死亡相关蛋白激酶C末端对谷氨酸诱导的SH-SY5Y细胞凋亡的抑制作用研究  

Inhibition of glutamate induced apoptosis of SH-SY5Y cells by C-terminal tail of DAPK

作　　者：张海涛[1] 汪亚君[1] 范大华[1] 陈小谊[1] 刘慧明[1] 

机构地区：[1]广东医学院生物化学与分子生物学教研室,广东湛江524023

出　　处：《解放军医学杂志》2010年第7期839-844,共6页Medical Journal of Chinese People's Liberation Army

基　　金：广东省卫生厅资助课题(A2006475);广东省科技计划项目(2008B030301025)

摘　　要：目的观察死亡相关蛋白激酶(DAPK)蛋白C-末端多肽(DCTP)对谷氨酸诱导的SH-SY5Y细胞凋亡的作用,探讨DCTP抑制DAPK功能的机制。方法采用PCR法扩增DCTP核酸片段,定向连接至质粒pEGFPN1、pIRES2-EGFP和pcDNA3.1(-),将重组质粒[pEGFPN1-DCTP、pIRES2-DCTP-EGFP和pcDNA3.1(-)-DCTP]转染至SH-SY5Y细胞中,筛选稳定细胞株。分析pEGFPN1-DCTP的表达产物在细胞中的定位。建立稳定表达DCTP的SH-SY5Y细胞。用MTT法、Hoechst33258染色法和流式细胞仪检测SH-SY5Y细胞对谷氨酸的敏感性,用Westernblotting检测细胞内DAPK的表达量以及磷酸化DAPK(p-DAPK)水平的变化。结果谷氨酸对SH-SY5Y细胞生长的抑制作用具有浓度依赖性,40mmol/L的谷氨酸对细胞的抑制率为51.7%。流式细胞仪检测结果显示,40mmol/L谷氨酸处理后细胞的凋亡率和坏死率分别为26.1%和27.7%,而正常培养细胞分别为0.08%和0%。谷氨酸诱导细胞凋亡时,p-DAPK表达下降。荧光显微镜观察可见DCTP定位于细胞质。用40mmol/L谷氨酸分别诱导转染pcDNA3.1(-)-DCTP和pcDNA3.1(-)质粒(对照质粒)的SH-SY5Y细胞,流式细胞仪检测结果显示,前者凋亡率为43.7%,而后者凋亡率为62.2%。Westernblotting检测结果显示,在谷氨酸诱导下,与转染pIRES2-EGFP质粒(对照质粒)的SH-SY5Y细胞相比,转染pIRES2-DCTP-EGFP质粒的细胞内DAPK和p-DAPK水平均无明显变化。结论 DCTP基因的表达产物定位于SH-SY5Y胞质。DCTP可以抵抗谷氨酸诱导的SH-SY5Y细胞的凋亡,但并不影响DAPK的磷酸化及其表达水平。Objective This study was to design a new recombinant plasmid expressing the carboxylic terminal peptide of DAPK （DCTP） to inhibit SH-SY5Y cells apoptosis induced by glutamate acid taking DAPK as the target.The mechanism of inhibition of DAPK by DCTP was explored.Methods The DCTP coded sequence was amplified by PCR with the cDNA of DAPK as template,and oriented to insert into the fusion expression plasmid pEGFPN1.The recombinant plasmid was transfected into SH-SY5Y cells.DCTP expression was detected with RT-PCR.The stably expressed DCTP cell line was established after the cells were screened by G418.MTT method,Hoechst33258-staining and flow cytometry were used to measure cell survival,apoptosis and necrosis after SH-SY5Y cells were treated with glutamate for 24h.The changes in DAPK expression and DAPK phosphorylation were deteced by Western bloting.Results Glutamate inhibited the growth of SH-SY5Y cells in a dose-dependent manner.The inhibition rate of 40mmol/L glutamic acid was 51.7%,the apoptotic rate and necrosis rate were 26.1% and 27.7% in the presence of 40mmol/L glutamate,respectively,while they were respectively 0.08% and 0% in control group.The phosphorylation levels of DAPK descreased in the presence of glutamate.Observation with fluorescence microscopy revealed that DCTP expression products were located in cytoplasm.When the cells of transfected DCTP and control cells were treated with 40mmol/L of glutamate for 24h,the apoptotic ratio of DCTP-transfected cell was 43.7%,while of control cell was 62.2%,as mearsured by flow cytometry.Compared with the vector-transfected control cells induced by glutamate,the expression of DAPK and the phosphorylation level of DAPK were not significantly changed in DCTP-transfected cells induced by glutamate.Conclusions It is verified that the DCTP expression products are located in the cytoplasm of SH-SY5Y cells.It can prevent apoptosis of SH-SY5Y cells induced by glutamate without affecting the expression and the phosphorylation level of DAPK.

关 键 词：死亡相关蛋白激酶 基因疗法 谷氨酸 细胞凋亡 DNA降解 坏死 

分 类 号：R394.114[医药卫生—医学遗传学]