细胞外组蛋白H4对淋巴细胞凋亡的影响及其与MAPKs信号通路的调控关系  

Effect of extracellular histone H4 on apoptosis of lymphocytes and its regulatory relationship with MAPKs signaling pathway

作　　者：骆婷婷 张瑞[1,2] 王志远 陈双[1,2] LUO Tingting;ZHANG Rui;WANG Zhiyuan;CHEN Shuang(Hematological Center,The First Affiliated Hospital of Xinjiang Medical University,Urumqi 830011,China)

机构地区：[1]新疆医科大学第一附属医院血液病中心,乌鲁木齐830011 [2]新疆维吾尔自治区血液病研究所 [3]新疆维吾尔自治区人民医院消化内科

出　　处：《山东医药》2023年第17期1-5,共5页Shandong Medical Journal

基　　金：新疆维吾尔自治区自然科学基金青年项目(2021D01C206);新疆维吾尔自治区自然科学基金医学联合基金项目(2015211C037)。

摘　　要：目的探讨细胞外组蛋白H4对小鼠淋巴细胞Raw264.7细胞凋亡的影响及其与促分裂素原活化蛋白激酶(MAPKs)信号通路的调控关系。方法收集Raw264.7细胞分为低浓度刺激组、高浓度刺激组及对照组,分别加入50μg/m L、100μg/m L组蛋白H4及不做任何处理,采用FCM法检测各组细胞凋亡率,选取既能诱导细胞凋亡又具有较低细胞毒性的组蛋白刺激浓度进行后续实验。组蛋白H4在去除脂多糖污染后,使用筛选浓度刺激Raw264.7细胞,分别在刺激开始0、5、10、30、60、120 min收取细胞,Western blotting法检测细胞磷酸化ERK(p-ERK)、p-JNK、p-p38蛋白表达。分别使用ERK通路抑制剂、JNK通路抑制剂、p38通路抑制剂、DMSO细胞冻存液预处理Raw264.7细胞1 h,作为ERK抑制组、JNK抑制组、p38抑制组、阴性对照组,另设空白对照组正常培养不做处理,每组分别给予或不给予筛选浓度的组蛋白刺激,采用FCM法检测各组给予或不给予组蛋白刺激的细胞凋亡率。结果低浓度刺激组、高浓度刺激组细胞凋亡率均高于对照组(P均<0.05),低浓度刺激组、高浓度刺激组细胞凋亡率比较差异无统计学意义,但高浓度刺激组晚期细胞凋亡率及死亡率较低浓度刺激组增高(P均<0.05)。考虑到细胞毒性,选取50μg/m L作为组蛋白H4的刺激浓度。p-ERK蛋白表达在组蛋白刺激10 min及30 min时均高于0 min时,且在30 min时达到高峰,在60 min及120 min时降至初始水平;p-JNK蛋白表达在组蛋白刺激10 min及30 min时均高于0 min时,且在10 min时达到高峰,在60 min及120 min时降至初始水平(P均<0.05);p-p38蛋白表达在各时点差异均无统计学意义。空白对照组、阴性对照组组蛋白刺激的Raw264.7细胞凋亡率均高于没有组蛋白刺激的细胞(P均<0.05),ERK1、JNK及p38抑制组组蛋白刺激、不刺激的细胞凋亡率比较差异均无统计学意义。结论细胞外组蛋白H4刺激可促进淋巴细胞凋亡,其作用可能Objective To investigate the influence of extracellular matrix protein H4 on the apoptosis of mouse lymphocytes,and to explore its regulatory relationship with the mitogen-activated protein kinase(MAPKs)signaling pathway.Methods Raw264.7 cells were collected and divided into three groups:the low-concentration stimulation group which was treated with 50μg/mL of histone H4,the high-concentration stimulation group which was treated with 100μg/mL of histone H4,and the control group which received no treatment.The apoptosis rate of each group was subsequently measured using the flow cytometry(FCM).For further experiment,the concentration of matrix protein stimulation that both induced apoptosis and maintained relatively low cytotoxicity was selected.After removing lipopolysaccharide contamination,histone H4 was used at the predetermined concentration to stimulate Raw264.7 cells.Cells were harvested at specific intervals:0,5,10,30,60,and 120 minutes following the onset of stimulation.The expression of phosphorylated ERK(p-ERK),p-JNK,and p-p38 proteins was detected by Western blotting.Raw264.7 cells were separately pre-treated with inhibitors for the ERK,JNK,and p38 pathways,and with DMSO cell freezing medium,which were taken as the ERK inhibition group,JNK inhibition group,p38 inhibition group,and negative control group,respectively.Additionally,a blank control group was established and was not treated.Each group was then either subjected to,or spared from histone stimulation.The apoptosis rate of cells in each group was detected by FCM.Results The apoptosis rates of the low-and highconcentration stimulation groups were significantly higher than that of the control group(both P<0.05).There was no significant difference in the apoptosis rate between the low-and high-concentration stimulation groups,but the rate of latestage apoptosis and death were significantly higher in the high-concentration stimulation group that those of the low-concentration stimulation group(both P<0.05).Considering cytotoxicity,50μg/mL was se

关 键 词：组蛋白 细胞凋亡 促分裂素原活化蛋白激酶 MAPKS信号通路 

分 类 号：R364.5[医药卫生—病理学]