DATS通过诱导HL-60细胞产生ROS激活JNK的分子机制
发布时间：2018-11-13 16:40
【摘要】： 目的:探讨DATS诱导HL-60细胞JNK活化的分子机制及活性氧在这一过程中的作用。 方法:用浓度为50、100、150μM的DATS处理HL-60细胞0、0.5、1、3、6、12h,流式细胞术检测细胞内的ROS水平。用抗氧化剂N-乙酰半胱氨酸(N-acetyl-L-cysteine,NAC)预孵育HL-60细胞30min后,再用150μM DATS处理HL-60细胞0、0.5、1、3、6、12h,流式细胞术检测细胞内的ROS水平;蛋白印迹法检测JNK、c-Jun、GST-π和JIP的表达及JNK、c-Jun和MLK3的磷酸化水平;免疫共沉淀和蛋白印迹技术分析GST-π与JNK、JIP与JNK的结合情况;用MLK特异性阻滞剂CEP-1347或JNK特异性阻滞剂SP600125预处理后,观察150μM DATS处理细胞3h后,HL-60细胞JNK、c-Jun磷酸化的变化。 结果:不同浓度DATS处理HL-60细胞不同时间后,细胞流式检测显示ROS的产生与DATS处理呈时间剂量依赖关系,在0.5-3h随着DATS处理时间和浓度的升高而升高,ROS的荧光强度在3h、150μM时达到最高峰,值为89.7±3.67,其后维持在一个较高水平。 DATS处理HL-60细胞在12h内,几乎不影响JNK蛋白的表达,c-Jun蛋白表达在前3h内有所增高,随后趋于稳定;JNK的磷酸化水平在DATS处理3h内显著增高,3h达到最高峰,随后稍有下降;c-Jun、MLK3的磷酸化水平随着时间的延长而增加,NAC能不同程度的抑制DATS诱导的c-Jun的表达及JNK、c-Jun的磷酸化;CEP-1347预处理细胞能降低DATS诱导的JNK的磷酸化水平,SP600125预处理细胞能显著降低DATS诱导的c-Jun的磷酸化水平。 DATS处理细胞6h内,几乎不影响GST-π的表达,12h时,DATS能抑制GST-π的表达;DATS处理几乎不影响JIP的表达;DATS处理HL-60细胞0-3h,DATS能诱导GST-π与JNK复合物的解离,3h时GST-π与JNK基本处于解离状态,3h后GST-π重新与JNK结合,NAC能显著抑制DATS诱导的GST-π与JNK复合物的解离;DATS处理HL-60细胞0-3h,DATS能诱导JIP与JNK复合物的形成,3小时以后JIP与JNK复合物逐渐解离。NAC对JIP与JNK复合物的形成无明显影响。 结论: 1. DATS能诱导HL-60细胞产生ROS及MLK3、JNK和c-Jun的磷酸化; 2. DATS诱导产生的ROS能介导MLK3/JNK/c-Jun磷酸化,并促进GST-π-JNK复合物的解离; 3. DATS可通过活化MLK3和解除GST-π对JNK的抑制作用,激活HL-60细胞JNK。
[Abstract]:Aim: to investigate the molecular mechanism of JNK activation induced by DATS in HL-60 cells and the role of reactive oxygen species in this process. Methods: HL-60 cells were treated with 50100150 渭 M DATS for 6 and 12 hours, and the level of ROS was detected by flow cytometry. HL-60 cells were preincubated with N-acetyl-L-cysteine (N-acetyl-L-cysteine NAC) and then treated with 150 渭 M DATS for 12 hours. The ROS level was detected by flow cytometry. The expression of JNK,c-Jun,GST- 蟺 and JIP and the phosphorylation of JNK,c-Jun and MLK3 were detected by Western blot, and the binding of GST- 蟺 to JNK,JIP and JNK was analyzed by immunoprecipitation and Western blotting. After pretreatment with MLK specific blocker CEP-1347 or JNK specific blocker SP600125, the phosphorylation of JNK,c-Jun in HL-60 cells was observed after treated with 150 渭 M DATS for 3 h. Results: after HL-60 cells were treated with different concentrations of DATS for different time, flow cytometry showed that the production of ROS was in a time-dose dependent manner with DATS treatment, and increased with the increase of DATS treatment time and concentration at 0.5-3h. The fluorescence intensity of ROS reached its peak at 150 渭 M (89.7 卤3.67) and remained at a high level. HL-60 cells treated with DATS for 12 h had little effect on the expression of JNK protein, but the expression of c-Jun protein increased in the first 3 hours and then became stable. The phosphorylation level of JNK increased significantly within 3 h of DATS treatment, reached its peak at 3 h, and then decreased slightly. The phosphorylation level of c-JunjianMLK3 increased with time. NAC could inhibit the expression of c-Jun and the phosphorylation of JNK,c-Jun induced by DATS to some extent. CEP-1347 pretreatment could decrease the phosphorylation level of JNK induced by DATS, while SP600125 pretreatment could significantly decrease the phosphorylation level of c-Jun induced by DATS. The expression of GST- 蟺 was almost unchanged in the cells treated with DATS for 6 h, DATS could inhibit the expression of GST- 蟺 at 12 h, and DATS treatment had little effect on the expression of JIP. HL-60 cells treated with DATS for 0-3 h could induce the dissociation of GST- 蟺 and JNK complex, GST- 蟺 and JNK were basically dissociated at 3 h, GST- 蟺 recombined with JNK 3 h later. NAC could significantly inhibit the dissociation of GST- 蟺 and JNK complexes induced by DATS. HL-60 cells treated with DATS for 0-3 h could induce the formation of JIP and JNK complex, and JIP and JNK complex dissociated gradually after 3 hours. NAC had no significant effect on the formation of JIP and JNK complex. Conclusion: 1. DATS could induce the phosphorylation of ROS, MLK3,JNK and c-Jun in HL-60 cells. ROS induced by DATS can mediate the phosphorylation of MLK3/JNK/c-Jun and promote the dissociation of GST- 蟺-JNK complex. 3. DATS can activate the JNK. of HL-60 cells by activating MLK3 and removing the inhibition of JNK by GST- 蟺.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R363
