Department of Orthopaedics, Shanghai Sixth People's Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai, China.
Cell Transplant. 2011;20(3):381-90. doi: 10.3727/096368910X524773. Epub 2010 Aug 18.
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, n = 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.
神经干细胞（NSCs）的治疗功效研究最近已成为热点。本研究通过探讨神经营养因子-3（NT-3）与来源于大鼠胚胎脊髓的 NSCs 联合应用对大鼠胫神经切断后失神经骨骼肌萎缩的延迟作用，为临床应用 NSCs 治疗肌萎缩提供实验依据。应用 PCR 扩增 NT-3 基因并亚克隆至慢病毒载体 pWPXL-MOD，构建慢病毒表达载体 pWPXL-MOD-NT-3，阳性克隆经测序鉴定正确后，转染 NSCs 并诱导分化，鉴定后用于移植。60 只成年大鼠切断胫神经后，随机分为实验组（n = 30）和对照组（n = 30），实验组植入 NSCs-NT-3，对照组植入空载 pWPXL-MOD 转染的 NSCs。分别通过免疫组化、RT-PCR、Western blot 检测细胞存活、分化情况，免疫荧光检测 NT-3 基因表达，电生理检测、腓肠肌横截面积（CSA）定量分析观察移植后对失神经骨骼肌萎缩的延迟作用。结果显示，克隆得到由 777bp 组成的 NT-3 基因，体外培养的 NSCs 和 NSCs-NT-3 中均有表达，且实验组明显高于对照组（P < 0.01）。移植后 4 周，实验组 ChAT 免疫阳性细胞数明显高于对照组（P < 0.01），12 周时仅实验组移植部位周围有 ChAT 免疫阳性细胞。电生理和腓肠肌 CSA 定量分析结果显示实验组有延迟失神经骨骼肌萎缩的作用。结论：表达 NT-3 的 NSCs 可能成为延迟失神经骨骼肌萎缩的更理想移植细胞。
