目的前期研究发现子宫肌瘤组织中存在甲基化CpG结合蛋白2(methyl-CpG binding domain protein 2,MBD2)基因和蛋白的差异性表达下调,本研究探讨MBD2过表达对子宫肌瘤生物学功能的影响,并分析子宫肌瘤组织中MBD2基因启动子区甲基化状态。...目的前期研究发现子宫肌瘤组织中存在甲基化CpG结合蛋白2(methyl-CpG binding domain protein 2,MBD2)基因和蛋白的差异性表达下调,本研究探讨MBD2过表达对子宫肌瘤生物学功能的影响,并分析子宫肌瘤组织中MBD2基因启动子区甲基化状态。方法采用慢病毒包装MBD2真核过表达载体,通过转染至293T细胞,检测293T细胞的增殖、凋亡及周期情况;利用飞行时间质谱技术检测子宫肌瘤(病例组)及瘤旁肌层组织(对照组)中MBD2的基因启动子区甲基化水平,分析MBD2基因CpG位点的甲基化率与MBD2mRNA水平的关系。结果①MBD2真核过表达载体成功转染293T细胞,上调293T细胞MBD2的表达对其增殖、凋亡和周期均无显著影响。②共46对标本的MBD2基因启动子区的10个CpG位点进行甲基化分析法发现,单点甲基化水平分析中,所有CpG位点上,MBD2 mRNA与MBD2甲基化的表达在病例组与对照组比较,差异均无统计学意义(P均>0.05)。结论过表达MBD2对子宫肌瘤细胞生物学功能并不产生影响,子宫肌瘤组织中MBD2基因的表达下调与其基因启动子区甲基化状可能无关。展开更多
该文探讨甲基化Cp G结合域蛋白2(methyl-Cp G-binding domain protein 2,MBD2)对人胰腺癌细胞增殖和迁移的影响。将干扰质粒MBD2 sh RNA转染入MBD2高表达的Pa Tu8988细胞和SW1990细胞中,运用CCK-8法检测细胞的增殖活性并绘制生长曲线,...该文探讨甲基化Cp G结合域蛋白2(methyl-Cp G-binding domain protein 2,MBD2)对人胰腺癌细胞增殖和迁移的影响。将干扰质粒MBD2 sh RNA转染入MBD2高表达的Pa Tu8988细胞和SW1990细胞中,运用CCK-8法检测细胞的增殖活性并绘制生长曲线,克隆形成实验检测细胞克隆形成率,Transwell检测细胞迁移率。结果显示,下调MBD2后,Pa Tu8988细胞中MBD2基因的表达量明显降低,细胞增殖减慢,克隆形成率降低,细胞迁移率减少;在SW1990细胞中下调MBD2基因后,得到类似结果。由此说明,沉默MBD2基因可以抑制胰腺癌细胞Pa Tu8988和SW1990的增殖和迁移,为进一步研究MBD2在胰腺癌细胞增殖与迁移中的作用机制奠定了基础。展开更多
目的:探讨Mbd2在EMs患者发病过程中的可能作用。方法:Trizol法提取病变组织的总RNA,反转录生成c DNA,Blast-primer设计Mbd2 m RNA引物,Real-time PCR方法检测各组间Mbd2 m RNA表达变化。结果:EMs患者异位内膜、在位内膜中Mbd2 m RNA表...目的:探讨Mbd2在EMs患者发病过程中的可能作用。方法:Trizol法提取病变组织的总RNA,反转录生成c DNA,Blast-primer设计Mbd2 m RNA引物,Real-time PCR方法检测各组间Mbd2 m RNA表达变化。结果:EMs患者异位内膜、在位内膜中Mbd2 m RNA表达显著高于子宫肌瘤患者在位内膜(P<0.01);且EMs患者异位内膜中Mbd2m RNA的表达显著高于在位内膜(P<0.01)。结论:Mbd2基因参与子宫内膜异位症的发生,还可能为子宫内膜异位症的早期发现做出贡献。展开更多
Introduction:Allergen-specific CD4+T cells play a central role in autoimmune disorders,allergies and asthma,with Th2-type immunity being the typical functional response of CD4+T cells.This study aimed to investigate t...Introduction:Allergen-specific CD4+T cells play a central role in autoimmune disorders,allergies and asthma,with Th2-type immunity being the typical functional response of CD4+T cells.This study aimed to investigate the role of MBD2 in regulating Th2 cell differentiation.Methods:Splenic mononuclear cells were extracted from C57BL/6 mice,and CD4+T cells were isolated using magnetic beads and confirmed through flow cytometry.Lentivirus was employed to construct MBD2-silenced CD4+T cells.In vitro experiments were performed to treat splenogenic mononuclear cells and CD4+T cells with Ovalbumin(OVA),and Th2 cell ratios and IL-4 levels were assessed using flow cytometry and ELISA.Results:The purity of the isolated CD4+T cells was 95.73%,confirming successful isolation of primary CD4+T cells.Compared to the control group,the Th2 cell ratio exhibited an increase in the Th2-induced group.Treatment with 5-Aza(concentrations,1-100μM)promoted Th2 cell differentiation and increased IL-4 levels.Notably,when combined with Th2 induction and 10μM 5-Aza treatment,silencing MBD2 further amplified Th2 cell ratios and elevated IL-4 levels in cell supernatants.Furthermore,OVA(concentration,200μg/mL)induced the differentiation of CD4+T cells into Th2 cells and increased IL-4 secretion.Interestingly,silencing MBD2 significantly increased the Th2 cell ratio and IL-4 levels in OVA-treated CD4+T cells.Conclusion:In summary,OVA promoted CD4+T cell differentiation into Th2 cells and enhanced IL-4 levels.MBD2 was identified as a mediator of Th2 cell differentiation in splenic-derived CD4+T cells,influenced by OVA or 5-Aza treatment.展开更多
