利用RNAi建立脊髓性肌萎缩症的细胞模型  

Establishment of spinal muscular atrophy cell model by RNAi

作　　者：杨晓苏[1] 胡益民[1] 肖波[1] 杨期东[1] 赵惠敏[1] 

机构地区：[1]中南大学湘雅医院神经内科,长沙410008

出　　处：《中南大学学报（医学版）》2008年第12期1108-1112,共5页Journal of Central South University ：Medical Science

摘　　要：目的:应用RNA干扰沉默SMN1基因的表达建立脊髓性肌萎缩症(SMA)的细胞模型。方法:将pshRNA-SMN1重组质粒转染人骨髓间充质干细胞(hMSCs),经G418筛选得到能稳定表达目的shRNA的单克隆细胞系后将其诱导分化为神经元样细胞(NLCs)建立SMA细胞模型组。同期设立转染重组质粒pshRNA-0的对照组和未转染重组质粒的空白对照组。观察NLCs的细胞形态,采用免疫细胞化学染色分析NLCs的NSE和NF蛋白表达,用RT-PCR和免疫印迹方法检测NLCs的SMN mRNA及其蛋白表达。结果:诱导后各组细胞呈典型的神经元样细胞形态且NSE和NF蛋白表达阳性;其fl-SMN mRNA,△7-SMN mRNA及fl-SMN蛋白表达均较诱导前增加(P<0.05),但SMA细胞模型组的fl-SMN mRNA及蛋白表达仍明显低于对照组(P<0.01);而诱导后△7-SMN mRNA的表达在各组间差异无统计学意义(P>0.05)。结论:SMN1 mRNA及其蛋白被抑制的MSCs诱导分化为NLCs后可以作为SMA的细胞模型。Objective To establish spinal muscular atrophy （SMA） cell model by blocking the expression of SMN1 gene with shRNA. Methods The recombinant SMN 1 shRNA expression vector was constructed. SMA cell model was established by human mesenchymal stem cells （ hMSCs ） that the vector was transfected into were differentiated to neuron like cells （NLCs）. At the same time the control groups were established that the shRNA - 0 vector was transfected into and no vector was transfected into. The expression of fl-SMN and △7-SMN mRNA was observed by RT-PCR analysis The expression of fl-SMN protein was detected by Western blot. Results The cells of all the groups were neuron like cells after being differentiated and the protein expression of NSE and NF was positive. The expression of fl-SMN and △7-SMN mRNA and protein of NLCs in each group was upregulated （P〈0.05）, but the expression of △7-SMN mRNA and protein in SMA model group was lower than that in the control group （ P 〈 0.05 ）. The expression of △ 7-SMN mRNA between the groups had no statistical difference （ P 〉 0.05 ）. Conclusion The NLCs, which recombinant SMN1 shRNA expression vector was transfected into, can be regarded as SMA cell model.

关 键 词：RNA干扰 运动神经元生存蛋白 脊肌萎缩症 人间充质干细胞 

分 类 号：R744[医药卫生—神经病学与精神病学]