5-HTR2C重组质粒电转染至大鼠海马神经元对细胞活性的影响  

Influence on Cell Activity of 5-HTR2C Recombinant Plasmid Transfection Electricity on Rat Hippocampal Neurons

作　　者：王杰琼[1,2] 高冬梅[2,3] 于艳红[2,3] 张媛媛[1,2] 张惠云[1,2] 

机构地区：[1]山东中医药大学药学院,济南250355 [2]山东中医药大学中医药经典理论教育部重点实验室,济南250355 [3]山东中医药大学中医基础理论研究所,济南250355

出　　处：《世界科学技术-中医药现代化》2015年第4期794-799,共6页Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology

基　　金：科学技术部国家973计划项目(2011CB505102):从肝论治抑郁症(轻;中度);经前期综合征(重度;PMDD)探讨肝主疏泄调畅情志的作用机制;负责人:张惠云

摘　　要：目的:5-羟色胺2C受体在抑郁症的发病及治疗机制中发挥重要的作用,模拟5-HTR2C表达的异常升高,建立5-HTR2C高表达的细胞模型,为抑郁症治疗药物的开发提供平台。方法:采用课题组前期已构建5-HTR2C真核表达载体;按试剂盒方法提取5-HTR2C重组质粒;并取新生24 h内Wistar乳鼠海马,制备海马神经元,按试剂盒方法对质粒进行电转染,并于荧光显微镜下观察转染效果;将所培养细胞分为:非转染细胞(NTC)和转染细胞(TC),转染细胞分为:转染组(TC+5-HTR2C质粒)、空质粒组(TC+空质粒)、激动剂组(TC+激动剂);为确定激动剂组细胞提取蛋白的时间,分别于加入激动剂后0、5、10、20、40、60 min提取蛋白,Western-blot检测;待各组细胞处理后,采用Western-blot检测5-HTR2C以及MEK/P-MEK的表达,以验证5-HTR2C重组质粒转染后是否仍有活性。结果:海马神经元电转染后48 h后,神经元数量较多,电转染率及生存率最高;体外培养的海马神经元中加入激动剂m CPP后5 min至60 min,MEK磷酸化水平逐渐升高,在20 min时MEK磷酸化水平已较高;与NTC相比,TC+5-HTR2C质粒和TC+激动剂组中P-MEK的蛋白水平明显升高。结论:5-HTR2C重组质粒导入海马神经元后48 h细胞生长良好,死亡率低,细胞饱满,电转染率较高,可于此时收集细胞。激动剂组可在m CPP与细胞共培养后20-60 min间收集细胞。通过比较TC+5-HTR2C质粒组及TC+激动剂组,发现加入激动剂的细胞中MEK磷酸化水平较前者要强。因此,5-HTR2C重组质粒转染至海马神经元中后仍具有生理活性,说明5-HTR2C重组质粒的转染比较成功。The 5-HTR2C played an important role in the pathogenesis and treatment mechanism of depression. The cell model of 5-HTR2C high expression was established for the simulation of abnormally elevated 5-HTR2C expression. The model provided platform for development of medication treatment for depression. The 5-HTR2C recombinant plasmid was extracted according to the kit method from 5-HTR2C eukaryotic expression vector that was built in the early stage by the team. And hippocampus of a new Wistar suckling rat was taken within 24 h, in order to prepare hippocampal neurons. Transfection plasmids were made according to the kit method. The transfection effect was observed under the fluorescent microscope. The cultured ceils can be divided into the notransfected cells （NTC） and transfected cells （TC）. And TC were divided into the transfection group （TC＋5-HTR2C plasmid）, the empty plasmid group （TC＋empty plasmid）, and the agonist group （TC＋agonist）. In order to detect the time for protein extraction after joining agonist, the protein was extracted at 0, 5, 10, 20, 40 and 60 rain by Western blot for determination of cell extraction time in agonist group. Western blot test was used in the detection of 5-HTR2C and MEK/P-MEK expression in groups of cells for the verification of hippocampal neurons activity after 5-HTR2C recombinant plasmid transfection. The results showed that the number of hippocampal neurons was more, and the electric transfection rate and survival rate were highest after 48 h electricity transfection. The MEK phospborylation level was gradually increased among in vitro cultured hippocampal neurons from 5 min to 60 min after adding agonist mCPP. MEK phosphorylation level was higher at 20 min. In addition, compared with the NTC group, P-MEK phosphorylation level was increased significantly in the TC＋5-HTR2C plasmid and TC＋agonist group. It was concluded that the cell growth was good in 48 h after 5-HTR2C recombinant plasmid into hippocampus neurons, with low cell mortality rate, ful

关 键 词：5-HTR2C重组质粒 电转染 大鼠海马神经元 抑郁症 

分 类 号：R285.5[医药卫生—中药学]