Methyl-CpG-binding domain （MBD） proteins can specifically recognize and bind methylated CpG sites of DNA, thus repress gene transcription. In this study, we designed and expressed two recombinant proteins, MBD2b and...Methyl-CpG-binding domain （MBD） proteins can specifically recognize and bind methylated CpG sites of DNA, thus repress gene transcription. In this study, we designed and expressed two recombinant proteins, MBD2b and SNAP-MBD2b, in E. coli. An optimized protocol was developed to purify the proteins using Ni-NTA affinity cartridge and cation exchange resin. The engineered proteins purified by this method exhibited more than 93% purity and high binding avidity. We found that both SNAP-MBD2b and MBD2b were prone to aggregate during dialysis. However, this could be prevented by the use of 0.3 mol/L NaCI. The fusion of SNAP-tag with MBD2b significantly enhanced the expression of MBD2b protein in E. coli and reduced the adsorption of MBD2b on solid interfaces involved in protein purification and immobilization. The engineered proteins can be used for the study of interaction with methylated DNA and the assays for DNA methylation.展开更多
To the Editor:Methyl CpG binding domain protein 2(MBD2)belongs to the histone deacetylase family,which promotes multi-gene expression by inhibiting methylation and participates in the pathological process of various d...To the Editor:Methyl CpG binding domain protein 2(MBD2)belongs to the histone deacetylase family,which promotes multi-gene expression by inhibiting methylation and participates in the pathological process of various diseases.Apoptosis and inflammatory response are the main mechanisms leading to a variety of immune diseases.MBD2,as an important methylation protein reader,has in recent years been shown to be indispensable in the inflammatory response.This paper summarizes the current understanding of inflammatory MBD2 biology and provides additional direction for the study of inflammatory mechanisms by summarizing the current understanding of the regulatory mechanisms of MBD2 in inflammatory diseases and the inflammatory pathways that may regulate dysfunction.PubMed,Embase,and Wanfang Data were searched up to May 2022 with no language restriction for reports on MBD2 in inflammatory pathways,immune cell differentiation,and macrophage polarization,among others.展开更多
文摘目的:探讨Mbd2在EMs患者发病过程中的可能作用。方法:Trizol法提取病变组织的总RNA,反转录生成c DNA,Blast-primer设计Mbd2 m RNA引物,Real-time PCR方法检测各组间Mbd2 m RNA表达变化。结果:EMs患者异位内膜、在位内膜中Mbd2 m RNA表达显著高于子宫肌瘤患者在位内膜(P<0.01);且EMs患者异位内膜中Mbd2m RNA的表达显著高于在位内膜(P<0.01)。结论:Mbd2基因参与子宫内膜异位症的发生,还可能为子宫内膜异位症的早期发现做出贡献。
基金supported by grants from the National Natural Science Foundation of China(Nos.81760009 and 81560007).
