【摘要】 背景:诱导骨髓基质干细胞成骨条件的优化与探讨是再生医学研究的热点。前期实验发现Wnt7b在骨发育中的作用,此次实验是前期工作的延续。目的:构建Wnt7b重组反转录病毒载体,观察其在C3H10T1/2中的表达。设计、时间及地点:细胞学体外观察,于2005-01/2008-08在华盛顿大学医学院和同济大学附属第十人民医院中心实验室完成。材料:pXy-Wnt7b购于ATCC公司,载体pCIG-IRES-EGFP,pLef-Luciferase,pCMV-Rennilla,反转录病毒载体pSFG和包装细胞GP293由华盛顿大学医学院提供。方法:用多步亚克隆技术构建目的基因Wnt7b和标记基因加强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)双表达重组反转录病毒载体pSFG-Wnt7b-IRES-EGFP,在条件培养液中经GP293包装,并在常规培养液中释放出假病毒并将其转染C3H10T1/2细胞,检测成骨活性诱导中的作用和荧光素酶报告基因检测信号转导通路。主要观察指标:①Wnt7b重组反转录病毒转染C3H10T1/2细胞的表达。②Wnt7b诱导C3H10T1/2碱性磷酸酶生成分析。③Wnt7b信号转导分析。结果:构建目的基因的Wnt7b和标记基因EGFP双表达重组反转录病毒载体能够高效地表达,转染C3H10T1/2细胞后能诱导碱性磷酸酶的生成。Wnt7b信号转导分析可见Wnt7b成骨作用通过Noncanonical途径。结论:Wnt7b在C3H10T1/2中能高效表达,能显著诱导骨发育的重要标志--碱性磷酸酶的生成,Noncanonical途径在其中起重要作用。更多还原

【Abstract】 BACKGROUND:Inducing mesenchymal stem cells(MSCs) to osteoblast is a hot field in regeneration medicine. This study is the continuous work of previous experiment,which had identified the effect of Wnt7b on osteogenesis. OBJECTIVE:To construct the Wnt7b recombinant retrovirus vector,in addition,to observe its expression in C3H10T1/2 cells. DESIGN,TIME AND SETTING:The in vitro cytology observation was performed at the Center Laboratory of Shanghai Tenth Hospital,Tongji University(China) and Washington University School of Medicine(America) from January 2005 to August 2008. MATERIALS:pXy-Wnt7b was purchased from ATCC Company. Vectors such as pCIG-IRES-EGFP,pLef-Luciferase,pCMV-Rennilla,pSFG and package cell line GP293 were supplied by Washington University School of Medicine. METHODS:Constructing wnt-7b and(enhanced green fluorescent protein) EGFP co-expression recombinant retroviral vector pSFG-Wnt7b-IRES-EGFP,packaged with GP293 followed by transfecting into C3H10T1/2 cells. The effects of Wnt7b on induce osteogenesis and signal transduction pathway was detected by luciferase report gene. MAIN OUTCOME MEASURES:Expression of C3H10T1/2 cells transfected with Wnt7b recombinant retrovirus; Analysis of alkaline phosphatase(AKP) generation,as well as analysis of signal transduction of Wnt7b. RESULTS:The recombinant retroviral vector pSFG-Wnt7b-IRES-EGFP could obtain the high expression and induce C3H 10T1/2 AKP expression. Wnt7b signal transduction assay showed that osteogenesis of Wnt7b passed the channel of Noncanonical. CONCLUSIONS:Wnt7b can effective express in C3H10T1/2 cells,can notably induce the generation of AKP,which is the important symbol of bone development,meanwhile,the signal transduction of Noncanonical plays a key role.更多还原