin. The purpose of this study is to search for better implant surface modification techniques, and to improve implant bone binding. On the other hand, in order to better explain the nature of the surface microstructure of biomaterials on cell behavior, so as to provide guidance for the surface structure design of biomaterials. Theoretical support. [Objective] to explore the feasibility and modification method of RNAi functional modification on the surface of titanium implant, explore the relationship between Si RNA transport and cell autophagy, and clarify the influence of the surface morphology of titanium implant on the organelles, and clarify the effect of the surface morphology of titanium implants on the autophagy. [Methods] Part 1: the RNAi function of the titanium implant surface The modified 1. uses micro arc oxidation, anodizing and other methods to construct micropore cavity and nanotube morphology on the surface of titanium implants. The transport carrier is mainly chitosan; MI RNA includes mi R-148b and negative control; Si RNA includes Si GFP and Si Ckip-1, etc., and uses a direct mixing method to form different transfection complexes and.2. use simple sucking. The chitosan /mi RNA transfected complex was coated on the surface of micro arc titanium oxide and vacuum freeze-drying, and the molecular self-assembly process was deposited on the smooth titanium surface alternately using the positive chitosan /si RNA complex and the negative hyaluronic acid. The cathodic electrodeposition method connected the nanotube titanium implant to the negative electrode and used the shell. The hydrochloric acid solution of chitosan and Si RNA formed complex as the electrolyte, graphite as the anode, and under low voltage to load.3. using scanning electron microscope, fluorescence microscopy, laser confocal, atomic force microscopy and other means to characterize the surface of the material; the fluorescence intensity was used to quantitatively detect the loading efficiency and release of the coating; the laser copolymerization after the transfection of the cells was used. Focus on transfection efficiency; flow cytometry and real-time quantitative PCR detection of gene silencing efficiency; osteogenic differentiation on the surface of the material, detection of ALP activity, collagen secretion and mineralization, and evaluation of the ability of the implant surface to promote osteogenesis differentiation.4. using liposome 2000 and chitosan and Si RNA to form lipoplex and polyplex complexes, respectively. Transfection of H1299 cells, MDC staining and Western blot to detect the change of autophagy; transfection of cells under the presence of autophagic regulation drugs, flow cytometry to detect cell uptake efficiency and gene silencing efficiency; immunofluorescence staining to observe the distribution of Si RNA and autophagic corpuscle in cells. The second part: signal transduction of the surface morphology of titanium implant 1. Using acid etching + anodic oxidation, alkali heat treatment, hydrogen peroxide corrosion and other methods to construct different morphology.2. on the surface of titanium implants, the osteoblast line MG63 was cultured on the surface of different morphologies, the cells were stained by the specific fluorescent probe of organelles, the distribution of the microcytometer was observed by laser confocal microscopy, and the fluorescence intensity was detected by flow cytometry. Degree; real-time quantitative PCR and Western blot detection of organelle specific identification expression; transmission electron microscope to observe the morphology of endoplasmic reticulum; real-time quantitative PCR detection of the expression of PERK pathway key molecules.3. to the osteoblast cell line MC3T3-E1 culture on the surface of different morphologies, using MDC method to stain the cells, detect autophagic activity; transmission electron microscope to observe the cell The formation and structural characteristics of autophagic vesicles; Western blot detection of autophagic related protein expression; Luo Danming tagged phimic cytoskeleton for cytoskeleton staining; scanning electron microscope to observe cell pseudo foot; calcium ion probe staining cytoplasmic calcium concentration; osteogenesis induced differentiation on the surface of the implant, intervention of autophagic activity, ALP activity and mineralization The first part: the first part: the surface of the titanium implant surface RNAi functionalized modification 1. micro arc oxidation and anodic oxidation can form micron level pore structure on the surface of pure titanium implant and the nanotube array.2. can use the simple adsorption process to realize the uniform loading of chitosan /mi RNA complex on the surface of micro arc oxidation. The complex can be deep. In the micropore cavity, the coating is evenly distributed under scanning electron microscope, the release of MI RNA in the early coating is rapid and then release slowly, and the transfection of bone marrow mesenchymal stem cells in the primary culture of rats can be realized; the bone differentiation can be promoted by Mi R-148b, and the activity dimension of the cell activity is more than 80%.3. molecular self-assembly technology can be in the titanium table. The surface formed a multilayer structure wrapped in chitosan /si RNA complex; atomic force microscopy showed that the surface roughness occurred alternately; the scanning electron microscope showed that the chitosan and hyaluronic acid formed a chimeric structure; the loading amount of Si RNA increased with the increase of the number of layers; the release rate was slow, so that GFP in H1299 cells after Si GFP was loaded. The expression was good, and the use of Si Ckip-1 could significantly promote the osteogenic differentiation of MG63.4. using cathodic electrodeposition to quickly load the chitosan /si RNA complex onto the surface of the nanotube. The fluorescence microscope and scanning electron microscope showed that the coating cloth covered the nanotube gradually with the increase of the current density; in a short time, Si RNA added. The load is proportional to loading voltage and loading time; Si RNA release patterns in different P H are different in acidic environment and slow in neutral and alkaline environments. After loading Si GFP, the target gene can be kept silent and transfected efficiently, and there is no significant effect on cell adhesion and activity; the use of Si Ckip-1 can significantly promote cells after the use of Si Ckip-1. The autophagy induced by lipoplex and polyplex transfected by.5. could induce the autophagy reaction of M TOR-independent, and there was no significant effect on the uptake efficiency of M TOR-independent by different autophagy regulating drugs; rapamycin and TG significantly promoted the efficiency of the Si RNA. The second part: the signal transduction of the surface morphology of titanium implant 1. by acid etching + anodic oxidation, alkali heat treatment, hydrogen peroxide corrosion and other methods can construct micropits nanotube, nanowire, nano pore morphology.2. micromorphology surface MG63 organelle on the surface of titanium implant, respectively. Distribution and quantity were not significantly affected. Compared with smooth titanium, the expression of organelle specific identification decreased at first days and recovered to normal level in third days; transmission electron microscope showed endoplasmic reticulum dilation; the expression of PERK pathway key molecules raised.3. nanotube morphologies to induce MC3T3-E1 temporary, reversible autophagy; morphologies induced autophagy. The type of reaction belongs to m TOR-independent type, the cytoskeleton is spread on the surface of the nanotube, the cell edge is rich in filamentous pseudo foot, the concentration of calcium ion in the cytoplasm increases, and the nanowires and nano holes can induce similar autophagy, and the nanotube structure can also induce autophagic reaction in the two tumor cell lines of Hela and H1299. After the application of autophagic tide inhibitor, the ALP activity of the osteoblast on the surface of the implant was significantly reduced; the cells were separated after the early contact with the nanotube morphology, and the ability to promote the osteogenic differentiation of the nanotubes could still be maintained. [Conclusion] the RNAi functionalization of the 1. titanium implant surface is feasible, simple adsorption, molecular self-assembly and cathodic electrodeposition. Methods the RNAi transfection complex can be loaded on the surface of titanium implant to promote the osteogenic differentiation of the cells. The surface micromorphology of.2. titanium implants has no significant influence on the number and distribution of organelles. However, the specific expression of organelles is suppressed in the early stage, and the surface morphology of the endoplasmic reticulum stress response.3. titanium implants can be stimulated. The autophagic reaction of live m TOR-independent has the characteristics of transient and reversible. It has important regulatory significance for cell osteogenesis.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R783
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本文编号：1993959