BMP9基因修饰的牙囊干细胞治疗大鼠牙周骨缺损的体内实验研究

发布时间：2018-08-26 19:17

【摘要】：背景:牙周病一直是人类口腔疾病中最常见的两大疾病之一[1],并且在我国,牙周病的患病率比口腔龋病的患病率更高[2].牙周炎是由牙周致病菌引起的慢性感染性疾病,以附着丧失和牙槽骨吸收为主要临床表现,牙周病治疗的最终目标是修复缺失的牙槽骨,恢复牙周组织生理结构和功能[3]。但就目前牙周治疗方法而言,牙周基础治疗能有效地控制牙周炎症,牙周手术、药物治疗、激光治疗等作为辅助手段依然不能有效地修复缺损的牙槽骨,恢复牙周组织结构和功能,在治疗伴牙槽骨缺失的牙周炎中,迫切的需要一种行之有效的方法,实现真正意义上的牙周组织再生,实现牙周生物学修复。近年组织工程技术迅猛发展,在口腔疾病研究中,牙周组织工程技术以具有促进成骨作用的生长因子、牙源性的干细胞和生物支架材料为三要素,为伴牙槽骨缺失的牙周炎的治疗提供了一种新思路[4-6]。牙源性干细胞中,牙囊干细胞是存在于牙胚周围牙囊组织中的干细胞,来源于间充质,能分化形成牙周支持组织,即牙槽骨、牙周膜和牙骨质[7]。牙囊干细胞具有自我更新和复制的能力,在特定诱导环境下能多向分化形成成骨细胞、成软骨细胞、成脂细胞和成神经细胞,作为牙周组织工程的种子细胞有一定优势。骨形成蛋白9(Bone Morphogenetic Protein 9,BMP9)作为骨形成蛋白(Bone Morphogenetic Proteins,BMPs)家族成员之一,在现已发现的有促成骨作用的BMPs中,拥有最强的促进成骨能力[8],将BMP9作为生长因子应用于牙周组织工程,具有极大地研究潜力和意义。CHA(Coralline Hydroxyapatite,CHA)来源于海洋珊瑚,具有天然的疏松多孔的结构,拥有良好的生物相容性[9],干细胞能在材料表面生长、增殖,并能作为骨材料用于修复骨组织缺损[10]。因此本研究以携带BMP9基因的腺病毒转染大鼠牙囊干细胞,在体外与CHA复合后形成组织工程化骨,探讨这种以BMP9、rDFCs、CHA为三要素的复合物治疗大鼠牙周骨缺损的作用。目的:1.应用酶组织块消化法和梯度离心法体外培养rDFCs,使携带BMP9基因的腺病毒稳定高效转染rDFCs。2.探讨CHA生物相容性,以及CHA对rDFCs成骨分化的作用。3.研究以BMP9、rDFCs和CHA作为组织工程三要素,治疗大鼠牙槽骨缺损的体内试验研究。4.探讨BMP9诱导rDFCs成骨分化过程中,Smad1/5/8信号通路的调控作用。方法:1.酶组织块消化法获得原代rDFCs,梯度离心消化法获得纯化的第三代rDFCs.2.电镜观察CHA表面形态,及rDFCs粘附与CHA表面生长、增殖状态,RT-q PCR探索CHA对rDFCs成骨因子表达、成骨分化的影响,探索CHA与rDFCs见生物相容性。3.Ad-BMP9转染rDFCs后与CHA形成组织工程化骨植入急性牙周骨缺损处,6周后通过Micro CT、组织切片染色观察牙槽骨新生情况。4.Smad1/5/8磷酸化阻断剂作用下,ALP染色、茜素红染色和及RT-q PCR观察BMP9诱导rDFCs成骨分化过程中,成骨相关因子表达情况。结果:1.原代牙囊干细胞形态呈多样性,以长梭形和多角形为主,经梯度消化离心传代后的第三代rDFCs主要以长梭形为主,内含1-3个细胞核,胞浆丰富,高倍镜下可见高密度颗粒影像。Ad-BMP9转染rDFCs后,镜下观察rDFCs稳定表达绿色荧光。2.扫描电镜下观察CHA结构,CHA呈现疏松多孔结构,相互联通,孔隙大小在100-600μm。rDFCs能稳定牢固地在CHA表面生长、增殖、分泌细胞外基质。RT-q PCR结果提示,CHA能促进rDFCs表达成骨因子:OPN、ALP、Osterix,促进rDFCs向成骨细胞分化。3.Micro CT数据分析结果及HE染色结果提示,BMP9转染的rDFCs与CHA复合后形成的组织工程化骨植入牙槽骨缺损处6周后,相较于单纯的CHA植入和rDFCs/CHA植入组,实现了更多的骨组织再生。4.Compound C作为Smad1/5/8磷酸化的阻断剂,也能阻断BMP9诱导rDFCs成骨分化过程中Smad1/5/8磷酸化。BMP9诱导rDFCs成骨分化及表达成骨因子过程中,受Smad1/5/8信号通路调控。结论:通过酶组织块消化法能获得呈多形性的牙囊干细胞,梯度消化离心法能获得纯化的第三代rDFCs,细胞呈长梭形,胞质内含1-3个细胞核,并可见胞浆内含有高密度颗粒。CHA有用良好的生物相容性,rDFCs能稳定牢固地在CHA表面粘附、增殖和分泌细胞外基质,并且CHA能促进rDFCs成骨向分化,促进成骨因子表达。以BMP9、rDFCs和CHA作为组织工程的三要素,Ad-BMP9基因转染rDFCs后与CHA复合形成组织工程化骨的方式能有效的促进牙周骨组织的生成。本实验研究为BMP9、rDFCs应用于牙周组织再生提供理论依据,也为组织工程技术应用于牙周组织再生提供支持。
[Abstract]:BACKGROUND: Periodontal disease has always been one of the two most common diseases in human oral diseases [1]. In China, the prevalence of periodontal disease is higher than that of oral caries [2]. The criterion is to repair the missing alveolar bone and restore the physiological structure and function of periodontal tissue [3].But as far as the current periodontal treatment is concerned, periodontal basic treatment can effectively control periodontitis, periodontal surgery, drug treatment, laser treatment and other auxiliary means still can not effectively repair the defective alveolar bone, restore the periodontal tissue structure and function. In the treatment of periodontitis with alveolar bone loss, an effective method is urgently needed to achieve real periodontal tissue regeneration and achieve periodontal biological restoration. Stem cells and biomaterial scaffolds provide a new way to treat periodontitis with alveolar bone loss [4-6].Dental follicle stem cells are stem cells that exist in periodontal dental follicle tissues, derived from mesenchyme and can differentiate into periodontal supporting tissues, namely alveolar bone, periodontal ligament and cementum [7]. Cystic stem cells have the ability of self-renewal and replication. They can differentiate into osteoblasts, chondrocytes, adipocytes and neuroblasts under specific induction conditions. They have certain advantages as seed cells for periodontal tissue engineering. Bone Morphogenetic Protein 9 (BMP9) is used as bone morphogenetic protein (BMP 9). C Proteins, one of the members of the BMPs family, has the strongest osteogenesis promoting ability among the BMPs found so far. It has great potential and significance to apply BMP9 as a growth factor in periodontal tissue engineering. CHA (Coralline Hydroxyapatite, CHA) is derived from marine corals and has natural loose and porous knots. Structures, good biocompatibility [9], stem cells can grow and proliferate on the surface of materials, and can be used as bone materials for repairing bone tissue defects [10].Therefore, in this study, BMP9 gene-carrying adenovirus transfected rat dental follicle stem cells in vitro and combined with CHA to form tissue-engineered bone, to explore the BMP9, rDFCs, CHA as three elements. Objective: 1. To investigate the biocompatibility of adenovirus carrying BMP9 gene and the effect of CHA on osteogenic differentiation of rDFCs. 