本文编号：2329713
[Abstract]:Aim: to investigate the molecular mechanism of JNK activation induced by DATS in HL-60 cells and the role of reactive oxygen species in this process. Methods: HL-60 cells were treated with 50100150 渭 M DATS for 6 and 12 hours, and the level of ROS was detected by flow cytometry. HL-60 cells were preincubated with N-acetyl-L-cysteine (N-acetyl-L-cysteine NAC) and then treated with 150 渭 M DATS for 12 hours. The ROS level was detected by flow cytometry. The expression of JNK,c-Jun,GST- 蟺 and JIP and the phosphorylation of JNK,c-Jun and MLK3 were detected by Western blot, and the binding of GST- 蟺 to JNK,JIP and JNK was analyzed by immunoprecipitation and Western blotting. After pretreatment with MLK specific blocker CEP-1347 or JNK specific blocker SP600125, the phosphorylation of JNK,c-Jun in HL-60 cells was observed after treated with 150 渭 M DATS for 3 h. Results: after HL-60 cells were treated with different concentrations of DATS for different time, flow cytometry showed that the production of ROS was in a time-dose dependent manner with DATS treatment, and increased with the increase of DATS treatment time and concentration at 0.5-3h. The fluorescence intensity of ROS reached its peak at 150 渭 M (89.7 卤3.67) and remained at a high level. HL-60 cells treated with DATS for 12 h had little effect on the expression of JNK protein, but the expression of c-Jun protein increased in the first 3 hours and then became stable. The phosphorylation level of JNK increased significantly within 3 h of DATS treatment, reached its peak at 3 h, and then decreased slightly. The phosphorylation level of c-JunjianMLK3 increased with time. NAC could inhibit the expression of c-Jun and the phosphorylation of JNK,c-Jun induced by DATS to some extent. CEP-1347 pretreatment could decrease the phosphorylation level of JNK induced by DATS, while SP600125 pretreatment could significantly decrease the phosphorylation level of c-Jun induced by DATS. The expression of GST- 蟺 was almost unchanged in the cells treated with DATS for 6 h, DATS could inhibit the expression of GST- 蟺 at 12 h, and DATS treatment had little effect on the expression of JIP. HL-60 cells treated with DATS for 0-3 h could induce the dissociation of GST- 蟺 and JNK complex, GST- 蟺 and JNK were basically dissociated at 3 h, GST- 蟺 recombined with JNK 3 h later. NAC could significantly inhibit the dissociation of GST- 蟺 and JNK complexes induced by DATS. HL-60 cells treated with DATS for 0-3 h could induce the formation of JIP and JNK complex, and JIP and JNK complex dissociated gradually after 3 hours. NAC had no significant effect on the formation of JIP and JNK complex. Conclusion: 1. DATS could induce the phosphorylation of ROS, MLK3,JNK and c-Jun in HL-60 cells. ROS induced by DATS can mediate the phosphorylation of MLK3/JNK/c-Jun and promote the dissociation of GST- 蟺-JNK complex. 3. DATS can activate the JNK. of HL-60 cells by activating MLK3 and removing the inhibition of JNK by GST- 蟺.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R363
【参考文献】
相关期刊论文 前2条
1 肖晓岚,向姝霖,唐国华,周秀田,谢娜,葛玲,苏琦;二烯丙基三硫对MGC-803细胞凋亡的影响[J];南华大学学报(医学版);2005年02期
2 姚淑琼;刘丽娟;黄泽香;雷小勇;朱炳阳;唐圣松;廖端芳;;二烯丙基二硫诱导人乳腺癌MCF-7细胞凋亡及机制的研究[J];现代生物医学进展;2007年03期
，本文编号：2329713
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2329713.html
最近更新
教材专著
