本文選題：純鈦種植體 + 骨髓間充質(zhì)干細(xì)胞膜片��； 參考：《浙江大學(xué)》2017年博士論文

【摘要】：種植牙被認(rèn)為是人類的第三副牙齒,種植義齒修復(fù)后,能最大程度地提高缺牙患者的咀嚼效率,延緩牙槽骨吸收,提高生活質(zhì)量。大量的研究報(bào)道了種植體表面改性的方法,然而相較于種植體表面處理,對(duì)于種植體骨結(jié)合的形成機(jī)理的研究較少。本研究通過(guò)在種植體表面構(gòu)建低密度脂蛋白受體-5(Low-density lipoprotein receptor-related protein 5,LRP5)過(guò)表達(dá)和 LRP5 干擾的細(xì)胞膜片,利用體內(nèi)、體外實(shí)驗(yàn)進(jìn)行生物學(xué)評(píng)價(jià),并探討該基因改性的細(xì)胞膜片在種植體骨結(jié)合中的作用。本研究中首先在二氧化鈦(Ti02)納米點(diǎn)石英晶片表面預(yù)鋪層黏連蛋白-521(Laminin-521,LN-521),利用光控細(xì)胞薄層技術(shù)制備出骨髓間充質(zhì)干細(xì)胞膜片(Bone marrow mesenchymal stem cell sheet,BMSC sheet),通過(guò)粘附、增殖、活性、三向分化等實(shí)驗(yàn)證實(shí)了BMSC膜片的成功構(gòu)建,并且LN-521明顯促進(jìn)了BMSCs的粘附,365nm紫外線(ultraviolet ray,UvV)對(duì)BMSC膜片的獲取是安全的,獲得的BMSC膜片具有良好的細(xì)胞活性,呈疊層生長(zhǎng)狀態(tài),并保持著細(xì)胞干性,可以向成骨、成脂、成軟骨方向分化。粘附實(shí)驗(yàn)的結(jié)果顯示當(dāng)LN-521的濃度為1200ng/mL時(shí),對(duì)BMCSs促粘附作用較強(qiáng),能夠穩(wěn)定的構(gòu)建BMSC膜片,因此后續(xù)實(shí)驗(yàn)中將選取1200ng/mL作為L(zhǎng)N-521的預(yù)鋪濃度。在構(gòu)建完成BMSC膜片的基礎(chǔ)上,利用慢病毒轉(zhuǎn)染技術(shù)制備載綠色熒光蛋白(GFP)和熒光素酶報(bào)告基因的BMSC膜片,從而實(shí)現(xiàn)膜片種植體的體內(nèi)追蹤。結(jié)果顯示,通過(guò)小動(dòng)物活體成像系統(tǒng)檢測(cè),攜帶熒光素酶報(bào)告基因的BMSC膜片種植體在植入后1個(gè)月內(nèi)可以檢測(cè)到信號(hào),2個(gè)月后信號(hào)消失,但GFP示蹤方法并不適合在體內(nèi)追蹤BMSC膜片種植體。然后在構(gòu)建完成BMSC膜片種植體的基礎(chǔ)上,制備Ti02納米點(diǎn)種植體。體內(nèi)外實(shí)驗(yàn)結(jié)果表明Ti02納米點(diǎn)表面能夠顯著促進(jìn)BMSC膜片的成骨向分化,有利于BMSCs的粘附和增殖,能有效提高成骨相關(guān)基因堿性磷酸酶(alkaline phosphatase,ALP)、骨鈣素(osteocalcin,OCN)、Ⅰ 型膠原(CollagenⅠ),骨形態(tài)發(fā)生蛋白 2(bonemorphogenicprotein2,BMP2)、Runx2、Osterix 的表達(dá),并能促進(jìn)種植體骨結(jié)合。最后通過(guò)腺病毒和慢病毒轉(zhuǎn)染技術(shù),以BMSC膜片為載體,本研究構(gòu)建了LRP5過(guò)表達(dá)和LRP5干擾型的BMSC膜片種植體。體內(nèi)外實(shí)驗(yàn)結(jié)果表明LRP5過(guò)表達(dá)后能夠明顯地促進(jìn)BMSCs向成骨細(xì)胞分化,經(jīng)典的Wnt信號(hào)通路激活,成骨相關(guān)基因Runx2、Osterix、ALP、OCN、BMP2表達(dá)增加,加快種植體骨結(jié)合的形成;相反地,LRP5干擾后會(huì)顯著降低BMSCs向成骨細(xì)胞分化,抑制經(jīng)典的Wnt信號(hào)通路,成骨相關(guān)基因Runx2、Osterix、ALP、OCN、BMP2表達(dá)減少,種植體骨結(jié)合形成減慢。臨床研究通過(guò)回顧浙江大學(xué)醫(yī)學(xué)院附屬口腔醫(yī)院2006-2011年間的種植患者資料,結(jié)果顯示種植體失敗發(fā)生率跟患者的年齡、性別、種植牙位、種植術(shù)式、種植醫(yī)生資歷等多種因素相關(guān),種植體周圍炎是種植體發(fā)生失敗的主因,種植醫(yī)師應(yīng)在治療過(guò)程中預(yù)防種植體周圍炎的發(fā)生發(fā)展,降低種植失敗率。
