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,...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。展开更多
目的在真核细胞中表达人Wnt7b基因并且观察对大鼠原代软骨细胞的退变作用。方法取出生24 h SD大鼠关节处软骨,Ⅱ型胶原酶多次消化后获得原代软骨细胞,取P1代细胞进行实验。扩增人Wnt7b基因及克隆至PCDH-GFP上,转染PCDH-GFP和PCDH-Wnt7b...目的在真核细胞中表达人Wnt7b基因并且观察对大鼠原代软骨细胞的退变作用。方法取出生24 h SD大鼠关节处软骨,Ⅱ型胶原酶多次消化后获得原代软骨细胞,取P1代细胞进行实验。扩增人Wnt7b基因及克隆至PCDH-GFP上,转染PCDH-GFP和PCDH-Wnt7b至293ft细胞中,48 h后收集细胞上清及转染细胞,Western blot鉴定Wnt7b在293ft细胞中的表达。收集的上清分别稀释10倍和50倍培养大鼠软骨细胞,24 h后观察细胞形态并收集细胞蛋白和抽提RNA,Western blot和定量PCR检测软骨退变指标MMP13、MMP3、Ⅱ型胶原、A-can、ADAMTS5、ColⅩ和SOX9的表达。结果成功克隆人Wnt7b基因至PCDH-GFP载体上且在293ft细胞中得到有效表达。含Wnt7b培养基干预软骨细胞24 h后,软骨细胞形态由原来的多角形变成长梭形,Wnt7b处理组软骨细胞MMP13和MMP3表达显著上调,Ⅱ型胶原的表达下调。PCR结果表明A-can和Sox9表达下降,ColⅩ和ADAMTS5表达增强。结论有效在真核细胞中表达Wnt7b基因并且促进大鼠软骨细胞退变,建立软骨细胞退变的体外模型。展开更多
There remain unmet clinical needs for safe and effective bone anabolic therapies to treat aging-related osteoporosis and to improve fracture healing in cases of nonunion or delayed union. Wnt signaling has emerged as ...There remain unmet clinical needs for safe and effective bone anabolic therapies to treat aging-related osteoporosis and to improve fracture healing in cases of nonunion or delayed union. Wnt signaling has emerged as a promising target pathway for developing novel bone anabolic drugs. Although neutralizing antibodies against the Wnt antagonist sclerostin have been tested,Wnt ligands themselves have not been fully explored as a potential therapy. Previous work has demonstrated Wnt7b as an endogenous ligand upregulated during osteoblast differentiation, and that Wnt7b overexpression potently stimulates bone accrual in the mouse. The earlier studies however did not address whether Wnt7b could promote bone formation when specifically applied to aged or fractured bones. Here we have developed a doxycycline-inducible strategy where Wnt7b is temporally induced in the bones of aged mice or during fracture healing. We report that forced expression of Wnt7b for 1 month starting at 15 months of age greatly stimulated trabecular and endosteal bone formation, resulting in a marked increase in bone mass. We further tested the effect of Wnt7b on bone healing in a murine closed femur fracture model. Induced expression of Wnt7b at the onset of fracture did not affect the initial cartilage formation but promoted mineralization of the subsequent bone callus. Thus, targeted delivery of Wnt7b to aged bones or fracture sites may be explored as a potential therapy.展开更多
Cholangiocarcinoma(CCA) is a relatively rare malignancy of the intra- or extra-hepatic bile ducts that is classified according to its anatomical localization as intrahepatic, perihilar or distal. Overall, CCA has a di...Cholangiocarcinoma(CCA) is a relatively rare malignancy of the intra- or extra-hepatic bile ducts that is classified according to its anatomical localization as intrahepatic, perihilar or distal. Overall, CCA has a dismal prognosis due to typical presentation at an advanced irresectable stage, lack of effective non-surgical treatments, and a high rate of disease recurrence. CCA frequently arises on a background of chronic liver inflammation and cholestasis. Chronic inflammation is accompanied by enhanced cell turnover with generation of additional inflammatory stimuli, and a microenvironment rich in pro-inflammatory mediators and proliferative factors that enable accumulation of mutations, transformation and expansion of mutated cells. A recent study by Boulter et al implicates the Wnt signaling cascade in cholangiocarcinogenesis. Wnt ligands Wnt7 B and Wnt10 A were found to be highly overexpressed in human CCA tissue. Wnt7 B protein was present throughout the tumor stroma, and often co-localized with a subset of CD68+ macrophages. To address in a direct manner whether Wnt signaling is engaged in development of CCA, Boulter et al explored the Wnt signaling pathway in an experimental model that recapitulates the multi-stage progression of human CCA.Wnt ligands found to be elevated in human CCA were also upregulated during the course of CCA development following thioacetamide treatment. Wnt10 a increased during the(pre-cancerous) regenerative phase, while Wnt7 b induction paralleled tumor growth. Along with upregulation of target genes, the findings demonstrate that the canonical Wnt pathway is progressively activated during cholangio-carcinogenesis. Macrophage depletion,eliminating a major source of Wnt7 b, prevented activation of the canonical Wnt cascade, and resulted in reduced number and volume of tumors in this model. Moreover,specific inhibitors of the canonical Wnt pathway(ICG-001 and C-59) caused reduction of tumor area and number,in xenograft and thioacetamide models of CCA. The aggregated findings show that experimental, and presumably human CCA, is a Wnt-driven tumor. Modulation of Wnt signaling, alone or in combination with surgicalor chemotherapy approaches, holds promise in the management of this fatal malignancy.展开更多
