利用IL-1A-eGFP慢病毒载体构建SASP细胞监测模型的研究  

Establishment of SASP-monitoring cell model by utilizing IL-1A-eGFP lentiviral vector

作　　者：彭健愉 孙明轩 姚琳 袁源 刘肖雨 赵威 林紫均 严詠恩 陈维春 刘新光 孙雪荣 PENG Jianyu;SUN Mingxuan;YAO Lin;YUAN Yuan;LIU Xiaoyu;ZHAO Wei;LIN Zijun;YAN Yongen;CHEN Weichun;LIU Xinguang;SUN Xuerong(Institute of Aging Research,Guangdong Medical University,Dongguan 523808,China;Key Laboratory for Medical Molecular Diagnostics of Guangdong Province;School of Medical Technology,Guangdong Medical University;Second Clinical School,Guangdong Medical University;School of Pharmacy,Guangdong Medical University;Institute of Biochemistry&Molecular Biology,Guangdong Medical University)

机构地区：[1]广东医科大学衰老研究所,东莞523808 [2]广东省医学分子诊断重点实验室 [3]广东医科大学医学技术学院 [4]广东医科大学第二临床医学院 [5]广东医科大学药学院 [6]广东医科大学生物化学与分子生物学研究所

出　　处：《山西医科大学学报》2021年第6期700-705,共6页Journal of Shanxi Medical University

基　　金：广东省自然科学基金资助项目(2020A1515010026);广东省普通高校青年创新人才项目(2018KQNCX098);湛江市科技发展专项资金竞争性分配项目(2020A01033);广东医科大学重点培育项目(GDMUZ2020007);大学生创新创业训练计划项目(S202010571051);广东医科大学创新实验项目(ZZDM006,FZDC001)。

摘　　要：目的本文拟建立基于IL-1A基因启动子驱动增强型绿色荧光蛋白(enhanced green fluorescent protein,eGFP)表达的细胞衰老相关分泌表型(senescence-associated secretory phenotype,SASP)监测模型,以便通过荧光判断SASP是否产生及其强度。方法构建含IL-1A-eGFP序列的慢病毒表达载体,经NcoⅠ及SalⅠ酶切及测序验证后,制备慢病毒颗粒,并感染人黑色素瘤M14细胞,经嘌呤霉素筛选、单克隆培养等获得整合IL-1A-eGFP序列的细胞株。用顺铂(以溶剂为阴性对照)诱导该细胞株衰老,连续观察荧光变化,并用qRT-PCR检测SASP因子IL-1A、IL-1B、IL-8的表达。结果经酶切和测序证实,IL-1A-eGFP慢病毒表达载体构建正确。用该慢病毒感染人黑色素瘤M14细胞并经过筛选后,得到形态均一、增殖迅速的M14-IL-1A-eGFP细胞株。该细胞常规状态下仅有微弱的绿色荧光背景,加入顺铂后,细胞增殖减慢,形态变大,同时绿色荧光明显增强,qRT-PCR检测发现SASP因子IL-1A,IL-1B和IL-8表达明显增强(P分别为0.002,0.028,0.056)。结论成功构建了SASP细胞监测模型,可通过绿色荧光的强度反映SASP状态,该模型有望为相关研究提供便利。Objective To establish a cell monitoring model of senescence-associated secretory phenotype(SASP)based on the expression of enhanced green fluorescent protein(eGFP)driven by IL-1A promoter,and to monitor SASP through fluorescence intensity.Methods The lentiviral expression vector containing IL-1A-eGFP sequence was constructed and then verified by NcoⅠand SalⅠdigestion and DNA sequencing.Then the lentiviruses were prepared and used to infect human melanoma M14 cells.After puromycin screening,monoclonal culture and so on,the cell line integrating IL-1A-eGFP sequence was obtained.The cell line was induced into senescence by cisplatin(vehicle served as negative control),and then the fluorescence was continuously observed,and the expression of SASP factors such as IL-1A,IL-1B,IL-8 was detected by qRT-PCR.Results Validity of IL-1A-eGFP lentiviral vector was confirmed by restriction enzyme digestion and DNA sequencing.After infected by lentiviruses and drug screening,the cell line M14-IL-1A-eGFP was obtained,which displayed uniform shape and rapid growth.The M14-IL-1A-eGFP cells had weak green fluorescence in normal culture condition.Compared with that of vehicle treatment,M14-IL-1A-eGFP cells treated with cisplain displayed enlarged cell morphology and suppressed proliferation.Meanwhile,these cells showed strong and bright green fluorescence with enhanced mRNA levels of SASP factors such as IL-1A(P=0.002),IL-1B(P=0.028)and IL-8(P=0.056).Conclusion The SASP-monitoring cell model is successfully established.SASP status can be evaluated by green fluorescence,which would provide convenience for the related research.

关 键 词：衰老 SASP 黑色素瘤 IL-1A GFP 

分 类 号：Q782[生物学—分子生物学]