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

Abstract:

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 remark⁃ ably 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 plas⁃ mid 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.

Key words:

death?associated protein kinase 1, lentiviral, plasmid construction, human neuroblasto? ma cells, apoptosis