Department of General Surgery, Qilu Hospital of Shandong University, Jinan 250012, China.
Cell Biol Int. 2013 May;37(5):458-63. doi: 10.1002/cbin.10060. Epub 2013 Mar 15.
Gene therapy and nerve stem cells isolated from the developing human enteric nervous system (ENS) are significant. They may open the route for the cell therapy of Hirschsprung's disease (HD). We have constructed the recombinant adenovirus-carrying glial cell line-derived neurotrophic factor (GDNF) and endothelin receptor B (EDNRB) gene, and investigated the exosomatic coexpression in neural stem cells. The recombinant adenovirus Ad-GE coexpressing GDNF and EDNRB gene was constructed by the AdEasy system and confirmed by the reverse transcription polymerase chain reaction (RT-PCR) method. Expression of exogenous genes in neural stem cells after transfection was confirmed by the flow cytometry and real-time fluorescence quantitative PCR. Fragments of pAd Track-CMV-GE were consistent with GDNF and EDNRB. The green fluorescence of the positive cells was followed by fluorescence microscopy at 24 h after NSCs had been transfected, reaching a peak at 72 h after transfection. Flow cytometry showed that the efficiency of transfection was 15.0, 23.6, and 25.4% at 24, 48 and 72 h respectively. Real-time fluorescence quantitative PCR showed the expression levels of mRNA of GDNF and EDNRB in 48 and 72 h groups were obviously higher than that in 24 and 96 h groups. Recombinant adenovirus carrying GDNF and EDNRB genes are coexpressed in neural stem cells, which may offer the possibility of a novel approach to local combination gene therapy for Hirschsprung's disease.
基因治疗和从发育中的人肠神经系统（ENS）中分离的神经干细胞是重要的。它们可能为先天性巨结肠症（HD）的细胞治疗开辟道路。我们构建了携带胶质细胞源性神经营养因子（GDNF）和内皮素受体 B（EDNRB）基因的重组腺病毒，并研究了其在神经干细胞中的体外共表达。通过 AdEasy 系统构建了携带 GDNF 和 EDNRB 基因的重组腺病毒 Ad-GE，并通过逆转录聚合酶链反应（RT-PCR）方法进行了验证。通过流式细胞术和实时荧光定量 PCR 证实了转染后神经干细胞中外源基因的表达。pAd Track-CMV-GE 片段与 GDNF 和 EDNRB 一致。转染后 24 小时，阳性细胞的绿色荧光通过荧光显微镜观察，72 小时后达到峰值。流式细胞术显示，转染 24、48 和 72 小时后，转染效率分别为 15.0%、23.6%和 25.4%。实时荧光定量 PCR 显示，48 和 72 小时组的 GDNF 和 EDNRB 基因 mRNA 表达水平明显高于 24 和 96 小时组。携带 GDNF 和 EDNRB 基因的重组腺病毒在神经干细胞中共同表达，这可能为先天性巨结肠症的局部联合基因治疗提供新的途径。
