本文選題：骨膜來源干細胞 + 成纖維細胞生長因子-2　； 參考：《南方醫(yī)科大學(xué)》2017年碩士論文

【摘要】：骨組織工程的目標是找一個更好的方案解決大段骨缺損、骨延遲愈合或不愈合的難題。由于越來越多的學(xué)者認識到骨膜在骨損傷修復(fù)過程中的重要作用,目前組織工程骨膜在不斷被研發(fā),研究者將組織工程的理念融入到組織工程骨膜中,制備了材料、細胞及細胞因子為三要素的組織工程膜,希望能從結(jié)構(gòu)或者功能上模擬自然的骨膜,并利用其促進骨損傷處的修復(fù)。從骨膜中提取到的細胞并被廣大學(xué)者習(xí)慣稱之為骨膜來源干細胞(periosteum derived stem cell,PDSC),已被證實其表面表達間充質(zhì)干細胞標記物,并具有成骨、成軟骨、成脂分化的潛能。所以PDSC可能是組織工程骨膜的優(yōu)秀的種子細胞。基于種子細胞的組織工程中仍然面臨一個關(guān)鍵問題,即在體外擴增過程中種子細胞的成骨能力逐步下降,這可能與成骨祖細胞干性下降有關(guān),而在細胞移植過程中通過模擬內(nèi)環(huán)境提供關(guān)鍵性因子有望解決這個難題,骨折的自然愈合早期,成纖維細胞生長因子-2(FGF-2)顯著增加,但在隨后的骨生成階段低表達,而骨形態(tài)發(fā)生蛋白-2(BMP-2)則在成骨分化階段大量表達。本課題制備了一種能仿生釋放FGF-2和BMP-2的殼聚糖-膠原支架膜,并聯(lián)合PDSC,進行了顱骨極量骨缺損修復(fù)實驗,證明了 PDSC在體內(nèi)外具有成骨分化能力,該仿生控釋膜材料能支持PDSC的體內(nèi)增殖、分化成骨及促進骨缺損處新骨形成。本課題采用主要方法如下:1.按以往方法制備膠原-殼聚糖支架及仿生控釋FGF-2和BMP-2的微囊,凍干后備用。2.提取PDSC,通過流式細胞儀檢測其表面干細胞標記物,并進行三系誘導(dǎo)分化實驗,鑒定其多能干性。PDSC與膠原-殼聚糖支架共培養(yǎng),檢測細胞在支架上的粘附、增殖、分化能力。3.制備組織工程膜支架,進行顱骨缺損造模并移植手術(shù),MicroCT和組織學(xué)檢查評價新骨生成,用EdU法檢測PDSC在移植部位的存活。4.統(tǒng)計學(xué)分析:應(yīng)用SPSS 20.0統(tǒng)計軟件進行數(shù)據(jù)分析,各組數(shù)值以Mean±SD表示,組間比較用單因素方差分析(one-wayANOVA),若方差齊用LSD檢驗、方差不齊則用DunnettT3檢驗作兩兩比較。設(shè)p值0.05具有統(tǒng)計學(xué)差異。主要結(jié)果:1.本實驗PDSC經(jīng)過流式鑒定,其表面表達間充質(zhì)干細胞標記物,并經(jīng)過誘導(dǎo)可向成骨、成軟骨、成脂等多向分化,證明其在該組織工程膜支架上具有增殖、成骨分化能力。2.MicroCT和組織學(xué)檢測證明,PDSC聯(lián)合仿生釋放的組織工程膜具有更優(yōu)秀的骨缺損修復(fù)能力。結(jié)論:骨膜來源的干細胞會是組織工程骨膜的優(yōu)秀種子細胞,并序貫釋放FGF-2和BMP-2為PDSC提供更好的增殖分化環(huán)境,從而提高了仿生組織工程骨膜的骨修復(fù)效能。
[Abstract]:The goal of bone tissue engineering is to find a better solution to the problem of large bone defect, delayed union or nonunion. As more and more scholars realize the important role of periosteum in the repair of bone injury, tissue engineering periosteum has been developed constantly. Researchers have integrated the concept of tissue engineering into tissue engineering periosteum and prepared materials. Cells and cytokines are three elements of tissue engineering membrane, hoping to simulate the natural periosteum structurally or functionally, and use it to promote the repair of bone injury. The cells extracted from periosteum and often referred to as periosteum derived stem cells (periosteum derived stem cells) have been confirmed to express mesenchymal stem cell markers on the surface and have the potential of osteogenesis, cartilage formation and adipogenic differentiation. So PDSC may be an excellent seed cell for tissue engineering periosteum. One of the key problems in tissue engineering based on seed cells is that the osteogenic ability of seed cells decreases gradually during the process of expansion in vitro, which may be related to the decline in dryness of osteoblast progenitor cells. In the process of cell transplantation, it is expected to solve this problem by providing key factors through simulating the internal environment. In the early stage of natural healing of fracture, fibroblast growth factor 2 (FGF-2) is increased significantly, but the expression of FGF-2 is low in the later stage of bone formation. Bone morphogenetic protein-2 (BMP-2) was highly expressed during osteogenic differentiation. In this paper, a chitosan collagen scaffold membrane which can release FGF-2 and BMP-2 by bionic method was prepared, and PDSC-PDSC was used to repair the maximal bone defect of the skull. It was proved that PDSC has the ability of osteogenic differentiation in vivo and in vitro. The biomimetic controlled-release membrane can support PDSC proliferation, differentiation and osteogenesis and promote the formation of new bone at bone defect. The main methods of this subject are as follows: 1. Collagen chitosan scaffolds and biomimetic controlled release microcapsules of FGF-2 and BMP-2 were prepared according to the previous methods. The PDSCs were extracted, the surface stem cell markers were detected by flow cytometry, and the differentiation was induced by three lines. The pluripotency. PDSC and collagen-chitosan scaffold co-culture were identified. The adhesion, proliferation and differentiation ability of the cells on the scaffold were detected. Tissue engineering membrane scaffolds were prepared to model the skull defect, and microCT and histological examination were performed to evaluate the new bone formation. The survival of PDSC in the graft site was detected by EdU method. Statistical analysis: SPSS 20.0 statistical software was used to analyze the data. The values of each group were expressed as mean 鹵SD. The comparison between groups was done by one-way ANOVA. If the variance was tested by LSD, the variance was compared by DunnettT3 test. Let p value 0.05 have statistical difference. The main result is 1: 1. In this experiment, PDSC expressed mesenchymal stem cell markers on the surface of PDSC by flow cytometry, and was induced to differentiate into osteogenesis, cartilage formation and lipid formation, which proved that PDSC could proliferate on the scaffold of tissue engineering membrane. 2. Osteogenic differentiation ability. 2. MicroCT and histological examination showed that PDSC combined with biomimetic release of tissue engineering membrane had better repair ability of bone defect. Conclusion: the stem cells derived from periosteum may be excellent seed cells for tissue engineering periosteum, and the sequential release of FGF-2 and BMP-2 provides a better environment for PDSC to proliferate and differentiate, thus improving the bone repair efficiency of biomimetic tissue engineered periosteum.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R318.08;R68

【參考文獻】

相關(guān)期刊論文 前1條

1 殷杰;邱素均;高浚淮;趙勝利;閔少雄;;FGF-2/PELA/BMP-2微囊支架促進大鼠骨膜來源干細胞的成骨分化[J];南方醫(yī)科大學(xué)學(xué)報;2017年01期

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本文編號：2103879