[Abstract]:Implant tooth is considered to be the third pair of human teeth. Implant denture can improve the masticatory efficiency, delay alveolar bone resorption and improve the quality of life. A large number of studies have reported the methods of surface modification of implants. However, compared with implant surface treatment, there are few studies on the formation mechanism of implant bone bonding. The aim of this study was to construct over expressed low density lipoprotein receptor low density lipoprotein receptor-related protein 5 LRP5 (LRP5) and LRP5 interference cell membranes on the surface of the implants for biological evaluation in vivo and in vitro. The role of the modified cell membrane in the osseous binding of implants was investigated. In this study, bone marrow mesenchymal stem cell sheettte (BMSCs) membranes were prepared by photo-controlled cell thin-layer technique, and bone marrow mesenchymal stem cell sheettes were prepared by adhesion, proliferation, and activity of bone mesenchymal stem cells (BMSCs), which were precoated with laminin-521 (laminin-521) and LN-521 on the surface of quartz wafers of titanium dioxide (Ti02). Three-way differentiation experiments confirmed the successful construction of BMSC membrane, and LN-521 significantly promoted the BMSCs adhesion to UV ultraviolet UV V). The obtained BMSC membrane had good cellular activity and was in a laminated growth state, and the obtained BMSC membrane was safe to obtain the BMSC membrane. And maintain the dry nature of the cell, can be osteogenic, adipogenic, cartilage differentiation. The results of adhesion experiment showed that when the concentration of LN-521 was 1200ng/mL, the adhesion to BMCSs was stronger, and the BMSC membrane could be constructed stably. Therefore, 1200ng/mL was chosen as the concentration of LN-521 in subsequent experiments. Based on the construction of BMSC membrane, the BMSC membrane carrying green fluorescent protein (GFP) and luciferase reporter gene was prepared by lentivirus transfection technique, so that the membrane implants could be traced in vivo. The results showed that the BMSC membrane implants carrying luciferase reporter gene could detect the signal within one month of implantation and then disappeared after 2 months. However, GFP tracer method is not suitable for tracking BMSC membrane implants in vivo. Then, on the basis of constructing BMSC film implant, Ti02 nano-dot implant was prepared. The results of in vitro and in vivo experiments showed that the surface of Ti02 nanoparticles could significantly promote the osteogenic differentiation of BMSC membranes and facilitate the adhesion and proliferation of BMSCs. It can effectively increase the expression of ALP, osteocalcinin, Collagen 鈪，

本文編號(hào)：1900596