3. To study the effects of BMP9, rDFCs and CHA as three key factors of tissue engineering in the treatment of rat periodontal bone defects. Objective: To investigate the regulation of Smad1/5/8 signaling pathway during BMP9-induced osteogenesis and differentiation of rDFCs in rats. Methods: 1. Primary rDFCs were obtained by enzymatic tissue block digestion and purified by gradient centrifugation. 2. The surface morphology of CHA, adhesion of rDFCs and growth and proliferation of CHA were observed by electron microscopy. In situ, RT-q PCR was used to explore the effect of CHA on the expression of osteogenic factors and osteogenic differentiation of rDFCs, and the biocompatibility between CHA and rDFCs. 3. After transfection of rDFCs with Ad-BMP9, tissue engineered bone was formed and implanted into the acute periodontal bone defect with CHA. After 6 weeks, alveolar bone regeneration was observed by micro-CT and histological staining. 4. Under the effect of Smad1/5/8 phosphorylation blocker, AL was implanted into alveolar bone. P staining, alizarin red staining and RT-q PCR were used to observe the expression of osteogenesis-related factors during BMP-9-induced osteogenic differentiation of rDFCs. Results: 1. Primary dental follicle stem cells showed diversity in morphology, mainly spindle-shaped and polygonal. After gradient digestion and centrifugation, the third generation of rDFCs mainly consisted of long spindle-shaped, containing 1-3 nuclei, rich cytoplasm. High-power microscopy showed high-density granular images. After transfection of rDFCs with Ad-BMP9, the stable expression of green fluorescence was observed. 2. The structure of CHA was observed by scanning electron microscopy. The pore size of CHA was 100-600 micron. The results of RT-q PCR suggested that CHA could grow, proliferate and secrete extracellular matrix steadily and firmly on the surface of CHA. Micro CT data analysis and HE staining showed that BMP-9-transfected rDFCs combined with CHA formed tissue-engineered bone after implantation into alveolar bone defects for 6 weeks, compared with the simple CHA implantation and rDFCs/CHA implantation group, more bone groups were achieved. 4. Compound C acts as an inhibitor of Smad1/5/8 phosphorylation and can also block Smad1/5/8 phosphorylation during BMP9-induced osteogenic differentiation of rDFCs. BMP9-induced osteogenic differentiation and expression of osteogenic factors in rDFCs are regulated by Smad1/5/8 signaling pathway. The third generation of purified rDFCs can be obtained by centrifugation. The cells are spindle-shaped with 1-3 nuclei in cytoplasm and high density particles in cytoplasm. CHA has good biocompatibility. rDFCs can adhere to, proliferate and secrete extracellular matrix on the surface of CHA stably and firmly, and CHA can promote the osteogenic differentiation and osteogenic factor surface of rDFCs. Da. Taking BMP9, rDFCs and CHA as the three key elements of tissue engineering, the method of tissue engineered bone formed by the combination of Ad-BMP9 gene transfected rDFCs and CHA can effectively promote the formation of periodontal bone tissue. This study provides a theoretical basis for the application of BMP9 and rDFCs in periodontal tissue regeneration, and also provides a theoretical basis for the application of tissue engineering technology in periodontal tissue regeneration. For support.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R781.4

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