慢病毒敲减质粒pLKO.1-shDAPK1的构建及其对细胞凋亡的影响  

Construction of lentiviral knockdown plasmid pLKO.1-shDAPK1 and its effect on cell apoptosis

作　　者：秦坤 徐绍业 韩雨 闫建国 夏春波[1] 邵晓云[1] QIN Kun;XU Shaoye;HAN Yu;YAN Jianguo;XIA Chunbo;SHAO Xiaoyun(Department of Human Anatomy,Guilin Medical College,Guilin 541100,China;不详)

机构地区：[1]桂林医学院人体解剖学教研室,广西桂林541100 [2]桂林医学院科学实验中心,广西桂林541100 [3]桂林医学院生理学教研室,广西桂林541100

出　　处：《实用医学杂志》2022年第6期715-720,共6页The Journal of Practical Medicine

基　　金：国家自然科学基金(编号:31660269);广西自然科学基金(编号:2021GXNSFAA075004,2017GXNSFAA198003,2016GXNSFBA380098);桂林医学院硕士研究生科研项目(编号:GYYK2021007);广西高等学校千名中青年骨干教师培育计划。

摘　　要：目的 构建敲减死亡相关蛋白激酶1(shDAPK1)基因的慢病毒质粒及稳转神经母细胞瘤细胞系(SH-SY5Y),并检测SH-SY5Y细胞中DAPK1敲减后对细胞凋亡的影响。方法 设计并合成特异性敲减DAPK1基因的上下游引物,将引物退火后与双酶切PLKO.1载体经T4连接酶连接、转化、提取质粒并进行DNA测序,获得重组正确的慢病毒干扰质粒pLKO.1-shDAPK1。将该重组的质粒与辅助质粒psPAX2和pMD2.G共转染HEK293T细胞,72 h后收集慢病毒上清液,感染SH-SY5Y细胞,利用嘌呤霉素进行阳性筛选,得到稳定敲减DAPK1的SH-SY5Y细胞系。利用Western blot检测SH-SY5Y稳转细胞系中DAPK1蛋白以及用1-甲基-4-苯基-吡啶离子(MPP+)处理后的Bax、Bcl-2及Caspase-3的蛋白表达水平,并应用流式细胞术检测SH-SY5Y细胞的凋亡情况。结果 重组pLKO.1-shDAPK1质粒的测序比对结果正确,嘌呤霉素筛选后获得shDAPK1稳转SH-SY5Y细胞系(shDAPK1-SY5Y),该稳转细胞系中DAPK1蛋白表达水平与对照组相比显著下降,此外还上调了Bcl-2蛋白的表达,下调Bax、Caspase-3蛋白的表达且流式检测结果显示该细胞系凋亡发生率明显下降。结论 成功构建了慢病毒敲减质粒pLKO.1-shDAPK1,并建立了DAPK1低表达的SH-SY5Y稳定感染细胞系,明显抑制了细胞凋亡。Objective To construct a lentiviral plasmid knocked down death-associated protein kinase 1(shDAPK1)gene and stably transfected neuroblastoma cell line(SH-SY5Y),and detect the effect of DAPK1 knockdown on cell apoptosis in SH-SY5Y cells.Methods An upstream and downstream primer specifically knocking down the DAPK1 gene was designed and synthesized.After the primers were annealed,they were connected with the double-enzyme-cleaved PLKO.1 vector by T4 ligase,followed by transformation and plasmid extraction.DNA sequencing was subsequently performed to confirm the availability of the recombinant correct lentiviral interfering plasmid pLKO.1-shDAPK1.The recombinant plasmid was co-transfected with the helper plasmids psPAX2 and pMD2.G into HEK293T cells.The supernatant of lentivirus was collected after 72 h and infected with SH-SY5Y cells.The SH-SY5Y cell line with stable DAPK1 knockout was obtained through positive screening with puromycin.Western blot was used to detect the DAPK1 protein in the SH-SY5Y stably transfected cell line and the protein expression levels of Bax,Bcl-2 and Caspase-3 after treatment with 1-methyl-4-phenylpyridinium(MPP+).Finally,the apoptosis of SH-SY5Y cells was detected by flow cytometry.Results The sequencing comparison result of the recombinant pLKO.1-shDAPK1 plasmid was correct.The shDAPK1-SY5Y cell line(shDAPK1-SY5Y)was stably transfected with puromycin.The expression level of DAPK1 protein in the stably transferred cell line was remarkably reduced compared with the control group.In addition,the expression level of Bcl-2 protein was up-regulated,while the expression levels of Bax and Caspase-3 proteins were down-regulated.Flow cytometry results showed that the incidence of apoptosis in the cell line was significantly reduced.Conclusions The lentivirus knock-down plasmid pLKO.1-shDAPK1 was successfully constructed and a stably infected SH-SY5Y cell line with low expression of DAPK1 was established,which significantly inhibited apoptosis.

关 键 词：死亡相关蛋白激酶1 慢病毒 质粒构建 人骨髓神经母细胞瘤细胞 细胞凋亡 

分 类 号：Q78[生物学—分子生物学]