稳定表达未折叠蛋白反应荧光报告基因的SH-SY5Y细胞系建立  

Stable establishment of SH-SY5Y cell line expressing unfolded protein response fluorescence reporter gene

作　　者：孙建强 朱伟[3] 陈昱 姜树原 刘晓蕾[4] 谢雅彬[4,5] 谢伟 巴德仁贵[4,5] 邵国[4,5] 贾小娥[1,5] 马宁 SUN Jian-Qiang;ZHU Wei;CHEN Yu(School of Basic Medicine and Forensic Medicine,Baotou Medical College,Baotou 014040,Inner Mongolia,China)

机构地区：[1]包头医学院基础医学与法医学院,内蒙古包头014040 [2]包头医学院第四附属医院,内蒙古包头014040 [3]包头医学院药学院,内蒙古包头014040 [4]包头医学院内蒙古自治区低氧转化医学重点实验室,内蒙古包头014040 [5]包头医学院医学技术与麻醉学院,内蒙古包头014040 [6]包头医学院图书馆,内蒙古包头014040

出　　处：《中国老年学杂志》2023年第23期5757-5763,共7页Chinese Journal of Gerontology

基　　金：国家自然科学基金项目(81901918,81660204);内蒙古自然科学基金(2019MS08060);2020年中央引导地方发展基金(2020ZY0040);2017年度内蒙古自治区“草原英才”工程青年创新创业人才(Q2017047);2022年度内蒙古自治区高等学校科学研究项目(NJZY22062)。

摘　　要：目的通过稳定表达70 kD热休克蛋白(HSP)4重组蛋白(HSPA4)-增强型绿色荧光蛋白(EGFP)的SH-SY5Y细胞系,直观实时反映细胞内未折叠蛋白反应(UPR)的变化。方法以SH-SY5Y细胞的cDNA为模板,克隆HSPA4,并连接EGFP。将HSPA4-EGFP连接入慢病毒载体中,慢病毒转染SH-SY5Y细胞。使用3μg/ml嘌呤霉素处理细胞1个月,筛选得到HSPA4-EGFP稳转系。通过实时荧光定量聚合酶链反应(QPCR)、Western印迹、共聚焦显微镜检测等方法,证实HSPA4-EGFP稳转系能有效且实时反映UPR的变化。结果扩增到HSPA4-EGFP,并成功构建HSPA4-EGFP稳定表达的UPR报告系统。QPCR检测表明,构建的报告系统可以指示UPR。在激光共聚焦显微镜下,可以观察到细胞内绿色EGFP的点状分布,其能够反映UPR错误折叠蛋白的多少及分布,分别加入UPR激活剂、抑制剂后,细胞内绿色EGFP的点状分布明显增多和减少,差异有统计学意义(P<0.05)。Western印迹检测到细胞中HSPA4-EGFP融合蛋白表达条带。结论成功构建HSPA4-EGFP稳转系,能够直观实时反映UPR变化,为深入研究UPR及其相关疾病(包括老年性疾病)提供工具。Objective To intuitively reflect the changes of unfolded protein response(UPR)in cells in real time by stably expressing 70 kD heat shock protein(HSP)4 recombinant protein(HSPA)4-enhanced green fluorescent protein(EGFP)SH-SY5Y cell line.Methods HSPA4 was cloned using cDNA of SH-SY5Y cells as template,and EGFP was ligated.HSPA4-EGFP was inserted into lentivirus vector,and lentivirus was transfected SH-SY5Y cells.The cells were treated with 3μg/ml purinomycin for one month to obtain stable HSPA4-EGFP lines.Real-time fluorescence quantitative polymerase chain reaction(QPCR),Western blot,confocal microscopy and other methods were used to confirm HSPA4-EGFP stable transformation system could effectively and in real time reflect the changes of UPR.Results HSPA4-EGFP was amplified,and the UPR reporting system for stable expression of HSPA4-EGFP was successfully constructed.QPCR detection showed that the constructed reporting system could indicate UPR.Under confocal laser microscope,the dot distribution of green EGFP in cells could be observed,which could reflect the number and distribution of UPR misfolded protein.After the addition of UPR activator and inhibitor,the dot distribution of green EGFP in cells was significantly increased and decreased,respectively(P<0.001).The expression bands of HSPA4-EGFP fusion protein in cells were detected by Western blot.Conclusions HSPA4-EGFP stable transformation system has been successfully constructed,which could directly reflect the changes of UPR in real time,and provide a tool for in-depth study of UPR and related diseases(including age-related diseases).

关 键 词：未折叠蛋白反应(UPR) 70 kD热休克蛋白4重组蛋白(HSPA4)-增强型绿色荧光蛋白(EGFP) SH-SY5Y细胞 

分 类 号：Q786[生物学—分子生物学]