目的：研究甘草苷对中波紫外线（UVB）诱导人皮肤角质形成细胞（Ha Ca T）凋亡的影响。方法：以不同浓度的甘草苷作用于Ha Ca T细胞,噻唑蓝（MTT）比色法筛选甘草苷的有效安全浓度;用30 m J·cm-2的UVB作用于Ha Ca T细胞,建立光老...目的：研究甘草苷对中波紫外线（UVB）诱导人皮肤角质形成细胞（Ha Ca T）凋亡的影响。方法：以不同浓度的甘草苷作用于Ha Ca T细胞,噻唑蓝（MTT）比色法筛选甘草苷的有效安全浓度;用30 m J·cm-2的UVB作用于Ha Ca T细胞,建立光老化模型,MTT比色法检测光老化Ha Ca T细胞增殖率;流式细胞术检测各组细胞中活性氧自由基（ROS）含量和总凋亡率;实时荧光定量PCR（Real-time PCR）检测光老化Ha Ca T细胞中半胱氨酸天冬氨酸-3（Caspase-3）,半胱氨酸天冬氨酸-9（Caspase-9）mRNA表达水平;蛋白免疫印记法（Western blot）检测光老化Ha Ca T细胞中B淋巴细胞瘤-2（Bcl-2）,Caspase-3,Caspase-9蛋白表达量。结果：筛选出甘草苷的最佳有效安全浓度为1×10-7,1×10-6,1×10-5mol·L-1。与空白组比较,UVB组细胞增殖率显著降低（P〈0.01）,细胞中ROS含量和总凋亡率显著升高（P〈0.01）,Caspase-3,Caspase-9 mRNA表达水平显著升高（P〈0.01）;Bcl-2蛋白表达量显著降低（P〈0.01）,Caspase-3,Caspase-9蛋白表达量显著升高（P〈0.01）。与UVB组比较,1×10-7,1×10-6,1×10-5mol·L-1甘草苷＋UVB组细胞增殖率显著升高（P〈0.01）;细胞中ROS含量和总凋亡率显著降低（P〈0.05,P〈0.01）,Caspase-3,Caspase-9 mRNA表达水平显著降低（P〈0.05,P〈0.01）;Bcl-2蛋白表达量显著升高（P〈0.05,P〈0.01）,Caspase-3,Caspase-9蛋白表达量显著降低（P〈0.05,P〈0.01）。结论：甘草苷能通过促进抗凋亡因子Bcl-2蛋白的表达,减少促凋亡相关细胞因子Caspase-3,Caspase-9 mRNA和蛋白的表达,从而抑制UVB诱导的Ha Ca T细胞凋亡。展开更多
Background Trichophyton rubrum （T. rubrum） represents the most important agent of dermatophytosis in humans. T. rubrum infection causes slight inflammation, and tends to be chronic and recurrent. It is suggested tha...Background Trichophyton rubrum （T. rubrum） represents the most important agent of dermatophytosis in humans. T. rubrum infection causes slight inflammation, and tends to be chronic and recurrent. It is suggested that it may result from the failure of epithelial cells to recognize T. rubrum effectively and initiate effective immune responses. The C-type lectin receptors （CLR） and toll-like receptors （TLR） are the two major pattern recognition receptors （PRRs） that recognize fungal components. Therefore, the purpose of the study was to analyze the expression of those PRRs and the cytokines in HaCaT cells stimulated with heat-inactivated T. rubrum conidia and hyphae, respectively. Methods HaCaT cells were unstimulated or stimulated with heat-inactivated T. rubrum conidia and hyphae （l×106 and 1.5×105 colony-forming unit （CFU） in 2 ml medium, respectively） for 6, 12 and 24 hours. The mRNA expression of PRRs involved in recognizing fungal pathogen-associated molecular patterns （PAMPs） and signaling molecules were measured by quantitative reverse transcription polymerase chain reaction （RT-PCR）. Meanwhile, surface toll-like receptor （TLR） 2, TLR4 and Dectin-1 were analyzed by fluorescence-activated cell sorter （FACS） 24 hours after treatment. The cytokines were detected in cell culture supernatants of HaCaT cells in 12 and 24 hours after treatment. Results HaCaT cells constitutively expressed mRNA of membrane-bound TLR1,2, 4 and 6, Dectinl and DC-SIGN, but not Dectin-2 or Mincle. Heat-killed T. rubrum did not significantly upregulate gene transcriptions of the PRRs of HaCaT cells. Heat-inactivated T. rubrum conidia significantly reduced the surface expression of TLR2 and Dectin-1, and suppressed the secretions of interferon-inducible protein-10 （IP-10） and monocyte chemotactic protein-1 （MCP-1） of HaCaT cells, while heat-killed T. rubrum hyphae significantly induced the secretions of IP-10 and MCP-I. Conclusion The cell-wall antigens of T. rubrum fail to activate transc展开更多
