DATS通過(guò)誘導(dǎo)HL-60細(xì)胞產(chǎn)生ROS激活JNK的分子機(jī)制
發(fā)布時(shí)間：2018-11-13 16:40
【摘要】： 目的:探討DATS誘導(dǎo)HL-60細(xì)胞JNK活化的分子機(jī)制及活性氧在這一過(guò)程中的作用。 方法:用濃度為50、100、150μM的DATS處理HL-60細(xì)胞0、0.5、1、3、6、12h,流式細(xì)胞術(shù)檢測(cè)細(xì)胞內(nèi)的ROS水平。用抗氧化劑N-乙酰半胱氨酸(N-acetyl-L-cysteine,NAC)預(yù)孵育HL-60細(xì)胞30min后,再用150μM DATS處理HL-60細(xì)胞0、0.5、1、3、6、12h,流式細(xì)胞術(shù)檢測(cè)細(xì)胞內(nèi)的ROS水平;蛋白印跡法檢測(cè)JNK、c-Jun、GST-π和JIP的表達(dá)及JNK、c-Jun和MLK3的磷酸化水平;免疫共沉淀和蛋白印跡技術(shù)分析GST-π與JNK、JIP與JNK的結(jié)合情況;用MLK特異性阻滯劑CEP-1347或JNK特異性阻滯劑SP600125預(yù)處理后,觀察150μM DATS處理細(xì)胞3h后,HL-60細(xì)胞JNK、c-Jun磷酸化的變化。 結(jié)果:不同濃度DATS處理HL-60細(xì)胞不同時(shí)間后,細(xì)胞流式檢測(cè)顯示ROS的產(chǎn)生與DATS處理呈時(shí)間劑量依賴關(guān)系,在0.5-3h隨著DATS處理時(shí)間和濃度的升高而升高,ROS的熒光強(qiáng)度在3h、150μM時(shí)達(dá)到最高峰,值為89.7±3.67,其后維持在一個(gè)較高水平。 DATS處理HL-60細(xì)胞在12h內(nèi),幾乎不影響JNK蛋白的表達(dá),c-Jun蛋白表達(dá)在前3h內(nèi)有所增高,隨后趨于穩(wěn)定;JNK的磷酸化水平在DATS處理3h內(nèi)顯著增高,3h達(dá)到最高峰,隨后稍有下降;c-Jun、MLK3的磷酸化水平隨著時(shí)間的延長(zhǎng)而增加,NAC能不同程度的抑制DATS誘導(dǎo)的c-Jun的表達(dá)及JNK、c-Jun的磷酸化;CEP-1347預(yù)處理細(xì)胞能降低DATS誘導(dǎo)的JNK的磷酸化水平,SP600125預(yù)處理細(xì)胞能顯著降低DATS誘導(dǎo)的c-Jun的磷酸化水平。 DATS處理細(xì)胞6h內(nèi),幾乎不影響GST-π的表達(dá),12h時(shí),DATS能抑制GST-π的表達(dá);DATS處理幾乎不影響JIP的表達(dá);DATS處理HL-60細(xì)胞0-3h,DATS能誘導(dǎo)GST-π與JNK復(fù)合物的解離,3h時(shí)GST-π與JNK基本處于解離狀態(tài),3h后GST-π重新與JNK結(jié)合,NAC能顯著抑制DATS誘導(dǎo)的GST-π與JNK復(fù)合物的解離;DATS處理HL-60細(xì)胞0-3h,DATS能誘導(dǎo)JIP與JNK復(fù)合物的形成,3小時(shí)以后JIP與JNK復(fù)合物逐漸解離。NAC對(duì)JIP與JNK復(fù)合物的形成無(wú)明顯影響。 結(jié)論: 1. DATS能誘導(dǎo)HL-60細(xì)胞產(chǎn)生ROS及MLK3、JNK和c-Jun的磷酸化; 2. DATS誘導(dǎo)產(chǎn)生的ROS能介導(dǎo)MLK3/JNK/c-Jun磷酸化,并促進(jìn)GST-π-JNK復(fù)合物的解離; 3. DATS可通過(guò)活化MLK3和解除GST-π對(duì)JNK的抑制作用,激活HL-60細(xì)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 鹵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.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R363
本文編號(hào)：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 鹵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.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R363
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，本文編號(hào)：2329713
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