目的探讨四氯二苯二噁英（2，3，7，8-tetrachlorodibenzo-p-dioxin，TCDD）致胎鼠先天性腭裂的可能作用机制。方法12只C57BL／6J孕鼠于妊娠第10天随机分为实验组和对照组，每组6只，实验组以TCDD64μg／kg灌胃，对照组以等量玉米油灌...目的探讨四氯二苯二噁英（2，3，7，8-tetrachlorodibenzo-p-dioxin，TCDD）致胎鼠先天性腭裂的可能作用机制。方法12只C57BL／6J孕鼠于妊娠第10天随机分为实验组和对照组，每组6只，实验组以TCDD64μg／kg灌胃，对照组以等量玉米油灌胃，于妊娠第18．5天在解剖显微镜下观察胎鼠腭裂的发生率。另取18只C57BL／6J孕鼠，于妊娠第10天随机分为实验组和对照组，每组9只，处理方法同前，根据标本采集时间不同，每组又分为妊娠第13．5、14．5和15．5天3个亚组，每个亚组3只，分别于妊娠第13．5、14．5和15．5天剪取胎鼠腭突组织提取RNA和DNA，采用RT—PCR检测Smad2～4及Smad7mRNA的表达情况、甲基化特异性PCR（methylmion specific PCR，MSP）检测转化生长因子-β3（transforming growthfactor—β3，TGF—β3）基因启动子甲基化水平。两样本间均数比较采用t检验。结果TCDD成功诱导建立C57BL／6J胎鼠先天性腭裂模型，实验组腭裂发生率为100％，对照组无腭裂发生。在妊娠第13．5、14．5和15．5天，RT—PCR显示实验组Smad2mRNA的相对表达值分别为0．263±0．088、0．296±0．016和0．159±0．027，对照组为0．180±0．042、0．282±0．029和0．165±0．018，差异无统计学意义（t=-1．474、-0．762、0．321，P〉0．05）；实验组Smad3mRNA的相对表达值分别为0．453±0．153、0．551±0．160和0．328±0．049，对照组为0．375±0．126、0．510±0．145和0．259±0．035，差异无统计学意义（t=-0．678、-0．336、-2．005，P〉0．05）；实验组Smad4mRNA的相对表达值分别为0．675±0．174、0．577±0．070和0．396±0．066，对照组为0．557±0．138、0．587±0．080和0．441±0．054，差异无统计学意义（t=-0．926、0．161、0．927，P〉0．05）；实验组Smad7mRNA的相对表达值分别为0．283±0．050、0．320±0．068和0．169±0．045，对照组为0．207±0．043、0．288±0．051和0．15展开更多
The periodontal ligament-derived mesenchymal stem cell is regarded as a source of adult stem cells due to its multipotency.However, the proof of chondrogenic potential of the cells is scarce.Therefore,we investigated ...The periodontal ligament-derived mesenchymal stem cell is regarded as a source of adult stem cells due to its multipotency.However, the proof of chondrogenic potential of the cells is scarce.Therefore,we investigated the chondrogenic differentiation capacity of periodontal ligament derived mesenchymal stem cells induced by transforming growth factor(TGF)-p3 and bone morphogenetic protein(BMP)-6.After isolation of periodontal ligament stem cells(PDLSCs) from human periodontal ligament,the cells were cultured in Dulbecco’s modified Eagle’s medium(DMEM) with 20%fetal bovine serum(FBS).A mechanical force initiated chondrogenic differentiation of the cells.For chondrogenic differentiation,10μg·LTGF-β3 or 100μg·LBMP-6 and the combination treating group for synergistic effect of the growth factors.We analyzed the PDLSCs by fluorescence-activated cell sorting and chondrogenesis were evaluated by glycosaminoglycans assay,histology,immunohistochemistry and genetic analysis.PDLSCs showed mesenchymal stem cell properties proved by FACS analysis.Glycosaminoglycans contents were increased 217%by TGF-β3 and 220%by BMP-6. The synergetic effect of TGF-β3 and BMP-6 were shown up to 281%compared to control.The combination treatment increased Sox9, aggrecan and collagen II expression compared with not only controls,but also TGF-P3 or BMP-6 single treatment dramatically.The histological analysis also indicated the chondrogenic differentiation of PDLSCs in our conditions.The results of the present study demonstrate the potential of the dental stem cell as a valuable cell source for chondrogenesis,which may be applicable for regeneration of cartilage and bone fracture in the field of cell therapy.展开更多
