热疗联合旋毛虫肌幼虫排泄分泌蛋白对小细胞肺癌H446凋亡及S期阻滞的影响  

Study on the apoptosis and S phase arrest of NCI-H446 small cell lung cancer cells by hyperthermia combined with Trichinella spiralis muscle larva excretory-secretory products

作　　者：吴合亮 安子熙 邵薪诺 李美辰 于莉 阴皓锋 李世昌 杜晶晶 潘靖丹 张嘉琦 李灿 张银广 杜娈英[1] WU He-liang;AN Zi-xi;SHAO Xin-nuo;LI Mei-chen;YU Li;YIN Hao-feng;LI Shi-chang;DU Jing-jing;PAN Jing-dan;ZHAN Jia-qi;LI Can;ZHANG Yin-guang;DU Luan-ying(Department of Pathogen Biology,Chengde Medical University,Chengde,China 067000;Department of Infection Management,Chifeng University Hospital)

机构地区：[1]承德医学院病原生物学教研室,河北承德067000 [2]赤峰学院附属医院感染管理科

出　　处：《中国病原生物学杂志》2019年第11期1250-1254,1260,共6页Journal of Pathogen Biology

基　　金：河北省教育厅重点项目(No.ZD2014049);河北省高校重点学科项目(冀教高[2013]4号);承德医学院重点项目(No.201709);国家级大学生创新创业训练计划项目(No.201704);省级大学生创新创业训练计划项目(No.201716);省级大学生创新创业训练计划项目(No.2018020)

摘　　要：目的观察热疗联合旋毛虫肌幼虫排泄分泌蛋白对小细胞肺癌H446凋亡及周期阻滞的影响。方法将对数生长期的H446细胞随机分为4组。对照组:H446细胞培养过程中不作任何处理;热处理组:将细胞消化后放置离心管中42℃水浴加热2 h;ESPs处理组:将提取的旋毛虫肌幼虫ESPs(0.30 mg/ml)与H446细胞共培养;热处理联合ESPs组:先经水浴处理后加入ESPs(0.30 mg/ml)与H446细胞共培养。采用流式细胞术(FCM)检测各组细胞凋亡率,分析细胞周期变化情况;采用Western blot法检测各组细胞凋亡相关蛋白Bax、Bcl-2、周期蛋白Cyclin D1、p27的表达情况。结果流式细胞术(FCM)检测显示,对照组细胞总凋亡率为11.39%,ESPs处理组细胞总凋亡率为15.94%,热处理组细胞总凋亡率为22.35%,热疗联合ESPs组细胞总凋亡率为29.32%。组间细胞凋亡率差异有统计学意义(P<0.01)。对照组S期细胞所占比例为27.01%, ESPs处理组S期细胞占39.83%,热处理组S期细胞占51.54%,热疗联合ESPs组S期细胞占51.83%。ESPs处理组、热处理组、热疗联合ESPs组S期细胞所占比例与对照组比较差异均有统计学意义(P<0.01)。Western blot检测热处理联合ESPs组与对照组比较Bax、Cyclin D1、p27蛋白表达上调,Bcl-2蛋白表达下调。结论热疗联合旋毛虫肌幼虫排泄分泌蛋白可诱导小细胞肺癌H446凋亡,存在S期细胞阻滞,可能与Bax、Cyclin D1、p27蛋白表达上调,Bcl-2蛋白表达下调有关。Objective To explore the effects of hyperthermia combined with Trichinella spiralis muscle larva(ML)excretory-secretory products(ESPs)on apoptosis and cell cycle arrest of NCI-H446 small cell lung cancer cells.Methods Logarithmically growing NCI-H446 cells were divided into a control group(group A),a hyperthermia treatment group(group B),an ESP treatment group(group C),and combined hyperthermia and ESP treatment group(group D).After digestion,the cells in group B were placed in a centrifuge tube and heated in a 42℃water bath for 2 h.The cells in group C were cultured with 0.30 mg/ml ESPs.The cells in group D were placed in a water bath before treatment with ESPs.The cells in group A were cultured on serum-free RPMI 1640 medium.After each group was cultured for 24 h,flow cytometry was used to detect the rate of apoptosis and the arrest of the cell cycle.Western blotting was used to detect changes in the apoptosis-related genes Bax and Bcl-2 and cell cycle genes Cyclin D1 and p27 in each group.Results Flow cytometry indicated apoptosis of NCI-H446 cells treated with hyperthermia and ESPs for 24 h.The rate of apoptosis was 11.39%in group A,15.94%in group B,22.35%in group C,and 29.32%in group D.The rate of apoptosis differed significant among the groups(P<0.01).Flow cytometry indicated that cells were arrested in the S phase by hyperthermia and ESPs.Twenty-seven-point-one percent of cells in group A were in the S-phase,39.83%of cells in group B were in the S-phase,51.54%of cells in group C were in the S-phase,and 51.83%of cells in group D were in the S-phase.Groups B,C,and D had a significantly higher proportion of cells in the S-phase than group A(P<0.01).Western blotting indicated increased expression of the genes Bax,Cyclin D1,and p27 compared to levels of expression in the control group and decreased expression of the gene Bcl-2.Conclusion Results suggested that hyperthermia and ESPs can induce apoptosis and S-phase arrest in the NCI-H446 small cell lung cancer line.This may be related to up-regulation of Bax,C

关 键 词：旋毛虫 排泄分泌蛋白 凋亡 热疗 小细胞肺癌 

分 类 号：R383.15[医药卫生—医学寄生虫学]