丙戊酸对脊髓性肌萎缩症患者神经元样细胞SMN2基因mRNA表达的影响

Effects of valproic acid on expression of SMN2 mRNA in neuron-like cells derived from patients with spinal muscular atrophy

目的探讨丙戊酸(VPA)对脊髓性肌萎缩症(SMA)患者神经元样细胞SMN2基因mRNA表达的影响。方法用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)的方法对SMA患者进行基因诊断。诱导患者的骨髓间质干细胞(MSCs)分化为神经元样细胞作为细胞模型,逆转录-聚合酶链反应(RT-PCR)和测序检测SMN2基因转录产物,半定量RT-PCR检测VPA干预前后SMN2mRNA的表达。结果RT-PCR扩增出266bp和212bp两条带,分别为全长转录产物(fl-SMNmRNA)和转录时跳过外显子7的产物(SMNΔ7mRNA);VPA干预后,fl-SMNmRNA和SMNΔ7mRNA表达均比干预前明显增加,有量效关系(干预前以及1、2、5、10mmol/LVPA干预后fl-SMNmRNA和SMNΔ7mRNA的表达分别为0·210±0·035、0·282±0·041、0·351±0·020、0·450±0·052、0·553±0·035,P<0·05和0·670±0·026、0·703±0·050、0·750±0·024、0·807±0·042、0·870±0·034,P<0·05);且fl-SMNmRNA增加的幅度大于SMNΔ7mRNA,fl-SMNmRNA/SMNΔ7mRNA的比值也逐渐增大。结论SMA患者神经元样细胞SMN2基因存在选择性剪接,VPA可能抑制选择性剪接并改善SMN2的转录,从而有望成为治疗SMA的药物。

Objective To study the effects of valproic acid （VPA） to the expression of SMN2 mRNA in neuron-like ceils （NLCs） derived from patients with spinal muscular atrophy （SMA）. Methods Polymerase chain reaction-restriction fragment length polyrnerphism （PCR-RFLP） was performed to select the patients with SMA. Mesenchymal stem cells （ MSCs ） derived from patients were induced into NLCs which were set as the model of neurons. The transcripts of SMN2 by reverse transcription-polymerase chain reaction （RT-PCR） combined with sequenceing were detected. Semi-quantitative RT-PCR analysis was performed to detect the changes of SMN2 mRNA expression between before and after the NLCs were treated by VPA. Results Two bands （266 bp and 212 bp） were found in the gel picture of RT-PCR, which were respectively the products of full length transcript （fl-SMN mRNA） and skipping exon 7 （SMN△7 mRNA）. NLCs had significantly increased fl-SMN mRNA and SMN△7 mRNA levels as compared with the untreated cells after treatment with VPA, and shown a dose effect（0. 210 ±0. 035,0. 282 ±0. 041,0. 351 ± 0. 020,0. 450 ± 0. 052,0. 553 ± 0. 035, P 〈 0. 05 ; 0. 670 ± 0. 026,0. 703 ± 0. 050,0. 750 ± 0. 024,0. 807 ± 0. 042,0. 870 ± 0. 034, P 〈 0. 05 ）. The amplitude of fl-SMN mRNA increase was higher than that of SMN△7 mRNA, and the ratios of fl-SMN mRNA/SMN△7 mRNA were increased. Conclusions The alternative splicing should exist in SMN2 transcription. Valproic acid might inhibit the alternative splicing and increase the full-length transcripts of SMN2, showing a promising treatment for SMA patients.