构建人SMARCAL1基因过表达慢病毒载体调控胚肾细胞增殖

Construction of human SMARCAL11 gene over-expressed lentiviral vector and its effect on proliferation of embryonic kidney cells

背景:研究发现SMARCAL1在人类发育和成熟肾脏的多种细胞类型中选择性表达,表明其在肾脏发育过程中发挥着重要的功能作用。目的:探讨SMARCAL1过表达对于胚肾细胞增殖功能的影响。方法:取293T细胞和HEK293细胞的第3代细胞用于实验。选用pHBLV-CMV-MCS-3FLAG-EF1-ZsGreen-T2A-PURO载体构建人SMARCAL1基因过表达慢病毒载体,转染293T细胞包装慢病毒。实验分为3组:空白组为无处理的HEK293细胞;阴性对照组为转染空载病毒的HEK293细胞;SMARCAL1过表达组为转染SMARCAL1过表达病毒的HEK293细胞。对SMARCAL1过表达慢病毒转染后用嘌呤霉素筛选获得稳定转染细胞株。通过观察转染率来检测病毒滴度;荧光定量PCR和Western印迹法验证稳转细胞株;CCK-8和EdU染色法检测细胞增殖情况;进一步通过荧光定量PCR探讨对Wnt通路的影响。结果与结论:①SMARCAL1过表达质粒基因序列与目的基因一致,经慢病毒包装后测得SMARCAL1过表达慢病毒滴度为1×108 TU/mL,空载慢病毒滴度为3×108 TU/mL;②与空白组和阴性对照组比较,SMARCAL1过表达慢病毒转染后细胞中SMARCAL1 mRNA和蛋白水平显著升高(P<0.05),细胞增殖活力明显下降(P<0.05),Wnt通路中CTNNB1和WNT4基因表达水平降低(P<0.05);③实验结果表明,SMARCAL1过表达慢病毒载体构建成功并能够在细胞中顺利表达;SMARCAL1基因可能通过Wnt通信号通路参与调节胚肾细胞增殖,进而影响肾脏发育。

BACKGROUND:Studies have found that SMARCAL1 is selectively expressed in a variety of cells in human developing and mature kidneys,indicating that it plays an important role in kidney development.OBJECTIVE:To investigate the effect of SMARCAL1 overexpression on the proliferation of embryonic kidney cells.METHODS:293T and HEK293 cells at passage 3 were selected.pHBLV-CMV-MCS-3FLAG-EF1-ZsGreen-T2A-PURO vector was used to construct lentiviral vector carrying human SMARCAL1 gene.The human SMARCAL1 gene was over-expressed using lentiviral vector and transfected into 293T cells.There were three groups:blank group untreated HEK293 cells;negative control group,in which HEK293 cells were transfected with empty virus;SMARCAL1 overexpression group,in which HEK293 cells were transfected with SMARCAL1 overexpressing virus.Stably transfected cell lines were screened using puromycin.Virus titer was determined based on the transfection rate.The stable transfection of SMARCAL1 into embryonic kidney cells was verified by quantitative real-time PCR and western blot.The effect of SMARCAL1 overexpression on the proliferation of HEK293 cells was assessed by cell counting kit-8 and EdU assays.Fluorogenic quantitative PCR was further used to evaluate the effect of SMARCAL1 overexpression on Wnt pathway.RESULTS AND CONCLUSION:The overexpressed SMARCAL1 plasmid gene sequence was consistent with the target gene.The titer of the SMARCAL1 overexpressed lentivirus was 1×108 TU/mL and 3×108 TU/mL for the control lentivirus.The mRNA and protein levels of SMARCAL1 in HEK293 cells were significantly increased after transfection with SMARCAL1 overexpressed lentivirus(P<0.05).Up-regulation of SMARCAL1 expression gradually inhibited the proliferation of HEK293 cells(P<0.05).The mRNA expression levels of CTNNB1 and WNT4 genes in the Wnt pathway were also decreased(P<0.05).The SMARCAL1 overexpressed lentiviral vector is successfully constructed and expressed in HEK293 cells.The SMARCAL1 gene may be involved in regulating the proliferation of embryonic kidney cells through the Wnt signaling pathway and may affect the development of kidney.