文摘目的探讨四氯二苯二噁英（2，3，7，8-tetrachlorodibenzo-p-dioxin，TCDD）致胎鼠先天性腭裂的可能作用机制。方法12只C57BL／6J孕鼠于妊娠第10天随机分为实验组和对照组，每组6只，实验组以TCDD64μg／kg灌胃，对照组以等量玉米油灌胃，于妊娠第18．5天在解剖显微镜下观察胎鼠腭裂的发生率。另取18只C57BL／6J孕鼠，于妊娠第10天随机分为实验组和对照组，每组9只，处理方法同前，根据标本采集时间不同，每组又分为妊娠第13．5、14．5和15．5天3个亚组，每个亚组3只，分别于妊娠第13．5、14．5和15．5天剪取胎鼠腭突组织提取RNA和DNA，采用RT—PCR检测Smad2～4及Smad7mRNA的表达情况、甲基化特异性PCR（methylmion specific PCR，MSP）检测转化生长因子-β3（transforming growthfactor—β3，TGF—β3）基因启动子甲基化水平。两样本间均数比较采用t检验。结果TCDD成功诱导建立C57BL／6J胎鼠先天性腭裂模型，实验组腭裂发生率为100％，对照组无腭裂发生。在妊娠第13．5、14．5和15．5天，RT—PCR显示实验组Smad2mRNA的相对表达值分别为0．263±0．088、0．296±0．016和0．159±0．027，对照组为0．180±0．042、0．282±0．029和0．165±0．018，差异无统计学意义（t=-1．474、-0．762、0．321，P〉0．05）；实验组Smad3mRNA的相对表达值分别为0．453±0．153、0．551±0．160和0．328±0．049，对照组为0．375±0．126、0．510±0．145和0．259±0．035，差异无统计学意义（t=-0．678、-0．336、-2．005，P〉0．05）；实验组Smad4mRNA的相对表达值分别为0．675±0．174、0．577±0．070和0．396±0．066，对照组为0．557±0．138、0．587±0．080和0．441±0．054，差异无统计学意义（t=-0．926、0．161、0．927，P〉0．05）；实验组Smad7mRNA的相对表达值分别为0．283±0．050、0．320±0．068和0．169±0．045，对照组为0．207±0．043、0．288±0．051和0．15
基金supported by the Bio & Medical Technology Development Program of the National Research Foundation(NRF) funded by the Korean government(MEST)(No.860-20110087)
文摘The periodontal ligament-derived mesenchymal stem cell is regarded as a source of adult stem cells due to its multipotency.However, the proof of chondrogenic potential of the cells is scarce.Therefore,we investigated the chondrogenic differentiation capacity of periodontal ligament derived mesenchymal stem cells induced by transforming growth factor(TGF)-p3 and bone morphogenetic protein(BMP)-6.After isolation of periodontal ligament stem cells(PDLSCs) from human periodontal ligament,the cells were cultured in Dulbecco’s modified Eagle’s medium(DMEM) with 20%fetal bovine serum(FBS).A mechanical force initiated chondrogenic differentiation of the cells.For chondrogenic differentiation,10μg·LTGF-β3 or 100μg·LBMP-6 and the combination treating group for synergistic effect of the growth factors.We analyzed the PDLSCs by fluorescence-activated cell sorting and chondrogenesis were evaluated by glycosaminoglycans assay,histology,immunohistochemistry and genetic analysis.PDLSCs showed mesenchymal stem cell properties proved by FACS analysis.Glycosaminoglycans contents were increased 217%by TGF-β3 and 220%by BMP-6. The synergetic effect of TGF-β3 and BMP-6 were shown up to 281%compared to control.The combination treatment increased Sox9, aggrecan and collagen II expression compared with not only controls,but also TGF-P3 or BMP-6 single treatment dramatically.The histological analysis also indicated the chondrogenic differentiation of PDLSCs in our conditions.The results of the present study demonstrate the potential of the dental stem cell as a valuable cell source for chondrogenesis,which may be applicable for regeneration of cartilage and bone fracture in the field of cell therapy.
