【作者】 张琦；

【导师】 袁晓；

【作者基本信息】 青岛大学 ， 口腔正畸学， 2021， 硕士

【摘要】 研究目的:骨性II类错畸形是临床上常见的一种正畸错类型,主要表现为患者的磨牙远中错位,前牙深覆、深覆盖以及下颌后缩等。由于此类患者的矢状向关系不调,严重者会导致颞下颌骨关节炎(temporomandibular joint osteoarthritis,TMJ OA)的发生。髁突关节重建术是TMJ OA严重者的一种手术手段。对于髁突骨缺损处的愈合,骨组织工程中的一种治疗方法是人工骨生物移植材料的替代。而生物移植材料表面的拓扑形貌可以模拟生物细胞的生长环境,促进细胞的迁移、分化、增殖等细胞行为。聚二甲基硅氧烷(polydimethylsiloxane,PDMS)作为可以很好模拟细胞外基质的聚合物材料,其表面形貌能够驱动细胞形态发生改变,特别是在细胞分化方面。已知不同维度拓扑形貌会影响成骨分化能力,但是与细胞核中β-catenin引导的成骨转录因子相关的功能尚未完全了解。自噬是细胞实现自我稳定的重要生理机制,近年来的研究表明自噬对成骨细胞自我更新和分化方面具有着重要的调控意义。而自噬在不同维度拓扑形貌对成骨细胞的成骨分化能力影响是否相关尚不清楚。本课题探究PDMS基底表面不同拓扑形貌对于成骨细胞自噬及成骨分化的影响,研究成骨的细胞对不同维度拓扑形貌的反应以及潜在的机制,寻找揭示表面不同维度拓扑形貌调控成骨作用中β-catenin的信号转导,该研究成骨将为材料界面改性和新型骨组织工程材料研究提供理论依据。从而为正畸临床中通过骨组织工程来治疗TMJ OA提供新理论。研究方法:本研究中,笔者主要探讨自噬在PDMS基底表面不同拓扑形貌对成骨分化影响中的作用。利用等离子体刻蚀技术制作具有不同拓扑形貌的硅片模板,复刻后得到具有不同拓扑形貌的PDMS基底表面,原子力显微镜检测所制备PDMS基底表面的不同拓扑形貌,在不同拓扑形貌的基底表面上培养小鼠胚胎成骨细胞前体细胞(MC3T3-E1)。使用倒置显微镜下观察在不同拓扑形貌上培养的MC3T3-E1形态,CCK-8试剂盒检测在不同拓扑形貌上培养的MC3T3-E1增殖情况。通过免疫荧光染色检测、茜素红染色、q RT-PCR、Western blot等方法检测不同维度拓扑形貌的PDMS基底表面MC3T3-E1成骨分化能力;通过构建Super8×TOPFlash质粒和Super8×FOPFlash质粒、免疫荧光染色、Western blot等方法检测β-catenin在MC3T3-E1内的表达变化;Western blot检测自噬相关基因p62、LC3II/I的蛋白表达;通过si RNA转染技术等方法分析自噬与β-catenin之间的相关性继而证明其与成骨分化的关系。研究结果:1.原子力显微镜对制备PDMS基底表面拓扑形貌进行检测,证明制备了不同维度的拓扑形貌:Flat、W0.5、W3、W10、W27(波长W:μm)。2.MC3T3-E1细胞粘附及增殖被证实受到PDMS基底表面不同维度拓扑形貌影响。肌动蛋白(细胞骨架)和细胞核的双标记荧光染色发现MC3T3-E1在W3上粘附更好。CCK-8分析证明不同维度拓扑形貌的基底上均显示在培养72h后依旧支持细胞的增殖,特别是W3表面上的细胞数量比其他表面上的细胞数量更多。3.PDMS基底表面不同拓扑形貌对MC3T3-E1细胞成骨分化影响不同。ALP免疫荧光染色结果以及RUNX2、OSX m RNA和蛋白表达量提示W3基底上的MC3T3-E1细胞成骨分化能力最强,W27基底上的MC3T3-E1细胞成骨分化能力最弱。4.MC3T3-E1细胞成骨分化与β-catenin进入细胞核,激活Wnt信号相关。荧光素酶基因报告分析显示β-catenin转录活性增加。Western blot及免疫荧光染色检测显示W3基底组的β-catenin入核含量最高。5.不同维度PDMS基底表面对MC3T3-E1细胞自噬影响不同。Western blot及GFP-LC3检测显示W3基底组的自噬水平更高。6.MC3T3-E1细胞自噬通过调控β-catenin来影响成骨分化。当W3基底组加入自噬抑制剂CQ后,β-catenin未沉默组入核含量降低,但高于β-catenin沉默组,成骨分化能力降低。加入激动剂RAPA后,β-catenin基因未沉默组入核含量增加,成骨分化能力增加。研究结论:本研究确定了不同维度的拓扑形貌对MC3T3-E1的成骨分化作用有影响。尤其是W3促进了β-catenin入核进而影响了成骨细胞的成骨分化,其中W3诱导的自噬发生是促进β-catenin入核的重要原因。该研究揭示了拓扑形貌介导的MC3T3-E1成骨分化的潜在机制,并为改善生物材料表面修饰提供了思路。这些见解有助于我们提高拓扑形貌与细胞成骨分化及自噬之间相互作用的理解,为通过骨组织工程和再生医学的应用治疗TMJ OA提供新理论。更多还原

