稳定表达hSOD1^(G93A)基因的肌萎缩侧索硬化体外细胞培养模型的建立与鉴定

Establishment and identification of a motor neuron-like cell line stably expressing hSOD1^(G93A)

目的建立并鉴定稳定表达G93A型突变人超氧化物歧化酶(hSOD1G93A)基因的肌萎缩侧索硬化体外细胞培养模型。方法利用活化的树突状聚合物将空质粒、hSOD1WT、hSOD1G93A基因转染入VSC4.1细胞内,G418抗性筛选,从而建立稳定的肌萎缩侧索硬化体外细胞模型。用免疫荧光技术检测VSC4.1细胞系运动神经元标志物。蛋白印迹实验鉴定hSOD1WT蛋白、hSOD1G93A蛋白的表达。MTT法检测细胞模型生长曲线。结果VSC4.1细胞分化前表达ClassⅢβ-Tubulin、MNR2,分化后表达ClassⅢβ-Tubulin、MNR2、NF200、MAP2等运动神经元标志物。VSC4.1-hSOD1WT、VSC4.1-hSOD1G93A细胞均过表达人来源的SOD1,而VSC4.1-mock则不表达。与VSC4.1-mock、VSC4.1-hSOD1WT相比,VSC4.1-hSOD1G93A生长缓慢,在48、72 h细胞活力均低于VSC4.1-mock(P=0.031,P=0.000)、VSC4.1-hSOD1WT(P=0.001,P=0.000),其余时间点无明显差异(P>0.05)。结论成功建立稳定表达hSOD1WT、hSOD1G93A基因的VSC4.1细胞系,为进一步研究肌萎缩侧索硬化的发病与治疗奠定了良好的基础。

Objective To establish and identify the motor neuron-likeVSC4. 1 cell line stably expressing hSOD1^G93A. Methods The pCI-neo-veetors, pCI-neo/WT-SOD1 and pCI-neo/G93A-SOD1 were transfected into VSC4.1 cells by activated-dendrimer structure. The stably transfected ceils were screened by G418. The expression of hSOD1 in VSC4. 1 cells was detected by Western blotting. The growth of the established cell line was assessed by MTT assay. Resuits Both VSC4.1-hSOD1^WT and VSC4. 1-hSOD1^G93A cells expressed hSOD1 detected by Western blotting, while the VSC4. 1-mock cells did not express that at all. The viability of VSC4. 1-hSOD1^G93A cells was lower than that of VSC4.1-mock cells （P =0. 031, P =0. 000） and VSC4. 1-hSOD1^WT cells （P =0. 001 ,P =0. 000） at 48 h and 72 h. There was no significant difference at other time points （ P 〉 0. 05 ）. Conclusions The VSC4. 1 cell line stably expressing hSOD1^WT and hSOD1^G93A is successfully established. It may provide a foundation for further exploration of the pathogenesis and therapy of amyotrophic lateral sclerosis （ALS）.