脂多糖对小鼠肺成纤维细胞增殖和P27蛋白表达的影响  

Effect of lipopolysaccharide on the proliferation of mouse lung fibroblast and the expression of P27 protein

作　　者：顾楠楠 邢顺鹏[2] 陈思涵[1] 周雨曦 蒋涛 焦英甫[1] 皋源[2] 俞卫峰 何征宇[2] 杭燕南[1] 闻大翔[1] GU Nannan;XING Shunpeng;OHEN Sihan;ZHOU Yuxi;JIANG Tao;JIAO Yingfu;GAO Yuan;YU Weifeng;HE Zhengyu;HANG Yannan;WEN Daxiang(Department of Anesthesiology,Renji Hospital,Shanghai Jiaotong University School of Medicine,Shanghai 200127,China)

机构地区：[1]上海交通大学医学院附属仁济医院麻醉科,上海200127 [2]上海交通大学医学院附属仁济医院重症医学科,上海200127

出　　处：《上海医学》2018年第8期477-482,共6页Shanghai Medical Journal

基　　金：国家自然科学基金(81700060)

摘　　要：目的观察原代培养的小鼠肺成纤维细胞在不同质量浓度的脂多糖(LPS)刺激下的增殖情况并检测P27蛋白的表达情况,以明确LPS对肺成纤维细胞增殖和P27蛋白表达的调控作用。方法将培养至4～7代的小鼠肺成纤维细胞分为对照组、100μg/L LPS组(LPS_(100)组)、500μg/L LPS组(LPS_(500)组)和1 000μg/L LPS组(LPS_(1000)组)。对照组加入PBS,各质量浓度LPS组加入相应终质量浓度的LPS。在LPS刺激后0、24、48、72h采用细胞计数试剂盒-8(CCK-8)法检测各组小鼠肺成纤维细胞的增殖情况。在LPS刺激后24h采用实时荧光定量PCR技术检测小鼠肺成纤维细胞P27的mRNA表达水平,并通过Western印迹法和免疫荧光技术比较小鼠肺成纤维细胞经不同质量浓度LPS刺激48h后P27的蛋白质表达情况。结果不同质量浓度的LPS刺激小鼠肺成纤维细胞后能引起细胞增殖,LPS刺激后24h,LPS1000组细胞增殖速率显著高于对照组(P<0.01);刺激后48h,LPS_(100)组、LPS_(500)组和LPS_(1000)组肺成纤维细胞的增殖速率均显著高于对照组(P值均<0.01);刺激后72h,LPS_(500)组和LPS_(1000)组肺成纤维细胞的增殖速率均显著高于对照组(P值均<0.01)。LPS刺激后24h,LPS_(500)组和LPS_(1000)组P27的mRNA表达水平均显著低于对照组(P值分别<0.05、0.01)。LPS刺激后48h,Western印迹法和免疫荧光实验结果显示,LPS_(100)组、LPS_(500)组和LPS_(1000)组的P27的蛋白质表达量和绿色荧光强度均显著低于对照组(P值分别<0.05、0.01、0.01)。结论肺成纤维细胞的增殖可能是LPS诱导的脓毒症相关性肺纤维化发生的机制之一,该过程可能与P27蛋白表达下调有关。Objective To evaluate the effect of lipopolysaccharide （LPS） on the proliferation of mouse lung fibroblasts and the expression of P27 protein in vitro. Methods Mouse lung fibroblasts cultured to 4 - 7 generations were randomly divided into 4 groups, and given phosphate buffer solution （PBS group）, 100 μg/L LPS （LPS100group）, 500 I^g/L LPS （LPS500 group） and 1 000 μg/L LPS （LPS1000 group）, respectively. The proliferation of mouse lung fibroblasts was detected by cell counting kit-8 （CCK-8） assay at 0, 24, 48, 72 h after LPS stimulation. The expression of P27 mRNA in mouse lung fibroblasts stimulated by different concentrations of LPS was detected by real-time fluorescence quantitative polymerase chain reaction （PCR） at 24 h after LPS stimulation. The expression of P27 protein was detected by Western blot and immunofluorescence at 48 h after LPS stimulation. Results The proliferation of mouse lung fibroblasts was accelerated after stimulation with different concentrations of LPS. The cell proliferation in LPS100 group was significantly faster than that in PBS group at 24 h after LPS stimulation （P〈0. 01 ）. The cell proliferation in LPS100 group, LPS500 group and LPS1000 group was significantly faster than that in PBS group at 48 h after LPS stimulation （all P〈0.01 ）. The cell proliferation in LPS500 group and LPS1000group was significantly faster than that in PBS group at 72 h after LPS stimulation （both P〈0.01）. The expression of P27 mRNA in LPS500 group and LPS1000 group was significantly down-regulated at 24 h after LPS stimulation as compared with PBS group （P〈0. 05, 0. 01 ）. The results of Western blot and immunofluorescence showed that the expression of P27 protein in LPS100 group, LPS500 group and LPS1000 group was significantly lower than that in PBS group at 48 h after LPS stimulation （P〈0.05, 0.01, 0.01）. Conclusion The proliferation of lung fibroblasts may be one of the mechanisms of sepsis-associated pulmonary fibrosis induced by LPS, which may

关 键 词：脂多糖类 肺纤维化 脓毒症 肺成纤维细胞 增殖 

分 类 号：R563[医药卫生—呼吸系统]