文摘目的：研究甘草苷对中波紫外线（UVB）诱导人皮肤角质形成细胞（Ha Ca T）凋亡的影响。方法：以不同浓度的甘草苷作用于Ha Ca T细胞,噻唑蓝（MTT）比色法筛选甘草苷的有效安全浓度;用30 m J·cm-2的UVB作用于Ha Ca T细胞,建立光老化模型,MTT比色法检测光老化Ha Ca T细胞增殖率;流式细胞术检测各组细胞中活性氧自由基（ROS）含量和总凋亡率;实时荧光定量PCR（Real-time PCR）检测光老化Ha Ca T细胞中半胱氨酸天冬氨酸-3（Caspase-3）,半胱氨酸天冬氨酸-9（Caspase-9）mRNA表达水平;蛋白免疫印记法（Western blot）检测光老化Ha Ca T细胞中B淋巴细胞瘤-2（Bcl-2）,Caspase-3,Caspase-9蛋白表达量。结果：筛选出甘草苷的最佳有效安全浓度为1×10-7,1×10-6,1×10-5mol·L-1。与空白组比较,UVB组细胞增殖率显著降低（P〈0.01）,细胞中ROS含量和总凋亡率显著升高（P〈0.01）,Caspase-3,Caspase-9 mRNA表达水平显著升高（P〈0.01）;Bcl-2蛋白表达量显著降低（P〈0.01）,Caspase-3,Caspase-9蛋白表达量显著升高（P〈0.01）。与UVB组比较,1×10-7,1×10-6,1×10-5mol·L-1甘草苷＋UVB组细胞增殖率显著升高（P〈0.01）;细胞中ROS含量和总凋亡率显著降低（P〈0.05,P〈0.01）,Caspase-3,Caspase-9 mRNA表达水平显著降低（P〈0.05,P〈0.01）;Bcl-2蛋白表达量显著升高（P〈0.05,P〈0.01）,Caspase-3,Caspase-9蛋白表达量显著降低（P〈0.05,P〈0.01）。结论：甘草苷能通过促进抗凋亡因子Bcl-2蛋白的表达,减少促凋亡相关细胞因子Caspase-3,Caspase-9 mRNA和蛋白的表达,从而抑制UVB诱导的Ha Ca T细胞凋亡。
基金This Work was supported by the grants from theFundamental Research Funds for the Central Universities （No. 10ykpy04） and the National Natural Science Foundation of China （No. 30600028）.
文摘Background Trichophyton rubrum （T. rubrum） represents the most important agent of dermatophytosis in humans. T. rubrum infection causes slight inflammation, and tends to be chronic and recurrent. It is suggested that it may result from the failure of epithelial cells to recognize T. rubrum effectively and initiate effective immune responses. The C-type lectin receptors （CLR） and toll-like receptors （TLR） are the two major pattern recognition receptors （PRRs） that recognize fungal components. Therefore, the purpose of the study was to analyze the expression of those PRRs and the cytokines in HaCaT cells stimulated with heat-inactivated T. rubrum conidia and hyphae, respectively. Methods HaCaT cells were unstimulated or stimulated with heat-inactivated T. rubrum conidia and hyphae （l×106 and 1.5×105 colony-forming unit （CFU） in 2 ml medium, respectively） for 6, 12 and 24 hours. The mRNA expression of PRRs involved in recognizing fungal pathogen-associated molecular patterns （PAMPs） and signaling molecules were measured by quantitative reverse transcription polymerase chain reaction （RT-PCR）. Meanwhile, surface toll-like receptor （TLR） 2, TLR4 and Dectin-1 were analyzed by fluorescence-activated cell sorter （FACS） 24 hours after treatment. The cytokines were detected in cell culture supernatants of HaCaT cells in 12 and 24 hours after treatment. Results HaCaT cells constitutively expressed mRNA of membrane-bound TLR1,2, 4 and 6, Dectinl and DC-SIGN, but not Dectin-2 or Mincle. Heat-killed T. rubrum did not significantly upregulate gene transcriptions of the PRRs of HaCaT cells. Heat-inactivated T. rubrum conidia significantly reduced the surface expression of TLR2 and Dectin-1, and suppressed the secretions of interferon-inducible protein-10 （IP-10） and monocyte chemotactic protein-1 （MCP-1） of HaCaT cells, while heat-killed T. rubrum hyphae significantly induced the secretions of IP-10 and MCP-I. Conclusion The cell-wall antigens of T. rubrum fail to activate transc
