Combined Application of Neutrophin-3 Gene and Neural Stem Cells Is Ameliorative to Delay of Denervated Skeletal Muscular Atrophy After Tibial Nerve Transection in Rats
Combined Application of Neutrophin-3 Gene and Neural Stem Cells Is Ameliorative to Delay of Denervated Skeletal Muscular Atrophy After Tibial Nerve Transection in Rats
复制标题
DOI：
10.3727/096368910x524773
复制
发表时间：
2011-01-01
影响因子：
3.3
通讯作者：
Gu, Yudong
中科院分区：
文献类型：
--
作者：
Lin, Sen;Xu, Jianguang;Gu, Yudong
检测神经干细胞(NSCs)的治疗效果最近已成为许多研究的焦点。本研究旨在探讨神经营养素-3(NT-3)与大鼠胚胎脊髓来源的神经干细胞联合应用对胫神经切断后失神经骨骼肌萎缩的延缓作用。通过聚合酶链式反应扩增NT-3基因，将其亚克隆到慢病毒载体pWPXL-MOD中，构建慢病毒表达载体pWPXL-MOD-NT-3。获得一株表达NT-3的阳性克隆(命名为NSCs-NT-3)，并用于体外分化和移植。取成年大鼠60只，切断胫神经，随机分为两组：神经干细胞移植组(实验组，n=30)和pWPXL-MOD转基因神经干细胞组(对照组，n=30)。通过免疫组织化学染色、RT-PCR、Western印迹、电生理分析和腓肠肌平均横截面积(CSA)检测细胞存活和分化、NT-3基因表达及延缓失神经骨骼肌萎缩的作用。结果表明，克隆的NT-3基因全长为777bp，在体外NSCs和NSCs-NT-3之间存在明显的差异表达。胆碱乙酰转移酶(ChAT)免疫阳性细胞定量分析显示，植入后4周，实验组的胆碱乙酰转移酶(ChAT)免疫阳性细胞明显多于对照组(P<；0.01)。移植后12周，仅实验组在移植区附近可见ChAT免疫阳性细胞。此外，在肌电检查和腓肠肌平均CSA中也显示了延缓失神经骨骼肌萎缩的作用。这些结果表明，内源性表达NT-3的神经干细胞可能是延缓失神经骨骼肌萎缩的较好的移植候选细胞。
Examination of the therapeutic efficacy of neural stem cells (NSCs) has recently become the focus of much investigation. In this study we present an insight of the effects of combined application with neurotrophin-3 (NT-3) and NSCs that derived from rat embryo spinal cord on delaying denervated skeletal muscular atrophy after tibial nerve was severed. NT-3 gene was amplified by PCR and subcloned into lentiviral vector pWPXL-MOD to construct a lentiviral expression vector pWPXL-MOD-NT-3. A positive clone expressing NT-3 (named NSCs-NT-3) was obtained and used for differentiation in vitro and transplantation. Sixty adult rats, whose tibial nerves were sectioned, were divided into two groups: one grafted with NSCs-NT-3 (experimental group, n = 30) and the other with NSCs transfected by pWPXL-MOD (control group, it = 30). The cell survival and differentiation, NT-3 gene expression, and effect of delaying denervated skeletal muscular atrophy were examined through immunohistostaining, RT-PCR, Western blot, electrophysiological analysis, and mean cross-sectional area (CSA) of gastrocnemius, respectively. The results show that the NT-3 gene, which is comprised of 777 bp, was cloned and significantly different expression were detected between NSCs and NSCs-NT-3 in vitro. Quantitative analysis of the choline acetyltransferase (ChAT) immunopositive cells revealed a significant increase in experimental group compared to the control group 4 weeks after implantation (p < 0.01). Twelve weeks after transplantation, the ChAT immunopositive cells were detected near the engrafted region only in experimental group. Furthermore, the effect in delaying denervated skeletal muscular atrophy is indicated in the EMG examination and mean CSA of gastrocnemius. These findings suggest that the neural stem cells expressing NT-3 endogenously would be a better graft candidate for the delay of denervated skeletal muscular atrophy.