目的探讨乙型肝炎病毒DNA聚合酶反式调节蛋白1(HBV DNA polymerase trans activated protein 1,HBVDNAPTP1)在羧酸酯酶1(carboxylesterase 1,CES1)介导的单核细胞凋亡信号通路中的作用机制。方法利用Phyre2在线工具预测得到CES1三级结构...目的探讨乙型肝炎病毒DNA聚合酶反式调节蛋白1(HBV DNA polymerase trans activated protein 1,HBVDNAPTP1)在羧酸酯酶1(carboxylesterase 1,CES1)介导的单核细胞凋亡信号通路中的作用机制。方法利用Phyre2在线工具预测得到CES1三级结构,分别用pCMV-Myc载体构建HBVDNAPTP1基因、pCMV-HA载体构建CES1基因及其截短体219-264aa和265-303aa的真核细胞表达质粒。分别转染细胞后通过免疫荧光和免疫共沉淀法确定HBVDNAPTP1和CES1互相作用的区段。后通过HBVDNAPTP1和CES1单独转染及共转染,检测其对细胞凋亡率的影响;同时在分离人外周血单个核细胞(Peripheral blood mononuclear cell,PBMC)上转染上述质粒,以确定CES1和HBVDNAPTP1对单核细胞形态的影响。利用qRT-PCR和Western blot检测不同质粒转染后下游Janus激酶-1(Janus kinase,JAK1)和信号传导及转录激活蛋白3(Signal transducer and activator of transcription 3,STAT3)的基因和蛋白的表达情况。结果HBVDNAPTP1与CES1有靶向关系,其中HBVDNAPTP1与CES1全长靶向结合效果最好,故后续实验以CES1全长序列为对象开展。CCK-8和PBMC电镜实验结果表明,与Control组和NC组相比,ov-HBVDNAPTP1组、ov-CES1组和ov-HBVDNAPTP1+ov-CES1组THP-1细胞的凋亡率均升高(P<0.001),细胞受损;与ov-HBVDNAPTP1组和ov-CES1组相比,ov-HBVDNAPTP1+ov-CES1组细胞凋亡率进一步升高(P<0.01),凋亡细胞、坏死细胞的数量增多。qRT-PCR和Western blot结果显示,HBVDNAPTP1和CES1均可以在转录水平和翻译水平上显著上调JAK1和STAT3的表达,且二者同时作用时上调趋势更加明显。结论本研究证实HBVDNAPTP1与CES1二者可协同抑制THP-1细胞增殖并诱导细胞损伤;还可协同上调JAK1/STAT3信号通路在转录和蛋白水平的表达来调控单核细胞凋亡。该发现为深入理解HBV感染相关免疫调控机制提供了新的理论依据。展开更多
HL-60 cells have been induced with differentiation index 16% by S-adenosyl-L-methionine (SAM) as in-ducer in the presence of optimum concentration of 10 μmol/L. The methylation level of genome DNA determined by HPLC ...HL-60 cells have been induced with differentiation index 16% by S-adenosyl-L-methionine (SAM) as in-ducer in the presence of optimum concentration of 10 μmol/L. The methylation level of genome DNA determined by HPLC is increased during cell differentiation. When restriction endonuclease Hae Ⅲ, Sma Ⅰ, Sal Ⅰ, Xho Ⅰ and Hind III which are sensitive to 5-methylcytosirte were used to cleave the genome DNA, a resistance effect was found. The interaction between DNA and DNA binding proteins is changed by using gel retarding test.展开更多
The emergence of single-cell genomic and transcriptomic sequencing accelerates the development of single-cell epigenomic technologies,providing an unprecedented opportunity for decoding cell fate decisions largely enc...The emergence of single-cell genomic and transcriptomic sequencing accelerates the development of single-cell epigenomic technologies,providing an unprecedented opportunity for decoding cell fate decisions largely encoded in the epigenome.Recent advances in single-cell multimodality epigenomic technologies facilitate directly interrogating the reg-ulatory relationship between multi-layer molecular information in the same cell.In this review,we discuss recent progress in development of single-cell multimodality epigenomic technologies and applications in elucidating cellular diversifications in development and diseases,with a focus on protein-DNA interactomics and regulatory links between epigenome and tran-scriptome.Further,we provide perspective on the future direction of single-cell multiomics tool development as well as challenges facing ahead.展开更多
