Objective To investigate the main proteinases responsible for CD16b shedding under different stimulators. Methods HEK293 cell line stably expressing CD16b was constructed by lentivirus system. The cell line was then o...Objective To investigate the main proteinases responsible for CD16b shedding under different stimulators. Methods HEK293 cell line stably expressing CD16b was constructed by lentivirus system. The cell line was then overexpressed with a disintegrin and metalloproteinase 10 (ADAM10) or ADAM17, suppressed with short hairpin RNA of ADAM10 or ADAM17, and reconstituted with ADAM10 or ADAM17, respectively. After each treatment, the cell line was stimulated with ionomycin or phorbol 12-myristate13-acetate (PMA) for 12 hours. The soluble CD16b released from cell membrane was detected by immunoprecipition and immunoblot. Quantitation was then implemented to compare the amount of soluble CD16b in cell supernatant after stimulation. Results HEK293 cell line stably expressing CD16b was successfully established. When CD16b expressing cell line was overexpressed with ADAM10, shedding of CD16b was increased after stimulation with ionomycin but not PMA; when the cell line overexpressed with ADAM17, shedding of CD16b was increased after stimulation with PMA but not ionomycin. Similarly, when ADAM10 was suppressed by short hairpin RNA, CD16b shedding was decreased after stimulation with ionomycin; when ADAM17 was suppressed by short hairpin RNA, CD16b shedding was decreased after stimulation with PMA. The shedding of CD16b was increased again when CD16b expressing cell line was reconstituted with ADAM10 and stimulated by ionomycin or reconstituted with ADAM17 and stimulated by PMA. Conclusions Both ADAM10 and ADAM17 could shed CD16b, but they possess differed preferences. ADAM10 is the main sheddase under stimulation of ionomycin, while ADAM17 is the main sheddase under stimulation of PMA.展开更多
目的探讨miR-25-3p靶向解整合素金属蛋白酶10(ADAM10)调控Notch信号通路对P19细胞向心肌细胞分化的影响。方法采用二甲基亚砜(DMSO)诱导P19细胞向心肌细胞分化,分别收集诱导分化第0、5、10天的细胞,实时荧光定量PCR检测诱导分化过程中...目的探讨miR-25-3p靶向解整合素金属蛋白酶10(ADAM10)调控Notch信号通路对P19细胞向心肌细胞分化的影响。方法采用二甲基亚砜(DMSO)诱导P19细胞向心肌细胞分化,分别收集诱导分化第0、5、10天的细胞,实时荧光定量PCR检测诱导分化过程中心肌分化标志物GATA结合蛋白4(GATA4)、心肌肌钙蛋白T(cTnT)、心房利钠尿多肽(ANP)的mRNA以及miR-25-3p水平, Western blot法检测诱导分化过程中ADAM10蛋白水平。通过逆转录病毒感染P19细胞过表达miR-25-3p,采用实时荧光定量PCR检测感染后P19细胞中miR-25-3p水平, Western blot法检测感染后P19细胞ADAM10蛋白水平。生物信息学软件预测并通过荧光素酶报告实验验证miR-25-3p与ADAM10之间的靶向结合作用。感染后的P19细胞经DMSO诱导分化10d后,免疫荧光实验检测细胞中心肌肌钙蛋白I(cTnI)蛋白的表达情况,并计算心肌细胞分化率;Western blot法检测GATA4、cTnT、ANP以及Notch信号通路相关蛋白Notch1、Hes家族bHLH转录因子1(Hes1)、Hairy相关转录因子1(Hey1)和Hey2蛋白水平。结果P19细胞向心肌细胞诱导分化的第0、5、10天过程中, GATA4、cTnT、ANP的mRNA水平及ADAM10蛋白水平均逐渐增加,而miR-25-3p水平逐渐降低;逆转录病毒感染后, P19细胞中miR-25-3p水平显著增加,而ADAM10蛋白水平显著降低;生物信息学分析证实ADAM10为miR-25-3p的靶基因;诱导分化10 d后,过表达miR-25-3p可显著降低心肌细胞分化率,下调GATA4、cTnT、ANP及Notch信号通路相关分子Notch1、Hes1、Hey1和Hey2的蛋白水平。结论miR-25-3p可显著抑制P19细胞向心肌细胞分化,其机制可能与其靶向抑制ADAM10表达,进而抑制Notch信号通路的激活有关。展开更多
目的探讨白介素-4(IL-4)和白介素-13(IL-13)对人肺成纤维细胞ADAM33(a disintegrin and a metal-loproteinase33,ADAM33)mRNA表达的影响。方法以不同浓度的IL-4或/和IL-13刺激培养的人肺成纤维细胞(MRC-5),然后用实时定量反转录多聚酶...目的探讨白介素-4(IL-4)和白介素-13(IL-13)对人肺成纤维细胞ADAM33(a disintegrin and a metal-loproteinase33,ADAM33)mRNA表达的影响。方法以不同浓度的IL-4或/和IL-13刺激培养的人肺成纤维细胞(MRC-5),然后用实时定量反转录多聚酶链反应(real-time RT-PCR)法测定ADAM33mRNA表达的变化。结果人肺成纤维细胞上存在着ADAM33mRNA的表达,当分别以10ng/mL的IL-4或IL-13刺激时,AD-AM33mRNA的表达增加呈现时间依赖性,至24h达到高峰。随着IL-4和IL-13刺激浓度的增加,ADAM33mRNA的表达也明显增加:以100ng/mL的IL-4和100ng/mL的IL-13刺激时,ADAM33mRNA的表达较未刺激细胞分别增加了(9.49±2.83)倍和(14.21±3.35)倍(P<0.01);而以10ng/mL的IL-4和10ng/mL的IL-13联合刺激时,ADAM33mRNA的表达较未刺激细胞增加了(15.06±4.57)倍(P<0.01)。结论IL-4和IL-13能明显促进肺成纤维细胞ADAM33mRNA的表达。展开更多