【Abstract】 Objective:Class II malocclusion is a common type of orthodontic malocclusion in clinical practice.The main manifestations are that the molars of the patients are distally related,the anterior teeth are deeply overlaid,and the mandible is retracted.Because the sagittal relationship of such patients is not adjusted,severe cases can lead to temporomandibular joint osteoarthritis(TMJ OA).Condylar joint reconstruction is a commonly used surgical method for patients with severe TMJ OA.For the healing of condyle bone defect,one of the treatment methods in bone tissue engineering is the replacement of artificial bone materials.Polydimethylsiloxane(PDMS)is a polymer material that can well mimic the extracellular matrix.Its topography drives the change of cell morphology,which greatly affects the cell biology process,especially in cell differentiation.It is known that the different dimensional surface topography arranged on the PDMS substrate will affect the osteogenic differentiation ability,but the osteogenic signal transduction related to the surface topography is not fully understood.The topography of the PDMS substrate surface is related to the function of β-cateninguided osteogenic transcription factors in the nucleus and autophagy in the cytoplasm.Autophagy is an important physiological mechanism for achieving the self-stability of osteoblasts.Recent studies have shown that autophagy has a significant regulatory role in osteoblast self-renewal and differentiation.However,the role of autophagy in the effect of different dimensional topography of PDMS substrate surface on the osteogenic differentiation ability of osteoblast is still unclear.This study explores the effect of PDMS substrate surface topography on the autophagy and osteogenic differentiation of osteoblasts and explores the response of osteoblast to topography and the underlying mechanism.Seeking to reveal the signal transduction in the regulation of osteogenesis by the topological topography of different dimensions on the surface,laying a theoretical foundation for material interface modification and the development of new bone tissue engineering materials.Methods:In this study,we mainly explored the role of autophagy in the influence of different topologies of PDMS substrate surface on osteogenic differentiation.Plasma etching technology is used to fabricate silicon wafer templates with different dimensions,and the PDMS substrate surface of different dimensions is re-engraved.The different dimensions of the prepared PDMS substrate are examined by atomic force microscope,and the osteoblast precursor cells MC3T3-E1 are cultured on the substrate surface of different dimensions.The morphology of MC3T3-E1 cultured on different topologies was observed by an inverted microscope,and the proliferation of MC3T3-E1 cultured on different topologies was detected by the CCK-8 kit.Detection of osteogenic differentiation ability of MC3T3-E1 on PDMS with different dimensions by immunofluorescence staining,alizarin red staining,q RT-PCR,Western blot and other methods;by constructing Super8 x TOPFlash plasmid and Super8 x FOPFlash plasmid,immunofluorescence staining,Western blot and other methods were used to detect the expression changes of β-catenin in MC3T3-E1;the protein expression of autophagy-related genes p62 and LC3II/I were detected by Western blot;transfection by si RNA technology and other methods analyze the correlation between autophagy and β-catenin and then prove its relationship with osteogenic differentiation.Results:1.Atomic force microscopy detects the topological morphology of the prepared PMDS substrate surface,and proves that the topological morphology of different dimensions is prepared: Flat,W0.5,W3,W10,W27(Wavelength W: μm)2.The adhesion and proliferation of MC3T3-E1 cells were confirmed to be affected by the topography of the PDMS substrate surface in different dimensions.Doublelabelled fluorescent staining of actin(cytoskeleton)and nucleus showed that MC3T3-E1 adhered better to W3.CCK-8 analysis proved that the substrates with different dimensions of topological morphology still support cell proliferation after 72 hours of culture,especially the number of cells on the surface of W10 is more than that on other surfaces.3.Different dimensions of PDMS substrate surface have different effects on the osteogenic differentiation of MC3T3-E1 cells.The results of ALP immunofluorescence staining and RUNX2,OSX m RNA and protein expression indicate that MC3T3-E1 cells on W27 substrate have the weakest osteogenic differentiation ability.4.The osteogenic differentiation of MC3T3-E1 cells is related to β-catenin entering the nucleus and activating Wnt signalling.Luciferase gene report analysis showed that β-catenin transcription activity increased.Western blot and immunofluorescence staining showed that the W3 basal group had the highest content of β-catenin into the nucleus.5.Different dimensions of PDMS substrate surface have different effects on the autophagy of MC3T3-E1 cells.Western blot and GFP-LC3 showed that the level of autophagy in the W3 basal group was higher.6.MC3T3-E1 cell autophagy affects osteogenic differentiation by regulating β-catenin.When the W3 basal group was added with the autophagy inhibitor CQ,the β-catenin non-silent group had a lower nuclear content,but it was higher than the β-catenin silent group,and the osteogenic differentiation ability was reduced.After the agonist RAPA was added,the β-catenin gene was not silenced and the nuclear content increased,and the osteogenic differentiation ability increased.Conclusions:This study determined that the topological morphology of different dimensions has an impact on the osteogenic differentiation of osteoblasts.In particular,W3 promotes β-catenin to enter the nucleus and thus affects the osteogenic differentiation of osteoblasts.W3-induced autophagy is an important reason for promoting β-catenin to enter the nucleus.This study reveals the potential mechanism of osteoblast differentiation mediated by topological morphology and provides ideas for improving the surface modification of biomaterials.These insights help us to improve our understanding of the interaction between cell osteogenic differentiation and topography,and provide new theories for the application of bone tissue engineering and regenerative medicine.更多还原