干扰和过表达热休克转录因子2对肠上皮细胞凋亡和迁移的影响  

Effect of knockdown or overexpression of heat shock transcription factor 2 on apoptosis and migration of human colonic epithelial cells

作　　者：杨刚[1] 牛俊坤[1] 李小玉[1] 张峰睿[1] 缪应雷[1,2] 

机构地区：[1]昆明医科大学第一附属医院消化内科,云南省昆明市650032 [2]云南省消化疾病研究所,云南省昆明市650032

出　　处：《世界华人消化杂志》2015年第24期3846-3859,共14页World Chinese Journal of Digestology

基　　金：国家自然科字基金资助项目;Nos;81160055;81260074;云南省科技厅-昆明医学院联合专项基金资助项目;Nos2011FB183;2007C0010R;云南省卫生厅卫生系统学科带头人培养计划基金资助项目;No.D-201215;云南省社会发展科技计划基金资助项目;No.2013CA021~~

摘　　要：目的:通过慢病毒载体介导的RNA干扰技术过表达和干扰肠上皮细胞热休克转录因子2(heat shock transcription factor 2,HSF2)表达,研究不同HSF2水平对肠上皮细胞凋亡和迁移的影响.方法:(1)选取HT-29(人结肠癌细胞株)作为研究材料,丁酸钠(设置不同时间点/浓度)诱导细胞凋亡,MTT法检测细胞增殖活性,流式细胞仪检测细胞凋亡、周期.按实验结果选取最佳丁酸钠浓度及时间位点;(2)慢病毒载体介导的RNA干扰技术过表达和干扰结肠上皮细胞HSF2表达;用倒置荧光显微镜、Western blot法检测目的基因在细胞中的转染效率;(3)细胞迁移实验(划痕法/Transwell小室)检测过表达和干扰HSF2后细胞迁移能力的改变;(4)经过表达和干扰HSF2后的细胞,给予丁酸钠诱导凋亡,MTT法检测细胞增殖活性,流式细胞仪检测细胞凋亡率、细胞周期.结果:(1)MTT实验结果显示:阴性对照组(NC组)的HT-29细胞增殖活性在实验观察期间(0-96 h)不断升高,48 h后,实验组的细胞对4个丁酸钠浓度组均表现出明显的生长抑制,相对于NC组之间差异具有统计学意义(P<0.01),故丁酸钠对HT-29细胞的生长抑制作用呈浓度、时间依赖性.流式细胞仪检测细胞凋亡率结果显示:NC组有少量凋亡,总凋亡率为0.548%±0.113%,而实验组经4个不同浓度丁酸钠处理48 h后,总凋亡率分别为51.588%±5.110%、77.732%±2.746%、90.115%±1.438%、94.247%±1.243%,与NC组之间差异具有统计学意义(P<0.01).而当丁酸钠浓度>2.5 mmol/L,凋亡率较1.25 mmol/L明显上升.细胞周期检测结果显示:与NC相比,1.25 mmol/L丁酸钠处理HT-29细胞48 h,G0/G1期HT-29细胞未显示出明显差异(P=1.00>0.05),即无明显细胞周期阻滞;而2.5、5.0、10 mmol/L以上浓度在各观察点均出现明显的细胞周期阻滞现象,呈明显的G1/G0期阻滞(P<0.01);(2)慢病毒载体转染效果检测:通过倒置荧光显微镜(×100)观察可发现,各组HT-29细胞经慢病毒载体转染后均�AIM： To explore the role of heat shock transcription factor 2（HSF2） in cell apoptosis and migration in human colonic epithelial cell line HT-29 by means of interference and overexpression. METHODS： Apoptosis of HT-29 cells was induced by incubation with sodium butyrate（SB） for different durations. Cytotoxicity was estimated by MTT assay, and the cell cycle and apoptosis were observed by flow cytometry to choose the optimal time and concentration of SB. HT-29 cells were then transfected with HSF2 si RNA or a lentiviral vector（Ubi-MCS-3FLAG-SV40-EGFP）. The overexpression or knockdown of HSF2 was detected by Leica DMIRB and Western blot. After transfection, cell migration ability was measured by wound healing assay and Transwell assay. Apoptosis of HT-29 cells was induced with SB after transfection, cell proliferation was studied by MTT assay, and cell cycle and apoptosis were observed by fl ow cytometry.RESULTS： Compared with the negative control（NC） group, SB at 2.5, 5.0, or 10 mmol/L could signif icantly cause growth inhibition after 48 hof incubation（P 〈0.01）, and the effect was timeand dose-dependent. The apoptosis rate was significantly higher in the SB treated groups（1.25, 2.5, 5.0, or 10 mmol/L for 48 h） than in the NC group（51.588% ± 5.110%, 77.732% ± 2.746%,90.115% ± 1.438%, 94.247% ± 1.243% vs 0.548%± 0.113%, P 〈0.01）. When the SB concentration was 〉2.5 mmol/L, the apoptosis rate increased signif icantly（P〈 0.01）. When treated with 1.25mmol/L SB for 48 h, the percentage of cells in G0/G1 phase cell did not show a signifi cant difference compared with the NC group; when the concentration was〉 2.5 mmol/L（5.0 or10.0 mmol/L）, SB could induce G1/G0 arrest（P〈 0.01）. After lentiviral transfection, a large number of HT-29 cells with green f luorescence was observed by Leica DMIRB（transfection efficiency 〉80%）. Lentiviral transfection of si RNA could effectively inhibit expression of HSF2, while lentiviral transfection of Ubi-MCS-3FLAG-SV40-EGFP

关 键 词：热休克转录因子2 RNA干扰 过表达 HT-29细胞 凋亡 迁移 

分 类 号：R574[医药卫生—消化系统]