PC12细胞SLC6A4基因沉默稳转细胞系的建立及SLC6A4基因沉默对缺氧PC12细胞凋亡的影响
投稿时间:2022-05-24  修订日期:2022-09-06  点此下载全文
引用本文:张冬梅,高迎春,何蕾,陈克明,李文斌,王荣,马慧萍.PC12细胞SLC6A4基因沉默稳转细胞系的建立及SLC6A4基因沉默对缺氧PC12细胞凋亡的影响[J].药学实践杂志,2022,40(5):389~394,463
摘要点击次数: 123
全文下载次数: 146
作者单位E-mail
张冬梅 中国人民解放军联勤保障部队第九四〇医院药剂科甘肃 兰州 730050  
高迎春 中国人民解放军联勤保障部队第九四〇医院药剂科甘肃 兰州 730050  
何蕾 中国人民解放军联勤保障部队第九四〇医院药剂科甘肃 兰州 730050  
陈克明 中国人民解放军联勤保障部队第九四〇医院药剂科甘肃 兰州 730050  
李文斌 中国人民解放军联勤保障部队第九四〇医院药剂科甘肃 兰州 730050  
王荣 中国人民解放军联勤保障部队第九四〇医院药剂科甘肃 兰州 730050 wangrong-69@163.com 
马慧萍 中国人民解放军联勤保障部队第九四〇医院药剂科甘肃 兰州 730050 mahuiping2022@aliyun.com 
基金项目:国家自然科学基金项目(81571847);军队后勤科研计划项目面上项目(CWH17J010);军队卫勤保障能力创新与生成专项计划(21WQ045)
中文摘要:目的 构建SLC6A4基因RNA干扰(RNAi)-短发夹RNA(shRNA)慢病毒表达载体,建立慢病毒介导沉默SLC6A4基因的PC12细胞稳转细胞系,检测SLC6A4基因沉默对缺氧诱导的细胞凋亡的影响。方法 设计合成3条针对SLC6A4基因的shRNA干扰序列,构建于慢病毒载体质粒GV248中,在293T细胞中共转染进行慢病毒包装。转染大鼠肾上腺髓质嗜铬细胞瘤PC12细胞,荧光显微镜观察GFP荧光,筛选最佳shRNA干扰序列,应用嘌呤霉素筛选出稳定转染的细胞株。应用RT-PCR方法检测SLC6A4基因的表达,Western blot方法检测蛋白5-HTT的表达变化,流式细胞术检测沉默SLC6A4基因对缺氧诱导的PC12细胞凋亡的影响。结果 成功构建SLC6A4-shRNA慢病毒载体并转染PC12细胞,与正常对照组及干扰对照组比较,干扰组细胞内SLC6A4基因和蛋白表达明显降低(P<0.01),证实慢病毒载体能够有效沉默SLC6A4基因,沉默SLC6A4基因可以逆转缺氧诱导的细胞凋亡。结论 通过shRNA慢病毒载体途径可有效沉默PC12细胞SLC6A4基因,沉默SLC6A4基因可以逆转缺氧诱导的细胞凋亡。
中文关键词:慢病毒载体  shRNA  RNA干扰  SLC6A4  细胞凋亡
 
Establishment of SLC6A4 gene silencing stable cell line in PC12 cells and the effect of SLC6A4 gene silencing on apoptosis of hypoxia PC12 cells
Abstract:Objective To construct SLC6A4-shRNA lentiviral vector, establish PC12 cells stable transformation cell line,and detect the effect of SLC6A4 gene silencing on hypoxia induced PC12 cells apoptosis. Methods Three specific targets sequence of SLC6A4 were designed and short hairpin RNA was synthesized, and then were recombined into shRNA expression vector GV248 plasmid, with non-homology shRNA sequence as negative control. The connection products were switched to competent cells. After dentification and sequencing, the vectors were co-transfected with the auxiliary vectors into 293T cells in order to produce recombinant shRNA lentiviral particles. Then, PC12 cells were infected with the recombinant lentiviral and screened by puromycin. The PC12 cells were divided into two groups: lentiviral negative control group (NC-shRNA) and SLC6A4-silenced group (SLC6A4-shRNA). The expression of SLC6A4 mRNA was detected by real-time fluorescence quantitative and the 5-HTT protein level was assayed by Western blot, The effect of SLC6A4 gene silencing on hypoxia induced apoptosis was detected by flow cytometry. Results The SLC6A4-shRNA lentiviral expression vector was constructed and the recombinant lentiviral particles by packaging the 293T cells were obtained, the stably infected PC12 cells were established after filtering. Compared with negative control group, the expression level of SLC6A4 gene and 5-HTT protein in SLC6A4-shRNA group was suppressed notably (P<0.01). It was confirmed that lentiviral vector could effectively silence SLC6A4 gene in PC12 cells and SLC6A4 gene silencing could decrease apoptosis rate of PC12 cells under hypoxia condition. Conclusion The SLC6A4 gene of PC12 cells could be effectively silenced by shRNA lentivirus vector,which could reverse hypoxia induced apoptosis.
keywords:lentivirus  shRNA  RNA interference  SLC6A4  apoptosis
查看全文  查看/发表评论  下载PDF阅读器
关闭

分享按钮