目的运用超高效液相色谱-四极杆静电场轨道阱质谱(ultra-high-performance liquid chromatography coupled with quadrupole electrostatic field orbital trap-mass spectrometry,UHPLC-QE-MS)非靶向代谢组学分析法探讨不同海拔高原鼠...目的运用超高效液相色谱-四极杆静电场轨道阱质谱(ultra-high-performance liquid chromatography coupled with quadrupole electrostatic field orbital trap-mass spectrometry,UHPLC-QE-MS)非靶向代谢组学分析法探讨不同海拔高原鼠兔肾脏低氧适应性代谢变化的潜在机制。方法捕捉青海省果洛藏族自治州玛多县星宿海地区海拔4360 m(MD组)和青海省海北藏族自治州门源地区海拔2900 m(MY组)处的高原鼠兔各10只,经麻醉后采集血清样本,经安死术后采集肾脏样本,分别进行一般生理生化指标测定和代谢组学分析。其中一部分血清样本用于血液学分析,另一部分用于血气分析,剩余部分检测生化指标。肾组织样本中代谢物提取后,进行UHPLC-QE-MS分析,运用代谢组学主成分分析(principal component analysis,PCA)和正交偏最小二乘判别分析(orthogonal partial least squares discriminant analysis,OPLS-DA)方法分析差异代谢物,筛选标准为变量投影重要度(variable Importance in the projection,VIP)>1.5且倍数变化(fold change,FC)>1.5或VIP>1.5且FC<1/1.5。利用相关性分析热力图、差异显著性分析火山图、信号通路识别气泡图和矩形图分别分析差异代谢物及相关信号通路。结果MD组高原鼠兔的红细胞计数、葡萄糖、尿素氮、尿酸和同型半胱氨酸含量高于MY组,而血红蛋白、血细胞比容、肌酐和二氧化碳结合力低于MY组,这表明不同海拔的高原鼠兔血液携氧能力存在明显差异。经主成分模式识别分析和OPLS-DA置换检验显示,MD组和MY组高原鼠兔肾脏代谢物具有明显的聚类型分布(R^(2)Y=0.930,Q^(2)=0.655)。按照筛选标准,并经数据库比对发现,不同海拔高原鼠兔的肾脏代谢物差异分子有46个,其中MD组高原鼠兔的蟾蜍二烯羟酸内酯、腺苷、腺嘌呤、薯蓣皂苷、盐酸小檗碱、鼠尾草酚和虾青素表达水平均显著升高(VIP>1.5,P<0.05),花生四烯酸、组胺和香豆素水平显著下降(VIP>1.5,P<0.05)。相关信号通路分析显示,缬氨酸、亮氨酸和异亮氨酸生物合成通路具有最大影响因子(P<0.05),而泛酸盐和辅酶A生物合成通路呈现最显著富集(P<0.05)。结论不同海拔高原鼠兔肾脏差异代谢物氨基酸、泛酸盐及辅酶A通路可能参与高原鼠兔高原低氧适应代谢机制。展开更多
The retroviral vector （RCAS） has been widely used in avian system to study development and diseases, but is not suitable for mammals which do not produce the retrovirus receptor TVA. In this review, we trace the cur...The retroviral vector （RCAS） has been widely used in avian system to study development and diseases, but is not suitable for mammals which do not produce the retrovirus receptor TVA. In this review, we trace the current uses of RCAS-TVA approach in mammalian system with improved strategies, including generation of tv-a transgenic mice, use of soluble TVA receptor and retroviral receptor-ligand fusion proteins, improvement of RCAS vectors, and compare a series of mammalian models in variant studies of gene function, development, oncogenesis and gene therapy. All those studies demonstrate that the RCAS-TVA based mammalian models are powerful tools for understanding the mechanisms and target treating of human diseases.展开更多
