旨在对基质金属蛋白酶组织抑制剂(tissue inhibitor of matrix metalloproteinases,TIMPs)家族成员中的TIMP1进行克隆和分析,通过过表达与敲减TIMP1,探究TIMP1对毛囊毛乳头细胞(dermal papilla cells,DPCs)毛囊生长相关基因的调控作用...旨在对基质金属蛋白酶组织抑制剂(tissue inhibitor of matrix metalloproteinases,TIMPs)家族成员中的TIMP1进行克隆和分析,通过过表达与敲减TIMP1,探究TIMP1对毛囊毛乳头细胞(dermal papilla cells,DPCs)毛囊生长相关基因的调控作用。本研究通过分子克隆TIMP1编码区(coding sequence,CDS)序列,并对其进行生物信息学功能分析。之后,构建过表达载体pcDNA 3.1-TIMP1并设计合成siRNA,在DPCs中过表达和敲低TIMP1,检测毛囊生长发育相关基因的表达,并通过EdU和CCK-8检测DPCs的增殖水平。结果显示,兔TIMP1基因的CDS区全长624 bp,共编码207个氨基酸。生物信息学分析表明TIMP1蛋白存在信号肽,不包含跨膜结构域,在不同哺乳动物中存在同源性。在DPCs中过表达TIMP1能够极显著上调BMP2(bone morphogenetic protein 2)、SFRP2(secreted frizzled-related protein 2)、TGFβ1(tranforming growth factorβ1)基因的mRNA表达水平(P<0.01),极显著下调WNT2(wnt family member 2)基因的mRNA表达水平(P<0.01),敲减TIMP1能显著下调BMP2、SFRP2基因的mRNA的表达(P<0.05),极显著下调TGFβ1基因的mRNA表达(P<0.01)。此外,EdU和CCK-8结果显示过表达TIMP1能够抑制DPCs的增殖,敲低TIMP1能够促进DPCs的增殖。本研究成功克隆家兔TIMP1基因CDS序列,并对其生物信息学功能进行预测,初步分析TIMP1对毛囊生长相关基因的调控作用,验证了其抑制DPCs增殖的作用,为阐明家兔毛囊生长发育的理论研究提供参考。展开更多
The hippocampus is essential for learning and memory,but it also plays an important role in regulating emotional behavior,as hippocampal excitability and plasticity affect anxiety and fear.Brain synaptic plasticity ma...The hippocampus is essential for learning and memory,but it also plays an important role in regulating emotional behavior,as hippocampal excitability and plasticity affect anxiety and fear.Brain synaptic plasticity may be regulated by tissue inhibitor of matrix metalloproteinase 1(TIMP1),a known protein inhibitor of extracellular matrix(ECM),and the expression of TIMP1 in the hippocampus can be induced by neuronal excitation and various stimuli.However,the involvement of Timp1 in fear learning,anxiety,and hippocampal synaptic function remains to be established.Our study of Timp1 function in vivo revealed that Timp1 knockout mice exhibit anxiety-like behavior but normal fear learning.Electrophysiological results suggested that Timp1 knockout mice showed hyperactivity in the ventral CA1 region,but the basic synaptic transmission and plasticity were normal in the Schaffer collateral pathway.Taken together,our results suggest that deletion of Timp1 in vivo leads to the occurrence of anxiety behaviors,but that Timp1 is not crucial for fear learning.展开更多
文摘旨在对基质金属蛋白酶组织抑制剂(tissue inhibitor of matrix metalloproteinases,TIMPs)家族成员中的TIMP1进行克隆和分析,通过过表达与敲减TIMP1,探究TIMP1对毛囊毛乳头细胞(dermal papilla cells,DPCs)毛囊生长相关基因的调控作用。本研究通过分子克隆TIMP1编码区(coding sequence,CDS)序列,并对其进行生物信息学功能分析。之后,构建过表达载体pcDNA 3.1-TIMP1并设计合成siRNA,在DPCs中过表达和敲低TIMP1,检测毛囊生长发育相关基因的表达,并通过EdU和CCK-8检测DPCs的增殖水平。结果显示,兔TIMP1基因的CDS区全长624 bp,共编码207个氨基酸。生物信息学分析表明TIMP1蛋白存在信号肽,不包含跨膜结构域,在不同哺乳动物中存在同源性。在DPCs中过表达TIMP1能够极显著上调BMP2(bone morphogenetic protein 2)、SFRP2(secreted frizzled-related protein 2)、TGFβ1(tranforming growth factorβ1)基因的mRNA表达水平(P<0.01),极显著下调WNT2(wnt family member 2)基因的mRNA表达水平(P<0.01),敲减TIMP1能显著下调BMP2、SFRP2基因的mRNA的表达(P<0.05),极显著下调TGFβ1基因的mRNA表达(P<0.01)。此外,EdU和CCK-8结果显示过表达TIMP1能够抑制DPCs的增殖,敲低TIMP1能够促进DPCs的增殖。本研究成功克隆家兔TIMP1基因CDS序列,并对其生物信息学功能进行预测,初步分析TIMP1对毛囊生长相关基因的调控作用,验证了其抑制DPCs增殖的作用,为阐明家兔毛囊生长发育的理论研究提供参考。
基金supported by grants from the Key-Area Research and Development Program of Guangdong Province(2019B030335001)the National Natural Science Foundation of China(81925011,92149304,and 32200636)+2 种基金the R&D Program of Beijing Municipal Education Commission(KM202310025028)the High-Level Teachers project(CIT&TCD20190334)the Youth Beijing Scholars Program(015).
文摘The hippocampus is essential for learning and memory,but it also plays an important role in regulating emotional behavior,as hippocampal excitability and plasticity affect anxiety and fear.Brain synaptic plasticity may be regulated by tissue inhibitor of matrix metalloproteinase 1(TIMP1),a known protein inhibitor of extracellular matrix(ECM),and the expression of TIMP1 in the hippocampus can be induced by neuronal excitation and various stimuli.However,the involvement of Timp1 in fear learning,anxiety,and hippocampal synaptic function remains to be established.Our study of Timp1 function in vivo revealed that Timp1 knockout mice exhibit anxiety-like behavior but normal fear learning.Electrophysiological results suggested that Timp1 knockout mice showed hyperactivity in the ventral CA1 region,but the basic synaptic transmission and plasticity were normal in the Schaffer collateral pathway.Taken together,our results suggest that deletion of Timp1 in vivo leads to the occurrence of anxiety behaviors,but that Timp1 is not crucial for fear learning.
