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Expression and Significance of Neuroligins in Myenteric Cells of Cajal in Hirschsprung's Disease

PLoS ONE (2013) 8(6)
DOI: 10.1371/journal.pone.0067205
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Abstract

Purpose:The aim of this study was to investigate the expression and significance of neuroligins in myenteric cells of Cajal (ICC-MY) in Hirschsprung's disease (HSCR).Methods:Longitudinal muscle with adherent myenteric plexus (LMMP) from surgical excision waste colon of HSCR children were prepared by peeling off the mucous layer, sub-mucosal layer and circular muscle. Neuroligins, c-Kit (c-Kit-immunoreactivity representing ICC) and their relationship were assessed by double labeling immunofluorescence staining. ICC-MY were dissociated and cultured from LMMP by enzymolysis method, and were purified and analyzed using a combination of magnetic-activated cell sorting (MACS) and flow cytometry (FCM). Western-blot analysis was applied to compare and evaluate the expression levels of neuroligins in ICC-MY which were dissociated from different segments of HSCR (ganglionic colonic segment, transitional colonic segment and aganglionic colonic segment).Results:Neuroligins and c-Kit were expressed on the same cells (ICC-MY); ICC-MY were dissociated, cultured and purified. For HSCR, neuroligins were expressed significantly in ICC-MY from ganglionic colonic segments, moderately in those from transitional colonic segments and down-regulated significantly in those from aganglionic colonic segments.Conclusions:Neuroligins were expressed in ICC-MY of human beings, and the expression varies from different segments of HSCR. This abnormal expression might play an important role in the pathogenesis of this disease through affecting the synaptic function of ICC-MY. © 2013 Wang et al.

Figures

  • Table 1. Primary antibody.
  • Table 2. Secondary antibody.
  • Figure 1. Immunohistochemical staining was performed on LMMP, light microscopy was used for observation. ICC-MY were identified by immunoreactivity for the tyrosine kinase receptor c-Kit(CD117) (brownish yellow, network structure) on LMMP. Figure 1A represented ganglionic colonic segment and Figure 1B represented aganglionic colonic segment. No obvious disruptions of ICC-MY network were showed in aganglionic segments. Magnification 406. doi:10.1371/journal.pone.0067205.g001
  • Figure 2. Double-labeled immunofluorescent staining of neuroligins and c-Kit was performed on LMMP. Laser confocal microscope was used for observation. Figure 2A, 2B and 2C represented that in ganglionic segments, neuroligins (Figure 2A, red) were expressed in the same cells (merged, Figure 2C, yellow) in which c-Kit was expressed (Figure 2B, green). Figure 2D, 2E and 2F represented that in aganglionic segments, neuroligins (Figure 2D, red) were expressed in the same cells (merged, Figure 2F, yellow) in which c-Kit was expressed (Figure 2E, green). In aganglionic segments, the expression of neuroligins was down-regulated and neuroligins network was disrupted (Figure 2D), but no obvious disruptions of ICC-MY network were showed (Figure 2E). Scale bars:50 mm. doi:10.1371/journal.pone.0067205.g002
  • Figure 3. ICC-MY percentages of ganglionic, transitional and aganglionic segments from one case of HSCR were shown by Figure 3A, 3B and 3C, respectively.
  • Figure 4. ICC-MY percentages of ganglionic, transitional and aganglionic segments from all 50 cases HSCR and the numerical data are presented as the mean6standard deviation (90.9863.24, 90.3063.09, 90.0163.11, n=50). Statistical analysis was performed using T test, and P,0.05 was considered statistically significant. doi:10.1371/journal.pone.0067205.g004
  • Figure 5. Immunofluorescence labeling was used to identify ICC-MY. c-Kit was expressed in ICC-MY, whose cell bodies and processes were obviously expressed. These cells interact and interwove with each other, whose shape was fusiform or triangular. Scale bars:25 mm. doi:10.1371/journal.pone.0067205.g005
  • Figure 6. Western blot analysis was used to compare and evaluate the expressing quantity of neuroligins in ICC-MY of different segments in HSCR. Neuroligins were expressed significantly in ICC-MY of ganglionic colonic segment, moderately in transitional segment, and obviously downed-regulated in aganglionic colonic segment. doi:10.1371/journal.pone.0067205.g006

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CITATION STYLE

APA

Wang, J., Mou, Y., Zhang, Q., Zhang, F., Yang, H., Zhang, W., & Li, A. (2013). Expression and Significance of Neuroligins in Myenteric Cells of Cajal in Hirschsprung’s Disease. PLoS ONE, 8(6). https://doi.org/10.1371/journal.pone.0067205

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