文摘目的运用超高效液相色谱-四极杆静电场轨道阱质谱(ultra-high-performance liquid chromatography coupled with quadrupole electrostatic field orbital trap-mass spectrometry,UHPLC-QE-MS)非靶向代谢组学分析法探讨不同海拔高原鼠兔肾脏低氧适应性代谢变化的潜在机制。方法捕捉青海省果洛藏族自治州玛多县星宿海地区海拔4360 m(MD组)和青海省海北藏族自治州门源地区海拔2900 m(MY组)处的高原鼠兔各10只,经麻醉后采集血清样本,经安死术后采集肾脏样本,分别进行一般生理生化指标测定和代谢组学分析。其中一部分血清样本用于血液学分析,另一部分用于血气分析,剩余部分检测生化指标。肾组织样本中代谢物提取后,进行UHPLC-QE-MS分析,运用代谢组学主成分分析(principal component analysis,PCA)和正交偏最小二乘判别分析(orthogonal partial least squares discriminant analysis,OPLS-DA)方法分析差异代谢物,筛选标准为变量投影重要度(variable Importance in the projection,VIP)>1.5且倍数变化(fold change,FC)>1.5或VIP>1.5且FC<1/1.5。利用相关性分析热力图、差异显著性分析火山图、信号通路识别气泡图和矩形图分别分析差异代谢物及相关信号通路。结果MD组高原鼠兔的红细胞计数、葡萄糖、尿素氮、尿酸和同型半胱氨酸含量高于MY组,而血红蛋白、血细胞比容、肌酐和二氧化碳结合力低于MY组,这表明不同海拔的高原鼠兔血液携氧能力存在明显差异。经主成分模式识别分析和OPLS-DA置换检验显示,MD组和MY组高原鼠兔肾脏代谢物具有明显的聚类型分布(R^(2)Y=0.930,Q^(2)=0.655)。按照筛选标准,并经数据库比对发现,不同海拔高原鼠兔的肾脏代谢物差异分子有46个,其中MD组高原鼠兔的蟾蜍二烯羟酸内酯、腺苷、腺嘌呤、薯蓣皂苷、盐酸小檗碱、鼠尾草酚和虾青素表达水平均显著升高(VIP>1.5,P<0.05),花生四烯酸、组胺和香豆素水平显著下降(VIP>1.5,P<0.05)。相关信号通路分析显示,缬氨酸、亮氨酸和异亮氨酸生物合成通路具有最大影响因子(P<0.05),而泛酸盐和辅酶A生物合成通路呈现最显著富集(P<0.05)。结论不同海拔高原鼠兔肾脏差异代谢物氨基酸、泛酸盐及辅酶A通路可能参与高原鼠兔高原低氧适应代谢机制。
文摘The retroviral vector （RCAS） has been widely used in avian system to study development and diseases, but is not suitable for mammals which do not produce the retrovirus receptor TVA. In this review, we trace the current uses of RCAS-TVA approach in mammalian system with improved strategies, including generation of tv-a transgenic mice, use of soluble TVA receptor and retroviral receptor-ligand fusion proteins, improvement of RCAS vectors, and compare a series of mammalian models in variant studies of gene function, development, oncogenesis and gene therapy. All those studies demonstrate that the RCAS-TVA based mammalian models are powerful tools for understanding the mechanisms and target treating of human diseases.
文摘目的本研究旨在建立一种实时荧光定量PCR方法,用于检测猕猴三磷酸腺苷结合盒转运蛋白G2(adenosine triphosphate-binding cassette transporter protein G2,ABCG2)mRNA的基因转录水平。方法使用NCBI上GenBank数据库猕猴(Macaca mulatta)的ABCG2核苷酸序列号NM_001032919.1及内参GAPDH核苷酸序列号NM_001195426.1,借助Primer premier 5.0软件设计PCR引物。提取猕猴新鲜肾组织的总RNA,并反转录合成cDNA。接着,利用PCR引物进行实时荧光定量PCR扩增,并根据反应体系中荧光的变化情况定量分析ABCG2的mRNA相对表达水平。结果PCR产物测序结果显示,扩增的ABCG2和GAPDH核苷酸序列与NCBI上猕猴的序列同源性分别为90.91%和91.14%。ABCG2和GAPDH的扩增效率均达到80%~120%,实时荧光定量PCR标准曲线的熔解曲线为单峰,R2接近1。结论本研究建立的检测猕猴ABCG2 mRNA实时荧光定量检测方法,为研究高尿酸血症的发病机制以及新药开发奠定基础。
