瘦素/ERK信号在云锡矿粉诱导大鼠II型肺泡上皮细胞转化中的作用  

Leptin-mediated ERK Signaling Pathway Promotes the Transformation of Rat Alveolar Type II Epithelial Cells Induced by Yunnan Tin Mine Dust

作　　者：胡雄 颜聪 张宇[1] 黎贵芸 周喆焱 阮永华 刘士岳 边莉[1] Xiong HU;Cong YAN;Yu ZHANG;Guiyun LI;Zheyan ZHOU;Yonghua RUAN;Shiyue LIU;Li BIAN(Department of Pathology,The First Affliated Hospital of Kunming Medical University,Kunming 650031,China;Basic Medicine of Kunming Medical University School of Basic Medicine,Kunming 650500,China;Department of Pathology,The Third People’s Hospital of Yunnan Province,Kunming 650011,China)

机构地区：[1]昆明医科大学第一附属医院病理科,昆明650031 [2]昆明医科大学基础医学院,昆明650500 [3]云南省第三人民医院病理科,昆明650011

出　　处：《中国肺癌杂志》2023年第10期732-740,共9页Chinese Journal of Lung Cancer

基　　金：国家自然科学基金项目(No.82360523、No.82060423);昆明医科大学肺部恶性肿瘤精准病理诊断科技创新团队项目(No.CXTD202210);兴滇人才计划“名医专项”(No.RLMY20220018)资助。

摘　　要：背景与目的目前,云南个旧锡矿有大量矿工从事开采工作,这种职业环境与接触粉尘颗粒、重金属、多环芳烃和放射性氡有关,大大增加了患肺癌的风险。本研究旨在探讨在云锡矿粉诱导大鼠肺泡Ⅱ型上皮细胞(immortalized rat alveolar cells type Ⅱ,RLE-6TN)恶性转化过程中,瘦素(leptin)及其介导的细胞外调节蛋白激酶(extracellular regulated protein kinase,ERK)信号通路所起的作用。方法采用200μg/mL的云锡矿粉隔代毒染RLE-6TN至第9代,建立毒染细胞模型,命名为R_(200)细胞,正常培养组命名为R细胞,通过Western blot法检测两种细胞leptin受体的表达情况。通过MTT法筛选出leptin及丝裂原活化蛋白激酶激酶(mitogen-activated protein kinase kinase,MEK)抑制剂(U0126)对R_(200)细胞的最佳作用浓度。自第20代起,将R组、R_(200)组细胞分别与leptin及MEK抑制剂U0126共培养,对各组细胞的形态改变进行观察,并利用苏木素-伊红(hematoxylin-eosin,HE)染色技术鉴别第40代细胞的形态学差异,通过刀豆凝集素A(concanavalin A,ConA)及锚着独立性生长实验法检测细胞恶性转化情况。通过Western blot法检测leptin作用后上皮细胞ERK信号通路的变化。结果R组和R_(200)组细胞均表达leptin受体(OB-R)。与R_(200)组比较,当leptin浓度达100 ng/mL时,其促增殖效应最为显著,30μmol/L U0126可抑制毒染细胞R_(200)增殖,与对照组相比具有统计学差异(P<0.05)。自第25代起,leptin诱导的R_(200)组(R_(200)L组)细胞形态发生变化,至第30代出现恶性转化,至第40代时恶性转化特征明显;而R_(200)组细胞及U0126诱导的R_(200)组(R_(200)LU组)细胞则在第40代时才出现恶性转化特征。R_(200)L组细胞凝集速度较R_(200)LU组快,其余各组细胞P30出现凝集,且随ConA浓度增加,细胞凝集速度加快。R_(200)L组细胞自P40可见克隆形成,克隆形成率为2.25‰±0.5‰,R_(200)LU组及R_(200)组未见克隆集落。R_(200)L组细胞pERK表�Background and objective Currently,a significant number of miners are involved in mining operations at the Gejiu tin mine in Yunnan.This occupational setting is associated with exposure to dust particles,heavy metals,polycyclic aromatic hydrocarbons,and radioactive radon,thereby significantly elevating the risk of lung cancer.This study aims to investigate the involvement of leptin-mediated extracellular regulated protein kinase(ERK)signaling pathway in the malignant transformation of rat alveolar type II epithelial cells induced by Yunnan tin mine dust.Methods Immortalized rat alveolar cells type Ⅱ(RLE-6TN)cells were infected with Yunnan tin mine dust at a concentration of 200μg/mL for nine consecutive generations to establish the infected cell model,which was named R_(200)cells.The cells were cultured normally,named as R cells.The expression of leptin receptor in both cell groups was detected using the Western blot method.The optimal concentration of leptin and mitogenactivated protein kinase kinase(MEK)inhibitor(U0126)on R_(200)cells was determined using the MTT method.Starting from the 20th generation,the cells in the R group were co-cultured with leptin,while the cells in the R_(200)group were co-cultured with the MEK inhibitor U0126.The morphological alterations of the cells in each group were visualized utilizing hematoxylin-eosin staining.Additionally,concanavalin A(ConA)was utilized to detect any morphological differences,and an anchorage-independent growth assay was conducted to assess the malignant transformation of the cells.The changes in the ERK signaling pathway in epithelial cells after the action of leptin were detected using the Western blot method.Results Both the cells in the R group and R_(200)group express leptin receptor OB-R.Compared to the R_(200)group,the concentration of leptin at 100 ng/mL shows the most significant pro-proliferation effect.The proliferation of R_(200)cells infected with the virus is inhibited by 30μmol/L U0126,and a statistically significant divergence was seen wh

关 键 词：瘦素 肺泡II型上皮细胞 转化 ERK信号通路 

分 类 号：R734.2[医药卫生—肿瘤]