本文選題：綠色熒光蛋白轉(zhuǎn)基因小鼠 + 骨髓間充質(zhì)干細(xì)胞��； 參考：《第四軍醫(yī)大學(xué)》2010年碩士論文

【摘要】： 干細(xì)胞分化為成牙本質(zhì)細(xì)胞是牙本質(zhì)缺損修復(fù)的關(guān)鍵環(huán)節(jié)。骨髓間充質(zhì)干細(xì)胞是一類(lèi)成體干細(xì)胞,在干細(xì)胞龕的調(diào)控下能夠定向分化為多種中胚層和神經(jīng)外胚層來(lái)源的組織細(xì)胞,但是對(duì)于這種非牙源性的干細(xì)胞能否通過(guò)分化為成牙本質(zhì)細(xì)胞的方式修復(fù)牙本質(zhì)缺損的研究鮮有報(bào)道。本實(shí)驗(yàn)采用綠色熒光蛋白轉(zhuǎn)基因小鼠的骨髓間充質(zhì)干細(xì)胞的體內(nèi)實(shí)驗(yàn)觀察BMMSCs能否分化為成牙本質(zhì)細(xì)胞并參與牙本質(zhì)損傷的修復(fù),為牙齒組織工程提供更具活力的種子細(xì)胞。 方法:(1)取綠色熒光蛋白轉(zhuǎn)基因小鼠的全骨髓,貼壁法體外培養(yǎng)綠色熒光蛋白轉(zhuǎn)基因小鼠骨髓間充質(zhì)干細(xì)胞,流式細(xì)胞術(shù)鑒定細(xì)胞表面分子標(biāo)記物,體外成骨成脂誘導(dǎo);(2)尾靜脈注射構(gòu)建綠色熒光骨髓間充質(zhì)干細(xì)胞嵌合普通C57BL/6小鼠模型;(3)制備嵌合小鼠牙本質(zhì)缺損修復(fù)模型,用免疫熒光檢測(cè)法觀察綠色熒光骨髓間充質(zhì)干細(xì)胞是否參與牙本質(zhì)缺損的修復(fù),探討骨髓間充質(zhì)干細(xì)胞作為修復(fù)牙本質(zhì)缺損種子細(xì)胞的可行性。 結(jié)果:(1)培養(yǎng)的綠色熒光蛋白轉(zhuǎn)基因小鼠骨髓間充質(zhì)干細(xì)胞具有很強(qiáng)的增殖能力并可持續(xù)傳代,P3代細(xì)胞流式細(xì)胞術(shù)檢測(cè)干細(xì)胞表面分子陽(yáng)性,綠色熒光表達(dá)穩(wěn)定,具有體外分化成骨成脂能力;(2)構(gòu)造的嵌合小鼠模型全骨髓再培養(yǎng),發(fā)現(xiàn)大量具有綠色熒光標(biāo)記的細(xì)胞存在,各組織臟器也有不同數(shù)量的綠色熒光細(xì)胞存在。(3)免疫組織熒光顯示牙本質(zhì)缺損模型嵌合小鼠牙髓腔內(nèi)出現(xiàn)大量自發(fā)綠色熒光的細(xì)胞,牙本質(zhì)缺損處的部分綠色熒光細(xì)胞表達(dá)牙本質(zhì)特異性蛋白DSP和DMP-1。 結(jié)論:本實(shí)驗(yàn)利用示蹤能力強(qiáng)的綠色熒光蛋白轉(zhuǎn)基因小鼠骨髓間充質(zhì)干細(xì)胞構(gòu)建熒光嵌合小鼠模型,驗(yàn)證骨髓間充質(zhì)干細(xì)胞體內(nèi)向成牙本質(zhì)細(xì)胞方向分化的能力,發(fā)現(xiàn)骨髓間充質(zhì)干細(xì)胞在體內(nèi)微環(huán)境的誘導(dǎo)下可以向成牙本質(zhì)細(xì)胞方向分化,并參與牙本質(zhì)缺損的修復(fù),可以作為牙齒組織工程的干細(xì)胞源。
[Abstract]:Differentiation of stem cells into odontoblast cells is a key link in dentin defect repair. Bone marrow mesenchymal stem cells (BMSCs) are a group of adult stem cells, which can differentiate into many mesodermal and neuroectodermal cells under the regulation of stem cell niche. However, there are few reports on whether the non-odontogenic stem cells can repair dentin defects by differentiation into odontoblast. In this study, bone marrow mesenchymal stem cells (BMSCs) of GFP transgenic mice were used to observe whether BMMSCs could differentiate into dentin cells and participate in the repair of dentin injury, and provide more active seed cells for tooth tissue engineering. Methods the whole bone marrow of transgenic mice with green fluorescent protein (GFP) was harvested. Bone marrow mesenchymal stem cells (BMSCs) of GFP transgenic mice were cultured in vitro by adherent method. Flow cytometry was used to identify the molecular markers on the surface of the cells. A green fluorescent bone marrow mesenchymal stem cell chimeric model of C57BL/6 mice was established by intravenous injection of green fluorescent bone marrow mesenchymal stem cells (C57BL/6) in vitro. Whether green fluorescent bone marrow mesenchymal stem cells were involved in the repair of dentin defect was observed by immunofluorescence assay, and the feasibility of using bone marrow mesenchymal stem cells as seed cells to repair dentin defects was discussed. Results the bone marrow mesenchymal stem cells (BMSCs) of GFP transgenic mice cultured with 1 / 1 were able to proliferate strongly. Flow cytometry was used to detect the positive surface molecules of stem cells and the expression of green fluorescence was stable. The whole bone marrow of the chimeric mouse model with the ability of adipogenic differentiation and osteogenesis in vitro was recultured and a large number of cells with green fluorescent labeling were found. There were also different numbers of green fluorescent cells in different tissues. The immunofluorescence showed that a large number of spontaneous green fluorescent cells appeared in dental pulp cavity of dental pulp of chimeric mouse model of dentin defect. Part of green fluorescent cells in dentine defect expressed dentin specific proteins DSP and DMP-1. Conclusion: in this study, a fluorescent chimeric mouse model of bone marrow mesenchymal stem cells (BMSCs) of green fluorescent protein transgenic mice with strong tracer ability was established to verify the ability of MSCs to differentiate into odontoblast cells in vivo. It was found that bone marrow mesenchymal stem cells could differentiate into odontoblast under the induction of microenvironment in vivo and participate in the repair of dentin defect, which could be used as stem cell source for tooth tissue engineering.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R329;R78

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

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本文編號(hào)：1910213