Wagner Justin P, Sullins Veronica F, Dunn James C Y
Department of Surgery, Division of Pediatric Surgery, University of California, Los Angeles, Los Angeles, CA 90095-1749, USA.
Department of Surgery, Division of Pediatric Surgery, University of California, Los Angeles, Los Angeles, CA 90095-1749, USA; Department of Bioengineering, University of California, Los Angeles, Los Angeles, CA 90095-7098, USA.
J Pediatr Surg. 2014 Dec;49(12):1809-14. doi: 10.1016/j.jpedsurg.2014.09.023. Epub 2014 Oct 1.
Skin-derived precursor cells (SKPs) may regenerate the enteric nervous system in Hirschsprung's disease. SKPs migrate and differentiate into myenteric ganglia in aganglionic intestine. We sought to characterize the time-course of SKP gangliogenesis and enteric neurotransmitter synthesis in vivo.
Adult Lewis rat jejunal segments were isolated and denervated with benzalkonium chloride (BAC). Denervation was evaluated by immunohistochemical (IHC) stains for markers of mature neuronal and glial cells. Green fluorescent protein (GFP)-expressing neonatal rat SKPs were cultured in neuroglial-selective medium. SKPs were transplanted into aganglionic segments 65-85days after BAC treatment. IHC was performed to identify glia, neurons, and neurotransmitter synthesis in GFP+cells between post-transplant days 1 and 28.
Aganglionosis was confirmed by IHC. On post-transplant days 1 and 2, GFP+cells were detected near injection sites within the muscularis propria. GFP+cell clusters were evident only between longitudinal and circular smooth muscle layers at post-transplant days 14, 21, and 28. These structures co-expressed markers of mature neurons and gliocytes. Several markers of neurotransmitter synthesis were detected in GFP+clusters at days 21 and 28.
SKPs are capable of enteric neuroglial differentiation in vivo. SKPs migrate to the intermuscular layer of aganglionic intestine within days of transplantation. Our observations suggest that SKPs are capable of generating enteric ganglia in aganglionic intestine.
皮肤来源的前体细胞（SKPs）可能会在先天性巨结肠病中使肠神经系统再生。SKPs在无神经节的肠段中迁移并分化为肌间神经节。我们试图在体内表征SKP神经节形成和肠神经递质合成的时间进程。
分离成年Lewis大鼠的空肠段，并用苯扎氯铵（BAC）去神经支配。通过对成熟神经元和神经胶质细胞标志物进行免疫组织化学（IHC）染色来评估去神经支配情况。将表达绿色荧光蛋白（GFP）的新生大鼠SKPs培养在神经胶质细胞选择性培养基中。在BAC处理后65 - 85天，将SKPs移植到无神经节段。在移植后第1天至第28天之间，进行IHC以鉴定GFP +细胞中的神经胶质细胞、神经元和神经递质合成。
通过IHC证实了无神经节症。在移植后第1天和第2天，在固有肌层内注射部位附近检测到GFP +细胞。在移植后第14天、21天和28天，GFP +细胞簇仅在纵行和环行平滑肌层之间明显可见。这些结构共表达成熟神经元和神经胶质细胞的标志物。在第21天和第28天，在GFP +细胞簇中检测到几种神经递质合成的标志物。
SKPs在体内能够分化为肠神经胶质细胞。SKPs在移植后数天内迁移到无神经节肠段的肌间层。我们的观察结果表明，SKPs能够在无神经节的肠段中生成肠神经节。
