【摘要】：目的：兔骨髓中分離獲得高純度間充質(zhì)干細胞(MSCs)進行體外擴增培養(yǎng)；pLEGFP-N1逆轉(zhuǎn)錄病毒基因轉(zhuǎn)染MSCs，檢測攜帶EGFP的MSCs體外蛋白表達能力，獲得高效表達細胞株；觀察研究MSCs和感染EGFP的MSCs體外生長規(guī)律和細胞生物學(xué)特性、體外成骨定向誘導(dǎo)能力和體內(nèi)骨缺損修復(fù)成骨能力。 方法：1、抽取兔髂骨骨髓，利用Percoll分離液(1.073g／ml)進行密度梯度離心法從骨髓中分離骨髓間充質(zhì)干細胞，體外培養(yǎng)擴增。 2、擴增重組逆轉(zhuǎn)錄病毒載體PLNCX-EGFP。重組逆轉(zhuǎn)錄病毒載體PLNCX-EGFP感染PT67包裝細胞，，MSCs轉(zhuǎn)染，進行蛋白表達，攜帶EGFP的MSCs體外培養(yǎng)檢測。 3、體外培養(yǎng)同生長期MSCs和轉(zhuǎn)染了EGFP的MSCs，通過細胞生長曲線、吸光度測定，檢測堿性磷酸酶表達、骨鈣素生成對其進行定性檢測和觀察；同時向成骨細胞定向誘導(dǎo)分化，觀察二者體外成骨能力。 4、兔下頜骨人造骨缺損模型，Ⅰ型膠原載體攜帶MSCs和轉(zhuǎn)染了EGFP的MSCs體內(nèi)骨缺損修復(fù)，進行對比研究。 結(jié)果：1、密度梯度離心結(jié)合貼壁培養(yǎng)獲取了較高純度的MSCs，而且較好地保持了細胞的活性。Percoll分離液(1.073g／ml)分離的骨髓單個核細胞24小時后貼壁，細胞呈圓形，48—72小時后展開呈紡錘形，梭形，迅速增值，原
[Abstract]:Objective: high purity mesenchymal stem cells (MSCs) were isolated from rabbit bone marrow and cultured in vitro, and the expression of MSCs protein carrying EGFP was detected by MSCs, gene transfer with pLEGFP-N1 retrovirus gene, and the high expression cell line was obtained. To observe and study the growth regularity and cell biological characteristics of MSCs infected with MSCs and EGFP in vitro, the ability of directional induction of osteogenesis in vitro and the ability of repairing osteogenesis of bone defects in vivo. Methods: 1. Rabbit ilium bone marrow was extracted and bone marrow mesenchymal stem cells were isolated from bone marrow by density gradient centrifugation with Percoll separation solution (1.073g/ml) and cultured in vitro. 2. Amplification of recombinant retrovirus vector PLNCX-EGFP. PT67 packaging cells were infected with recombinant retrovirus vector PLNCX-EGFP. MSCs was transfected and protein expression was carried out. MSCs carrying EGFP was cultured and detected in vitro. 3. The expression of alkaline phosphatase (ALP) was detected by cell growth curve, absorbance measurement and osteocalcin production by cell growth curve, absorbance assay and osteocalcin production, respectively. 3. The co-culture of MSCs in vitro and the MSCs, transfected with EGFP were detected and observed qualitatively by cell growth curve and absorbance. At the same time, osteoblasts were induced to differentiate into osteoblasts, and the osteogenic ability of osteoblasts in vitro was observed. 4. Rabbit mandibular artificial bone defect model, type I collagen carrier carrying MSCs and MSCs transfected with EGFP were used to repair the bone defect in vivo. Results: 1. High purity MSCs, was obtained by density gradient centrifugation combined with adherent culture, and the cell activity was well maintained. Bone marrow mononuclear cells isolated by Percoll separation solution (1.073g/ml) adhered to the wall 24 hours later, and the cells were round. 48 hours later, it was spindles, fusiform, rapidly adding value, original.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R329.2

【參考文獻】

相關(guān)期刊論文 前2條

1 何悅,張志愿;骨組織工程技術(shù)及其在頜骨修復(fù)重建中的應(yīng)用[J];口腔材料器械雜志;2005年02期

2 曾暉 ,康斌 ,劉國平 ,杜靖遠 ,鄭啟新 ,劉勇;堿性成纖維細胞生長因子對成骨細胞中轉(zhuǎn)化生長因子-β1和堿性成纖維細胞生長因子mRNA表達的影響[J];中華實驗外科雜志;2002年06期

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