文摘Introduction:Allergen-specific CD4+T cells play a central role in autoimmune disorders,allergies and asthma,with Th2-type immunity being the typical functional response of CD4+T cells.This study aimed to investigate the role of MBD2 in regulating Th2 cell differentiation.Methods:Splenic mononuclear cells were extracted from C57BL/6 mice,and CD4+T cells were isolated using magnetic beads and confirmed through flow cytometry.Lentivirus was employed to construct MBD2-silenced CD4+T cells.In vitro experiments were performed to treat splenogenic mononuclear cells and CD4+T cells with Ovalbumin(OVA),and Th2 cell ratios and IL-4 levels were assessed using flow cytometry and ELISA.Results:The purity of the isolated CD4+T cells was 95.73%,confirming successful isolation of primary CD4+T cells.Compared to the control group,the Th2 cell ratio exhibited an increase in the Th2-induced group.Treatment with 5-Aza(concentrations,1-100μM)promoted Th2 cell differentiation and increased IL-4 levels.Notably,when combined with Th2 induction and 10μM 5-Aza treatment,silencing MBD2 further amplified Th2 cell ratios and elevated IL-4 levels in cell supernatants.Furthermore,OVA(concentration,200μg/mL)induced the differentiation of CD4+T cells into Th2 cells and increased IL-4 secretion.Interestingly,silencing MBD2 significantly increased the Th2 cell ratio and IL-4 levels in OVA-treated CD4+T cells.Conclusion:In summary,OVA promoted CD4+T cell differentiation into Th2 cells and enhanced IL-4 levels.MBD2 was identified as a mediator of Th2 cell differentiation in splenic-derived CD4+T cells,influenced by OVA or 5-Aza treatment.
基金supported by the National Basic Research Program of China(21077129,20877091,20890112,21125523,20921063)the National Natural Science Foundation of China(2009CB421605,2010CB933502)
文摘Methyl-CpG-binding domain （MBD） proteins can specifically recognize and bind methylated CpG sites of DNA, thus repress gene transcription. In this study, we designed and expressed two recombinant proteins, MBD2b and SNAP-MBD2b, in E. coli. An optimized protocol was developed to purify the proteins using Ni-NTA affinity cartridge and cation exchange resin. The engineered proteins purified by this method exhibited more than 93% purity and high binding avidity. We found that both SNAP-MBD2b and MBD2b were prone to aggregate during dialysis. However, this could be prevented by the use of 0.3 mol/L NaCI. The fusion of SNAP-tag with MBD2b significantly enhanced the expression of MBD2b protein in E. coli and reduced the adsorption of MBD2b on solid interfaces involved in protein purification and immobilization. The engineered proteins can be used for the study of interaction with methylated DNA and the assays for DNA methylation.
基金the Foundation of Guizhou Science and Technology Cooperation(No.[2020]1Z061)the National Natural Science Foundation of China(No.82160370).
文摘To the Editor:Methyl CpG binding domain protein 2(MBD2)belongs to the histone deacetylase family,which promotes multi-gene expression by inhibiting methylation and participates in the pathological process of various diseases.Apoptosis and inflammatory response are the main mechanisms leading to a variety of immune diseases.MBD2,as an important methylation protein reader,has in recent years been shown to be indispensable in the inflammatory response.This paper summarizes the current understanding of inflammatory MBD2 biology and provides additional direction for the study of inflammatory mechanisms by summarizing the current understanding of the regulatory mechanisms of MBD2 in inflammatory diseases and the inflammatory pathways that may regulate dysfunction.PubMed,Embase,and Wanfang Data were searched up to May 2022 with no language restriction for reports on MBD2 in inflammatory pathways,immune cell differentiation,and macrophage polarization,among others.
