BMP9基因修飾的牙囊干細胞治療大鼠牙周骨缺損的體內實驗研究
發(fā)布時間：2018-08-26 19:17
【摘要】：背景:牙周病一直是人類口腔疾病中最常見的兩大疾病之一[1],并且在我國,牙周病的患病率比口腔齲病的患病率更高[2].牙周炎是由牙周致病菌引起的慢性感染性疾病,以附著喪失和牙槽骨吸收為主要臨床表現(xiàn),牙周病治療的最終目標是修復缺失的牙槽骨,恢復牙周組織生理結構和功能[3]。但就目前牙周治療方法而言,牙周基礎治療能有效地控制牙周炎癥,牙周手術、藥物治療、激光治療等作為輔助手段依然不能有效地修復缺損的牙槽骨,恢復牙周組織結構和功能,在治療伴牙槽骨缺失的牙周炎中,迫切的需要一種行之有效的方法,實現(xiàn)真正意義上的牙周組織再生,實現(xiàn)牙周生物學修復。近年組織工程技術迅猛發(fā)展,在口腔疾病研究中,牙周組織工程技術以具有促進成骨作用的生長因子、牙源性的干細胞和生物支架材料為三要素,為伴牙槽骨缺失的牙周炎的治療提供了一種新思路[4-6]。牙源性干細胞中,牙囊干細胞是存在于牙胚周圍牙囊組織中的干細胞,來源于間充質,能分化形成牙周支持組織,即牙槽骨、牙周膜和牙骨質[7]。牙囊干細胞具有自我更新和復制的能力,在特定誘導環(huán)境下能多向分化形成成骨細胞、成軟骨細胞、成脂細胞和成神經細胞,作為牙周組織工程的種子細胞有一定優(yōu)勢。骨形成蛋白9(Bone Morphogenetic Protein 9,BMP9)作為骨形成蛋白(Bone Morphogenetic Proteins,BMPs)家族成員之一,在現(xiàn)已發(fā)現(xiàn)的有促成骨作用的BMPs中,擁有最強的促進成骨能力[8],將BMP9作為生長因子應用于牙周組織工程,具有極大地研究潛力和意義。CHA(Coralline Hydroxyapatite,CHA)來源于海洋珊瑚,具有天然的疏松多孔的結構,擁有良好的生物相容性[9],干細胞能在材料表面生長、增殖,并能作為骨材料用于修復骨組織缺損[10]。因此本研究以攜帶BMP9基因的腺病毒轉染大鼠牙囊干細胞,在體外與CHA復合后形成組織工程化骨,探討這種以BMP9、rDFCs、CHA為三要素的復合物治療大鼠牙周骨缺損的作用。目的:1.應用酶組織塊消化法和梯度離心法體外培養(yǎng)rDFCs,使攜帶BMP9基因的腺病毒穩(wěn)定高效轉染rDFCs。2.探討CHA生物相容性,以及CHA對rDFCs成骨分化的作用。3.研究以BMP9、rDFCs和CHA作為組織工程三要素,治療大鼠牙槽骨缺損的體內試驗研究。4.探討B(tài)MP9誘導rDFCs成骨分化過程中,Smad1/5/8信號通路的調控作用。方法:1.酶組織塊消化法獲得原代rDFCs,梯度離心消化法獲得純化的第三代rDFCs.2.電鏡觀察CHA表面形態(tài),及rDFCs粘附與CHA表面生長、增殖狀態(tài),RT-q PCR探索CHA對rDFCs成骨因子表達、成骨分化的影響,探索CHA與rDFCs見生物相容性。3.Ad-BMP9轉染rDFCs后與CHA形成組織工程化骨植入急性牙周骨缺損處,6周后通過Micro CT、組織切片染色觀察牙槽骨新生情況。4.Smad1/5/8磷酸化阻斷劑作用下,ALP染色、茜素紅染色和及RT-q PCR觀察BMP9誘導rDFCs成骨分化過程中,成骨相關因子表達情況。結果:1.原代牙囊干細胞形態(tài)呈多樣性,以長梭形和多角形為主,經梯度消化離心傳代后的第三代rDFCs主要以長梭形為主,內含1-3個細胞核,胞漿豐富,高倍鏡下可見高密度顆粒影像。Ad-BMP9轉染rDFCs后,鏡下觀察rDFCs穩(wěn)定表達綠色熒光。2.掃描電鏡下觀察CHA結構,CHA呈現(xiàn)疏松多孔結構,相互聯(lián)通,孔隙大小在100-600μm。rDFCs能穩(wěn)定牢固地在CHA表面生長、增殖、分泌細胞外基質。RT-q PCR結果提示,CHA能促進rDFCs表達成骨因子:OPN、ALP、Osterix,促進rDFCs向成骨細胞分化。3.Micro CT數(shù)據(jù)分析結果及HE染色結果提示,BMP9轉染的rDFCs與CHA復合后形成的組織工程化骨植入牙槽骨缺損處6周后,相較于單純的CHA植入和rDFCs/CHA植入組,實現(xiàn)了更多的骨組織再生。4.Compound C作為Smad1/5/8磷酸化的阻斷劑,也能阻斷BMP9誘導rDFCs成骨分化過程中Smad1/5/8磷酸化。BMP9誘導rDFCs成骨分化及表達成骨因子過程中,受Smad1/5/8信號通路調控。結論:通過酶組織塊消化法能獲得呈多形性的牙囊干細胞,梯度消化離心法能獲得純化的第三代rDFCs,細胞呈長梭形,胞質內含1-3個細胞核,并可見胞漿內含有高密度顆粒。CHA有用良好的生物相容性,rDFCs能穩(wěn)定牢固地在CHA表面粘附、增殖和分泌細胞外基質,并且CHA能促進rDFCs成骨向分化,促進成骨因子表達。以BMP9、rDFCs和CHA作為組織工程的三要素,Ad-BMP9基因轉染rDFCs后與CHA復合形成組織工程化骨的方式能有效的促進牙周骨組織的生成。本實驗研究為BMP9、rDFCs應用于牙周組織再生提供理論依據(jù),也為組織工程技術應用于牙周組織再生提供支持。
[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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R781.4
本文編號：2205904
[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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R781.4
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