pLEGFP-N1基因转染骨髓基质干细胞的体外稳定性和体内体外成骨能力应用研究
发布时间：2019-05-15 14:02
【摘要】：目的：兔骨髓中分离获得高纯度间充质干细胞(MSCs)进行体外扩增培养；pLEGFP-N1逆转录病毒基因转染MSCs，检测携带EGFP的MSCs体外蛋白表达能力，获得高效表达细胞株；观察研究MSCs和感染EGFP的MSCs体外生长规律和细胞生物学特性、体外成骨定向诱导能力和体内骨缺损修复成骨能力。 方法：1、抽取兔髂骨骨髓，利用Percoll分离液(1.073g／ml)进行密度梯度离心法从骨髓中分离骨髓间充质干细胞，体外培养扩增。 2、扩增重组逆转录病毒载体PLNCX-EGFP。重组逆转录病毒载体PLNCX-EGFP感染PT67包装细胞，，MSCs转染，进行蛋白表达，携带EGFP的MSCs体外培养检测。 3、体外培养同生长期MSCs和转染了EGFP的MSCs，通过细胞生长曲线、吸光度测定，检测碱性磷酸酶表达、骨钙素生成对其进行定性检测和观察；同时向成骨细胞定向诱导分化，观察二者体外成骨能力。 4、兔下颌骨人造骨缺损模型，Ⅰ型胶原载体携带MSCs和转染了EGFP的MSCs体内骨缺损修复，进行对比研究。 结果：1、密度梯度离心结合贴壁培养获取了较高纯度的MSCs，而且较好地保持了细胞的活性。Percoll分离液(1.073g／ml)分离的骨髓单个核细胞24小时后贴壁，细胞呈圆形，48—72小时后展开呈纺锤形，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.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R329.2
本文编号：2477557
[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.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R329.2
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相关期刊论文 前2条
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2 曾晖 ,康斌 ,刘国平 ,杜靖远 ,郑启新 ,刘勇;碱性成纤维细胞生长因子对成骨细胞中转化生长因子-β1和碱性成纤维细胞生长因子mRNA表达的影响[J];中华实验外科杂志;2002年06期
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