基金Supported by the National Natural Science Foundation of China (30872287)
文摘Objective To investigate the main proteinases responsible for CD16b shedding under different stimulators. Methods HEK293 cell line stably expressing CD16b was constructed by lentivirus system. The cell line was then overexpressed with a disintegrin and metalloproteinase 10 (ADAM10) or ADAM17, suppressed with short hairpin RNA of ADAM10 or ADAM17, and reconstituted with ADAM10 or ADAM17, respectively. After each treatment, the cell line was stimulated with ionomycin or phorbol 12-myristate13-acetate (PMA) for 12 hours. The soluble CD16b released from cell membrane was detected by immunoprecipition and immunoblot. Quantitation was then implemented to compare the amount of soluble CD16b in cell supernatant after stimulation. Results HEK293 cell line stably expressing CD16b was successfully established. When CD16b expressing cell line was overexpressed with ADAM10, shedding of CD16b was increased after stimulation with ionomycin but not PMA; when the cell line overexpressed with ADAM17, shedding of CD16b was increased after stimulation with PMA but not ionomycin. Similarly, when ADAM10 was suppressed by short hairpin RNA, CD16b shedding was decreased after stimulation with ionomycin; when ADAM17 was suppressed by short hairpin RNA, CD16b shedding was decreased after stimulation with PMA. The shedding of CD16b was increased again when CD16b expressing cell line was reconstituted with ADAM10 and stimulated by ionomycin or reconstituted with ADAM17 and stimulated by PMA. Conclusions Both ADAM10 and ADAM17 could shed CD16b, but they possess differed preferences. ADAM10 is the main sheddase under stimulation of ionomycin, while ADAM17 is the main sheddase under stimulation of PMA.
文摘目的探讨miR-25-3p靶向解整合素金属蛋白酶10(ADAM10)调控Notch信号通路对P19细胞向心肌细胞分化的影响。方法采用二甲基亚砜(DMSO)诱导P19细胞向心肌细胞分化,分别收集诱导分化第0、5、10天的细胞,实时荧光定量PCR检测诱导分化过程中心肌分化标志物GATA结合蛋白4(GATA4)、心肌肌钙蛋白T(cTnT)、心房利钠尿多肽(ANP)的mRNA以及miR-25-3p水平, Western blot法检测诱导分化过程中ADAM10蛋白水平。通过逆转录病毒感染P19细胞过表达miR-25-3p,采用实时荧光定量PCR检测感染后P19细胞中miR-25-3p水平, Western blot法检测感染后P19细胞ADAM10蛋白水平。生物信息学软件预测并通过荧光素酶报告实验验证miR-25-3p与ADAM10之间的靶向结合作用。感染后的P19细胞经DMSO诱导分化10d后,免疫荧光实验检测细胞中心肌肌钙蛋白I(cTnI)蛋白的表达情况,并计算心肌细胞分化率;Western blot法检测GATA4、cTnT、ANP以及Notch信号通路相关蛋白Notch1、Hes家族bHLH转录因子1(Hes1)、Hairy相关转录因子1(Hey1)和Hey2蛋白水平。结果P19细胞向心肌细胞诱导分化的第0、5、10天过程中, GATA4、cTnT、ANP的mRNA水平及ADAM10蛋白水平均逐渐增加,而miR-25-3p水平逐渐降低;逆转录病毒感染后, P19细胞中miR-25-3p水平显著增加,而ADAM10蛋白水平显著降低;生物信息学分析证实ADAM10为miR-25-3p的靶基因;诱导分化10 d后,过表达miR-25-3p可显著降低心肌细胞分化率,下调GATA4、cTnT、ANP及Notch信号通路相关分子Notch1、Hes1、Hey1和Hey2的蛋白水平。结论miR-25-3p可显著抑制P19细胞向心肌细胞分化,其机制可能与其靶向抑制ADAM10表达,进而抑制Notch信号通路的激活有关。
文摘目的探讨白介素-4(IL-4)和白介素-13(IL-13)对人肺成纤维细胞ADAM33(a disintegrin and a metal-loproteinase33,ADAM33)mRNA表达的影响。方法以不同浓度的IL-4或/和IL-13刺激培养的人肺成纤维细胞(MRC-5),然后用实时定量反转录多聚酶链反应(real-time RT-PCR)法测定ADAM33mRNA表达的变化。结果人肺成纤维细胞上存在着ADAM33mRNA的表达,当分别以10ng/mL的IL-4或IL-13刺激时,AD-AM33mRNA的表达增加呈现时间依赖性,至24h达到高峰。随着IL-4和IL-13刺激浓度的增加,ADAM33mRNA的表达也明显增加:以100ng/mL的IL-4和100ng/mL的IL-13刺激时,ADAM33mRNA的表达较未刺激细胞分别增加了(9.49±2.83)倍和(14.21±3.35)倍(P<0.01);而以10ng/mL的IL-4和10ng/mL的IL-13联合刺激时,ADAM33mRNA的表达较未刺激细胞增加了(15.06±4.57)倍(P<0.01)。结论IL-4和IL-13能明显促进肺成纤维细胞ADAM33mRNA的表达。