重组CC16蛋白质抑制香烟烟雾提取物诱导人支气管上皮细胞衰老的初步研究  

Recombinant human CC16 inhibits senescence in human bronchial epithelial cells induced by cigarette smoke extract

作　　者：李婷[1] 杨晓雪 高睿 栗馨洋 王海龙[2] 庞敏[1] LI Ting;YANG Xiaoxue;GAO Rui;LI Xinyang;WANG Hailong;PANG Min(Department of Pulmonary and Critical Care Medicine,Shanxi Provincial Key Laboratory of Respiratory Diseases,First Hospital of Shanxi Medical University,Taiyuan,Shanxi Province,030001;Laboratory Center for Basic Medicine,Shanxi Medical University,Jinzhong,Shanxi Province,030600,China)

机构地区：[1]山西医科大学第一医院呼吸与危重症医学科,呼吸疾病防治与基础研究山西省重点实验室,太原030001 [2]山西医科大学基础医学研究中心,山西晋中030600

出　　处：《陆军军医大学学报》2024年第12期1410-1416,共7页Journal of Army Medical University

基　　金：山西省省筹资金资助回国留学人员科研项目(2022-191);山西省应用基础研究计划项目(202103021223433);山西医科大学第一医院136专项经费科研项目(2021-07)。

摘　　要：目的探讨重组人CC16蛋白质(rhCC16)对香烟烟雾提取物(cigarette smoke extract,CSE)诱导的人支气管上皮细胞(human bronchial epithelial cells,HBECs)衰老的抑制作用及机制。方法CCK-8法检测CSE(0%、1%、2.5%、5%、7.5%和10%)及rhCC16(0、10、100、250和500 ng/mL)对HBECs细胞活力的影响;β-半乳糖苷酶染色试剂盒检测对照组、CSE组以及CSE+rhCC16组细胞β-半乳糖苷酶活性;实时荧光定量PCR(qPCR)检测各组细胞P16、P21 mRNA表达水平;Western blot检测各组细胞P16、P21、p-P53、P38、p-P38、ERK1/2、p-ERK1/2蛋白表达水平。结果与对照组相比,5%CSE刺激细胞72 h对HBECs细胞活力无显著影响;500 ng/mL及以下浓度rhCC16对HBECs细胞活力无显著影响;与对照组相比,5%CSE刺激HBECs细胞72 h致β-半乳糖苷酶染色阳性细胞率明显升高(P<0.05),且P16和P21蛋白和mRNA表达水平明显升高(P<0.05),p-P53、p-P38与p-ERK1/2蛋白表达量显著增加(P<0.05);与CSE组相比,250 ng/mL rhCC16干预下,HBECs细胞β-半乳糖苷酶染色阳性细胞率显著下降(P<0.05),P16和P21蛋白和mRNA表达水平显著下降(P<0.05),p-P53、p-p38和p-ERK1/2的表达量降低(P<0.05)。结论rhCC16蛋白质抑制香烟烟雾诱导的HBECs衰老,抑制P38 MAPK和ERK1/2信号通路可能是其作用机制。Objective To investigate the effect and mechanism of recombinant human CC16 protein(rhCC16)on cigarette smoke extract(CSE)-induced senescence of human bronchial epithelial cells(HBECs).Methods CCK-8 assay was used to evaluate the cell viability in HBECs after treated with 0%,1%,2.5%,5%,7.5%and 10%CSE and/or 0,10,100,250 and 500 ng/mL rhCC16.β-galactosidase staining was used to observe the activity of the enzyme in control,CSE,and CSE+rhCC16 treated HBECs.The mRNA levels of P16 and P21 in above cell groups were detected by real-time fluorescence quantitative PCR(qPCR),and the protein levels of P16,P21,p-P53,P38,p-P38,ERK1/2 and p-ERK1/2 were detected with Western blot in control group,CSE and CSE+rhCC16 groups.Results Compared with the control group,5%CSE stimulation for 72 h or the treatment of 500 ng/mL or lower dose of rhCC16 had no significant effect on cell viability of HBECs.Stimulation 5%CSE for 72 h resulted in significantly higher positive rate toβ-galactosidase staining(P<0.05),elevated mRNA and protein levels of P16 and P21(P<0.05),and enhanced protein levels of p-P53,p-P38 and p-ERK1/2(P<0.05)when compared with the control group.Compared with the simple CSE stimulation,250 ng/mL rhCC16 treatment decreased positive rate toβ-galactosidase staining(P<0.05),reduced mRNA and protein levels of P16 and P21(P<0.05)and protein levels of p-P53,p-P38 and p-ERK1/2(P<0.05).Conclusion rhCC16 inhibits CSE-induced cellular senescence of HBECs,which may due to its suppression in P38 MAPK and ERK1/2 signaling pathway.

关 键 词：细胞衰老 香烟烟雾提取物 CC16蛋白质 P38 MAPK/ERK1/2 

分 类 号：R322.34[医药卫生—人体解剖和组织胚胎学] R329.28[医药卫生—基础医学] R977.6