Huwe1-shRNA靶向沉默L2.3神经干细胞的Huwe1表达

Silencing of huwe1 gene expression in neural stem L2.3 cells transfected with GIPI shRNA lentiviral vectors

目的探讨Huwe1-shRNA通过慢病毒载体转染L2.3神经干细胞,是否可以靶向沉默L2.3神经干细胞的Huwe1基因。方法将体外培养的L2.3神经干细胞分成3组:huwe1-shRNA未转染组(control组)、阴性序列转染组(control shRNA组)、huwe1-shRNA转染组(Huwe1shRNA组)。Control组:仅常规体外培养L2.3神经干细胞。不将阴性序列和Huwe1-shRNA通过慢病毒载体转染L2.3神经干细胞。Control shRNA组:阴性序列通过慢病毒转染体外培养的L2.3神经干细胞。Huwe1shRNA组:Huwe1-shRNA通过慢病毒载体成功感染体外培养L2.3神经干细胞。分别收集三组体外培养的L2.3神经干细胞,应用western blot方法分别检测三组L2.3神经干细胞Huwe1蛋白的表达。结果与control组、control shRNA组比,Huwe1shRNA组体外培养的L2.3神经干细胞huwe1蛋白表达条带显著减弱、变细(P<0.05)。与control组Huwe1蛋白的相对表达量比,Huwe1shRNA组Huwe1蛋白的相对表达量是control组的25%(P<0.05)。与control组比,control shRNA组和Huwe1shRNA组在慢病毒载体转染L2.3神经干细胞,L2.3神经干细胞存活率无明显下降(P>0.05)结论 Huwe1shRNA通过慢病毒载体转染L2.3神经干细胞,可以靶向沉默L2.3神经干细胞Huwe1基因,却不影响L2.3神经干细胞的存活率。

Objective To investigate the slice effects of huwel shRNA on huwel gene in neuronal stem L2.3 cells. Methods The neuronal stem L2.3 cells were divided control group （control group）, control shRNA group （control non- silencing shRNA group） and huwel sliced group （huwel shRNA group）. L2.3 cells in control group were cultured in Se- rum-free Medium. Control non-silencing and rat huwel shRNA vectors were obtained from open Brosystems. Lentivival packaging was done in TLA-HEK293T cells. L2.3 cells were transduced with lectiviral stock. All the neural stern cells in different groups were collected, and the expression of huwel protein was detected by western blot technology. Results Compared those in control group and control non-silencing shRNA group, the expression of huwel protein huwel shRNA group were significantly decreased. Conclusions Knockdown huwel by GIPZ shRNA lentiviral vector decreased the ex- pression of huwel protein in L2.3 cells.