文摘目的探讨乙型肝炎病毒DNA聚合酶反式调节蛋白1(HBV DNA polymerase trans activated protein 1,HBVDNAPTP1)在羧酸酯酶1(carboxylesterase 1,CES1)介导的单核细胞凋亡信号通路中的作用机制。方法利用Phyre2在线工具预测得到CES1三级结构,分别用pCMV-Myc载体构建HBVDNAPTP1基因、pCMV-HA载体构建CES1基因及其截短体219-264aa和265-303aa的真核细胞表达质粒。分别转染细胞后通过免疫荧光和免疫共沉淀法确定HBVDNAPTP1和CES1互相作用的区段。后通过HBVDNAPTP1和CES1单独转染及共转染,检测其对细胞凋亡率的影响;同时在分离人外周血单个核细胞(Peripheral blood mononuclear cell,PBMC)上转染上述质粒,以确定CES1和HBVDNAPTP1对单核细胞形态的影响。利用qRT-PCR和Western blot检测不同质粒转染后下游Janus激酶-1(Janus kinase,JAK1)和信号传导及转录激活蛋白3(Signal transducer and activator of transcription 3,STAT3)的基因和蛋白的表达情况。结果HBVDNAPTP1与CES1有靶向关系,其中HBVDNAPTP1与CES1全长靶向结合效果最好,故后续实验以CES1全长序列为对象开展。CCK-8和PBMC电镜实验结果表明,与Control组和NC组相比,ov-HBVDNAPTP1组、ov-CES1组和ov-HBVDNAPTP1+ov-CES1组THP-1细胞的凋亡率均升高(P<0.001),细胞受损;与ov-HBVDNAPTP1组和ov-CES1组相比,ov-HBVDNAPTP1+ov-CES1组细胞凋亡率进一步升高(P<0.01),凋亡细胞、坏死细胞的数量增多。qRT-PCR和Western blot结果显示,HBVDNAPTP1和CES1均可以在转录水平和翻译水平上显著上调JAK1和STAT3的表达,且二者同时作用时上调趋势更加明显。结论本研究证实HBVDNAPTP1与CES1二者可协同抑制THP-1细胞增殖并诱导细胞损伤;还可协同上调JAK1/STAT3信号通路在转录和蛋白水平的表达来调控单核细胞凋亡。该发现为深入理解HBV感染相关免疫调控机制提供了新的理论依据。
文摘HL-60 cells have been induced with differentiation index 16% by S-adenosyl-L-methionine (SAM) as in-ducer in the presence of optimum concentration of 10 μmol/L. The methylation level of genome DNA determined by HPLC is increased during cell differentiation. When restriction endonuclease Hae Ⅲ, Sma Ⅰ, Sal Ⅰ, Xho Ⅰ and Hind III which are sensitive to 5-methylcytosirte were used to cleave the genome DNA, a resistance effect was found. The interaction between DNA and DNA binding proteins is changed by using gel retarding test.
基金supported by the National Key Research and Development Program of China(2021YFA1100101 and 2017YFA0103402)the National Natural Science Foundation of China(32025015 and 31771607)the Peking-Tsinghua Center for Life Sciences.
文摘The emergence of single-cell genomic and transcriptomic sequencing accelerates the development of single-cell epigenomic technologies,providing an unprecedented opportunity for decoding cell fate decisions largely encoded in the epigenome.Recent advances in single-cell multimodality epigenomic technologies facilitate directly interrogating the reg-ulatory relationship between multi-layer molecular information in the same cell.In this review,we discuss recent progress in development of single-cell multimodality epigenomic technologies and applications in elucidating cellular diversifications in development and diseases,with a focus on protein-DNA interactomics and regulatory links between epigenome and tran-scriptome.Further,we provide perspective on the future direction of single-cell multiomics tool development as well as challenges facing ahead.