文摘背景: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。
基金supported by AR060456 (F.L.), AR047867 (M.J.S.)the Washington University Musculoskeletal Research Center (NIH P30 AR057235)
文摘There remain unmet clinical needs for safe and effective bone anabolic therapies to treat aging-related osteoporosis and to improve fracture healing in cases of nonunion or delayed union. Wnt signaling has emerged as a promising target pathway for developing novel bone anabolic drugs. Although neutralizing antibodies against the Wnt antagonist sclerostin have been tested,Wnt ligands themselves have not been fully explored as a potential therapy. Previous work has demonstrated Wnt7b as an endogenous ligand upregulated during osteoblast differentiation, and that Wnt7b overexpression potently stimulates bone accrual in the mouse. The earlier studies however did not address whether Wnt7b could promote bone formation when specifically applied to aged or fractured bones. Here we have developed a doxycycline-inducible strategy where Wnt7b is temporally induced in the bones of aged mice or during fracture healing. We report that forced expression of Wnt7b for 1 month starting at 15 months of age greatly stimulated trabecular and endosteal bone formation, resulting in a marked increase in bone mass. We further tested the effect of Wnt7b on bone healing in a murine closed femur fracture model. Induced expression of Wnt7b at the onset of fracture did not affect the initial cartilage formation but promoted mineralization of the subsequent bone callus. Thus, targeted delivery of Wnt7b to aged bones or fracture sites may be explored as a potential therapy.
文摘Cholangiocarcinoma(CCA) is a relatively rare malignancy of the intra- or extra-hepatic bile ducts that is classified according to its anatomical localization as intrahepatic, perihilar or distal. Overall, CCA has a dismal prognosis due to typical presentation at an advanced irresectable stage, lack of effective non-surgical treatments, and a high rate of disease recurrence. CCA frequently arises on a background of chronic liver inflammation and cholestasis. Chronic inflammation is accompanied by enhanced cell turnover with generation of additional inflammatory stimuli, and a microenvironment rich in pro-inflammatory mediators and proliferative factors that enable accumulation of mutations, transformation and expansion of mutated cells. A recent study by Boulter et al implicates the Wnt signaling cascade in cholangiocarcinogenesis. Wnt ligands Wnt7 B and Wnt10 A were found to be highly overexpressed in human CCA tissue. Wnt7 B protein was present throughout the tumor stroma, and often co-localized with a subset of CD68+ macrophages. To address in a direct manner whether Wnt signaling is engaged in development of CCA, Boulter et al explored the Wnt signaling pathway in an experimental model that recapitulates the multi-stage progression of human CCA.Wnt ligands found to be elevated in human CCA were also upregulated during the course of CCA development following thioacetamide treatment. Wnt10 a increased during the(pre-cancerous) regenerative phase, while Wnt7 b induction paralleled tumor growth. Along with upregulation of target genes, the findings demonstrate that the canonical Wnt pathway is progressively activated during cholangio-carcinogenesis. Macrophage depletion,eliminating a major source of Wnt7 b, prevented activation of the canonical Wnt cascade, and resulted in reduced number and volume of tumors in this model. Moreover,specific inhibitors of the canonical Wnt pathway(ICG-001 and C-59) caused reduction of tumor area and number,in xenograft and thioacetamide models of CCA. The aggregated findings show that experimental, and presumably human CCA, is a Wnt-driven tumor. Modulation of Wnt signaling, alone or in combination with surgicalor chemotherapy approaches, holds promise in the management of this fatal malignancy.
