APPswe基因慢病毒表达质粒的构建及对其SH-SY5Y细胞生长和凋亡的影响  

Construction of Lentiviral Expression Plasmid of APPswe Gene and Its Effect on the Cell Growth and Apoptosis of SH-SY5Y Cells

作　　者：宋喜君 周鹤妍 常平[1] 陶鹏飞 劳凤学 黄汉昌[1] Song Xijun;Zhou Heyan;Chang Ping;Tao Pengfei;Lao Fengxue;Huang Hanchang(Beijing Key Laboratory of Bioactive Substances and Functional Foods,Beijing Union University,Beijing,100191)

机构地区：[1]北京联合大学,生物活性物质与功能食品北京市重点实验室,北京100191

出　　处：《基因组学与应用生物学》2021年第1期287-295,共9页Genomics and Applied Biology

基　　金：国家自然科学基金项目(31471587);北京联合大学科研项目(JZ10202001);北京联合大学校级课题(ZK40201902);北京联合大学“启明星”大学生科技创新项目(202011417SJ125)共同资助。

摘　　要：为考察APPswe基因的表达对细胞生长和凋亡作用的影响,本研究采用gateway分子重组技术构建表达APPswe基因的慢病毒质粒,并将该质粒转染至SH-SY5Y细胞中。用RT-PCR和Western blot技术分别测定APPswe基因的mRNA转录和蛋白翻译水平。活细胞计数法和细胞外乳酸脱氢酶法分别测定细胞存活力和细胞膜损伤程度。Annexin V-FITC/PI染色流式细胞术测定细胞凋亡水平,Hoescht 33342染色观察细胞核形态变化,DCFH-DA染色法测定胞内活性氧水平。转录和翻译水平的验证表明,APPswe基因能够在SH-SY5Y细胞中相对稳定地表达。表达APPswe基因后,细胞的存活能力降低,损伤程度增加,细胞内活性氧水平上升,细胞核浓缩聚集,细胞发生凋亡反应。试验结果表明,APPswe基因的表达对SH-SY5Y细胞生长产生抑制作用,造成细胞损伤和凋亡,表达APPswe基因的SH-SY5Y细胞系可应用于体外AD病理发生机制和药物作用的研究。In order to investigate the effect of APPswe gene expression on cell growth and apoptosis, the lentiviral plasmid expressing APPswe gene(APPswe plasmid) was constructed by Gateway recombination cloning technology, and the plasmid was transfected into SH-SY5Y cells(APPswe cells). The mRNA transcription and the protein translation of APPswe gene were measured by RT-PCR and Western blot, respectively. The cell viability and the cell membrane damage were assessed through viable cell counting and extracellular LDH assays. Cell apoptosis was determined with flow cytometry by staining with Annexin V-FITC/PI and the changes of nuclear morphology was detected through staining with Hoescht 33342 dye. The intracellular level of reactive oxygen species(ROS)was assayed by DCFH-DA staining. The results of transcription and translation of APPswe gene showed that the plasmid constructed in this study could be stably expressed in SH-SY5Y cells. After the expression of APPswe gene, the cell viability decreased and the degree of membrane damage increased. After the expression of APPswe gene, the cell apoptosis occurred with increasing of early and late apoptosis. In addition, the cell nu cleus condensed and gathered, and intracellular ROS increased in APPswe cells. These results showed that the expression of APPswe gene can inhibit the growth of SH-SY5Y cells and cause cell damage and apoptosis. The APPswe cells can be used in the study of the pathological mechanism and of drug therapy of AD in vitro.

关 键 词：慢病毒表达质粒 Β-淀粉样前体蛋白 细胞活力 细胞凋亡 

分 类 号：Q78[生物学—分子生物学]