目的用直接酪胺信号放大(tyramine signal amplification,TSA)方法显示Y染色体荧光原位杂交信号,以提高检测脑组织切片Y染色体荧光原位杂交信号的灵敏度。方法以小鼠Y染色体重复序列pY353/B cDNA为模板标记RNA探针,以雌性小鼠为对照,用...目的用直接酪胺信号放大(tyramine signal amplification,TSA)方法显示Y染色体荧光原位杂交信号,以提高检测脑组织切片Y染色体荧光原位杂交信号的灵敏度。方法以小鼠Y染色体重复序列pY353/B cDNA为模板标记RNA探针,以雌性小鼠为对照,用原位杂交方法杂交Y染色体特异性基因区域,分别用直接TSA方法和"三步抗体法"显示荧光原位杂交信号。通过计数Y染色体阳性细胞核占所有细胞核的百分率来计算TSA法和"三步抗体法"检测脑组织切片Y染色体荧光原位杂交信号的灵敏度,比较分析两种方法灵敏度的差异。结果"三步抗体法"在脑切片检测Y染色体阳性信号的灵敏度和特异度分别为85.67%和100%;直接TSA法在脑切片检测Y染色体阳性信号的灵敏度和特异度分别为92.82%和100%。结论TSA方法检测脑组织切片Y染色体荧光原位杂交信号的灵敏度比"三步抗体"法有显著的提高,可以用于脑内追踪和分析骨髓干细胞的迁移规律。展开更多
Objective Tyro-3 and Axl receptors are expressed in brain in a region-specific manner and their bioactivities in the central nervous system remain still elusive.The aim of the present study was to investigate their fu...Objective Tyro-3 and Axl receptors are expressed in brain in a region-specific manner and their bioactivities in the central nervous system remain still elusive.The aim of the present study was to investigate their functions in neuronal differentiation.Methods PC12 cells overexpressing Tyro-3 or Axl were established by transfection with full-length CMV-Tyro3-eCFP or CMV-Axl-eGFP plasmid,respectively.CMV-eGFP plasmid served as a control vector.After that,the fluorescence intensity and distributions of green fluorescent protein （GFP） and cyan fluorescent protein （CFP） in the cells with or without nerve growth factor （NGF） treatment were real-time monitored.Results Expressions of Tyro-3 and Axl receptors were under the regulation of NGF and associated with neuronal differentiation.This was not observed in CMV-eGFP-transfected PC12 cells.Besides,confocal microscopy revealed that NGF affected intracellular localization of full-length Axl-eGFP and Tyro-3eCFP in PC12 cells.Moreover,the development of outgrowth of differentiated PC12 cells under stimulation of NGF was promoted by overexpression of Tyro-3 or Axl.Conclusion Expressions of Tyro-3 and Axl receptors are under the regulation of NGF and are involved in NGF-induced neuronal differentiation of PC12 cells.展开更多
文摘目的用直接酪胺信号放大(tyramine signal amplification,TSA)方法显示Y染色体荧光原位杂交信号,以提高检测脑组织切片Y染色体荧光原位杂交信号的灵敏度。方法以小鼠Y染色体重复序列pY353/B cDNA为模板标记RNA探针,以雌性小鼠为对照,用原位杂交方法杂交Y染色体特异性基因区域,分别用直接TSA方法和"三步抗体法"显示荧光原位杂交信号。通过计数Y染色体阳性细胞核占所有细胞核的百分率来计算TSA法和"三步抗体法"检测脑组织切片Y染色体荧光原位杂交信号的灵敏度,比较分析两种方法灵敏度的差异。结果"三步抗体法"在脑切片检测Y染色体阳性信号的灵敏度和特异度分别为85.67%和100%;直接TSA法在脑切片检测Y染色体阳性信号的灵敏度和特异度分别为92.82%和100%。结论TSA方法检测脑组织切片Y染色体荧光原位杂交信号的灵敏度比"三步抗体"法有显著的提高,可以用于脑内追踪和分析骨髓干细胞的迁移规律。
基金supported by the National Basic Research Development Program of China(No.2006CB500700)the National Natural Science Foundation of China(No. 30900421/c090201)
文摘Objective Tyro-3 and Axl receptors are expressed in brain in a region-specific manner and their bioactivities in the central nervous system remain still elusive.The aim of the present study was to investigate their functions in neuronal differentiation.Methods PC12 cells overexpressing Tyro-3 or Axl were established by transfection with full-length CMV-Tyro3-eCFP or CMV-Axl-eGFP plasmid,respectively.CMV-eGFP plasmid served as a control vector.After that,the fluorescence intensity and distributions of green fluorescent protein （GFP） and cyan fluorescent protein （CFP） in the cells with or without nerve growth factor （NGF） treatment were real-time monitored.Results Expressions of Tyro-3 and Axl receptors were under the regulation of NGF and associated with neuronal differentiation.This was not observed in CMV-eGFP-transfected PC12 cells.Besides,confocal microscopy revealed that NGF affected intracellular localization of full-length Axl-eGFP and Tyro-3eCFP in PC12 cells.Moreover,the development of outgrowth of differentiated PC12 cells under stimulation of NGF was promoted by overexpression of Tyro-3 or Axl.Conclusion Expressions of Tyro-3 and Axl receptors are under the regulation of NGF and are involved in NGF-induced neuronal differentiation of PC12 cells.
