本文選題：鈦種植體 + 表面修飾�。� 參考：《第四軍醫(yī)大學(xué)》2017年博士論文

【摘要】：【背景】鈦種植體是目前應(yīng)用于口腔牙齒缺失較為理想的修復(fù)材料。雖然大量的臨床病例證實該技術(shù)成功率較高,然而仍存在由于骨結(jié)合不良造成的失敗案例。因此,如何提高種植體骨結(jié)合能力仍是研究的熱點。利用生物分子進行種植體的表面活化具有生物活性強、生物相容性好等優(yōu)勢,是目前較為常用的手段。其中,利用RNAi技術(shù)對種植體表面活化具有作用靶向、穩(wěn)定、持久等優(yōu)點,是生物活化中的研究熱點。種植體的表面物理形貌優(yōu)化也具有很強的應(yīng)用前景,而對于形貌的信號轉(zhuǎn)導(dǎo)機制尚不明確。我們課題組在前期進行了大量的關(guān)于微米坑結(jié)構(gòu)和納米管結(jié)構(gòu)影響成骨分化相關(guān)的分子信號通路進行了初步闡釋,然而尚有諸多問題未得到深入研究。因此,深入探索種植體表面形貌的信號轉(zhuǎn)導(dǎo)機制也是研究的重點方向。為此,本研究共分為兩個部分,第一部分是采用相關(guān)物理化學(xué)手段對鈦種植體表面進行RNAi功能化修飾,主要包括簡單吸附、分子自組裝和陰極電沉積等方法;第二部分是研究鈦種植體表面形貌的信號轉(zhuǎn)導(dǎo),主要從亞細胞結(jié)構(gòu)變化和細胞應(yīng)激反應(yīng)角度進行闡釋。本研究旨在尋找更為優(yōu)良的種植體表面修飾技術(shù),進而提高種植體骨結(jié)合;另一方面,為了更好的闡釋生物材料表面微觀結(jié)構(gòu)對細胞行為影響的本質(zhì),從而為指導(dǎo)生物材料的表面結(jié)構(gòu)設(shè)計提供理論支持�！灸康摹刻剿麾伔N植體表面RNAi功能化修飾的可行性和修飾方法;探索si RNA轉(zhuǎn)運與細胞自噬的關(guān)系;闡明鈦種植體表面形貌對細胞器的影響;闡明鈦種植體表面形貌對細胞自噬的影響。【方法】第一部分:鈦種植體表面RNAi功能化修飾1.采用微弧氧化、陽極氧化等方法在鈦種植體表面構(gòu)建微米級孔洞和納米管形貌;使用的轉(zhuǎn)運載體主要為殼聚糖;mi RNA包括mi R-148b和陰性對照;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轉(zhuǎn)染復(fù)合物。2.利用簡單吸附過程,將殼聚糖/mi RNA轉(zhuǎn)染復(fù)合物涂布在微弧氧化鈦表面,真空冷凍干燥;分子自組裝過程利用帶正電的殼聚糖/si RNA復(fù)合物和帶負電的透明質(zhì)酸交替孵育沉積在光滑鈦表面;陰極電沉積方法將納米管化的鈦種植體連接到負極,使用殼聚糖的鹽酸溶液與si RNA形成復(fù)合物作為電解液,石墨做正極,低電壓下進行加載。3.使用掃描電鏡、熒光顯微鏡、激光共聚焦、原子力顯微鏡等手段進行材料表面表征;利用熒光強度定量檢測涂層加載效率和釋放規(guī)律;細胞轉(zhuǎn)染后激光共聚焦觀察轉(zhuǎn)染效率;流式細胞儀和實時定量PCR檢測基因沉默效率;在材料表面進行成骨分化實驗,檢測ALP活性、膠原分泌以及礦化狀況,評估種植體表面促進成骨分化的能力。4.分別使用脂質(zhì)體2000和殼聚糖與si RNA形成lipoplex和polyplex復(fù)合物轉(zhuǎn)染H1299細胞,MDC染色和western blot檢測自噬水平變化;在自噬調(diào)節(jié)藥物存在情況下轉(zhuǎn)染細胞,流式細胞儀檢測細胞攝取效率和基因沉默效率;免疫熒光染色觀察細胞內(nèi)si RNA和自噬小體的分布。第二部分:鈦種植體表面形貌的信號轉(zhuǎn)導(dǎo)1.采用酸蝕+陽極氧化、堿熱處理、過氧化氫腐蝕等方法在鈦種植體表面構(gòu)建不同的形貌。2.將成骨細胞系MG63培養(yǎng)在不同形貌材料表面,采用細胞器特異性熒光探針對細胞進行染色,激光共聚焦觀察細胞器的分布狀況;流式細胞儀檢測熒光強度;實時定量PCR和western blot檢測細胞器特異性標(biāo)識表達;透射電鏡觀察內(nèi)質(zhì)網(wǎng)形態(tài);實時定量PCR檢測PERK通路關(guān)鍵分子表達。3.將成骨細胞系MC3T3-E1培養(yǎng)在不同形貌材料表面,采用MDC方法對細胞進行染色,檢測自噬活性;透射電鏡觀察細胞內(nèi)自噬泡的形成和結(jié)構(gòu)特征;western blot檢測自噬相關(guān)蛋白表達變化;羅丹明標(biāo)記鬼筆環(huán)肽進行細胞骨架染色;掃描電鏡觀察細胞偽足;鈣離子探針染色胞漿鈣離子濃度;在種植體表面進行成骨誘導(dǎo)分化,干預(yù)自噬活性,檢測ALP活性和礦化形成�！窘Y(jié)果】第一部分:鈦種植體表面RNAi功能化修飾1.微弧氧化和陽極氧化分別能夠在純鈦種植體表面形成微米級孔洞結(jié)構(gòu)和納米管陣列。2.利用簡單吸附過程能夠?qū)崿F(xiàn)殼聚糖/mi RNA復(fù)合物在微弧氧化表面的均勻加載,復(fù)合物能夠深入微米孔洞內(nèi)部;掃描電鏡下涂層分布均勻;早期涂層中mi RNA釋放迅速,隨后釋放緩慢;可以實現(xiàn)大鼠原代培養(yǎng)的骨髓間充質(zhì)干細胞的轉(zhuǎn)染;使用mi R-148b后能夠顯著促進其成骨分化;細胞活性維持在80%以上。3.分子自組裝技術(shù)能夠在鈦表面形成多層結(jié)構(gòu)包裹殼聚糖/si RNA復(fù)合物;原子力顯微鏡顯示表面粗糙度發(fā)生交替波動;掃描電鏡顯示殼聚糖與透明質(zhì)酸形成嵌合結(jié)構(gòu);si RNA的加載量隨著層數(shù)的增加而增加;釋放速率緩慢;使用si GFP加載后可持續(xù)的沉默H1299細胞中GFP的表達,細胞相容性良好;使用si Ckip-1后能夠顯著促進MG63的成骨分化。4.利用陰極電沉積可迅速的將殼聚糖/si RNA復(fù)合物加載到納米管表面;熒光顯微鏡和掃描電鏡顯示隨著電流密度的增加,涂層分布逐漸覆蓋納米管口;在短時間內(nèi)si RNA加載量與加載電壓和加載時間成正比;在不同p H中si RNA釋放模式不同,在酸性環(huán)境中較快,中性和堿性環(huán)境中較慢;加載si GFP后能夠?qū)崿F(xiàn)靶基因持續(xù)性沉默和細胞高效轉(zhuǎn)染;對細胞粘附和活力無顯著影響;使用si Ckip-1后能夠顯著促進細胞成骨分化。5.使用lipoplex和polyplex轉(zhuǎn)染H1299細胞后能夠誘發(fā)m TOR-independent自噬反應(yīng);不同自噬調(diào)節(jié)藥物對細胞攝取效率無顯著影響;rapamycin和TG顯著促進si RNA沉默效率,Li Br和3-MA顯著減弱si RNA沉默效率;不同自噬調(diào)節(jié)藥物對細胞內(nèi)游離si RNA的比例產(chǎn)生不同影響,與沉默效率相對應(yīng)。第二部分:鈦種植體表面形貌的信號轉(zhuǎn)導(dǎo)1.采用酸蝕+陽極氧化、堿熱處理、過氧化氫腐蝕等方法分別能在鈦種植體表面構(gòu)建出微米坑-納米管、納米線、納米孔洞形貌。2.微納米形貌表面MG63細胞器分布和數(shù)量未受到顯著影響;與光滑鈦相比,細胞器特異性標(biāo)識表達均在第1天時下降,第3天后恢復(fù)正常水平;透射電鏡顯示內(nèi)質(zhì)網(wǎng)腔擴張;PERK通路關(guān)鍵分子表達上調(diào)。3.納米管形貌誘發(fā)MC3T3-E1暫時性、可逆性的自噬反應(yīng);形貌誘發(fā)的自噬反應(yīng)類型屬于m TOR-independent類型;細胞骨架分布在納米管表面鋪展明顯,細胞邊緣絲狀偽足豐富,胞漿內(nèi)鈣離子濃度上升;納米線、納米孔洞均可誘發(fā)類似的自噬反應(yīng);納米管結(jié)構(gòu)同時也能夠在Hela和H1299兩種腫瘤細胞系中誘發(fā)自噬反應(yīng);應(yīng)用自噬潮抑制劑后種植體表面成骨細胞ALP活性顯著下調(diào);細胞在與納米管形貌早期接觸后分離,納米管促進成骨分化的能力仍能夠保持。【結(jié)論】1.鈦種植體表面RNAi功能化修飾具有可行性,利用簡單吸附、分子自組裝和陰極電沉積的方法均可以在鈦種植體表面加載RNAi轉(zhuǎn)染復(fù)合物,促進細胞的成骨分化能力。2.鈦種植體表面微納米形貌對細胞器的數(shù)量和分布未產(chǎn)生顯著影響,但在早期抑制了細胞器特異性標(biāo)識表達,誘發(fā)內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng)。3.鈦種植體表面形貌能夠激活m TOR-independent自噬反應(yīng),具有暫時性和可逆性的特點,對細胞成骨分化具有重要的調(diào)控意義。
[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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R783
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本文編號：1993959