- Title
-
Down-Regulation of Double C2 Domain Alpha Promotes the Formation of Hyperplastic Nerve Fibers in Aganglionic Segments of Hirschsprung's Disease
- Authors
- Xiao, J., Meng, X., Chen, K., Wang, J., Wu, L., Chen, Y., Yu, X., Feng, J., Li, Z.
- Source
- Full text @ Int. J. Mol. Sci.
Intestinal innervation in the HSCR colon. (A) Intestinal innervation network. (B) Intrinsic nervous system and exogenous nerve fibers in the HSCR colon. Abbreviations: HSCR, Hirschsprung’s disease; T, thoracic vertebrae; L, lumbar vertebrae; S, sacral vertebrae; TS, thoracic spinal; LS, lumbar spinal; SS, sacral spinal; CG, celiac ganglion; SMG, superior mesenteric ganglion; IMG, inferior mesenteric ganglion; PP, pelvic nerve; ENS, enteric nervous system; MP, myenteric plexus; SMP, submucosal plexus. |
Aganglionic segments of HSCR patients and model mice were divided into four grades according to AChE+ nerves. (A–D) Different grades of AChE+ in colonic aganglionic segments of HSCR patients. (E–H) Different grades of AChE+ in colonic aganglionic segments of HSCR model mice. (I,J) The ratio of AchE+ crypts in the colons of HSCR patients and model mice. **, p < 0.01; ***, p < 0.001, one-way ANOVA with Bonferroni post hoc, n = 10. |
Relative expression levels of DOC2A in different grades. (A,C) Quantification of mRNA expression levels of DOC2A in different grades determined by RT-qPCR. (B,D) Protein expression of the DOC2A in different grades determined by western blots. **, p < 0.01; ***, p < 0.001, one-way ANOVA with Bonferroni post hoc, n = 3. Abbreviations: DOC2A, Double C2 domain alpha; RT-qPCR, real-time quantitative PCR. |
Comparison of neuron polarity among different N-2a cell groups. (A–C) Double immunofluorescent labeling (Tuj1) of N-2a cell lines. (D,E) Comparison of mean area of per cell/um2 and amount of neurite outgrowth per cell in different groups. ***, p < 0.001, one-way ANOVA with Bonferroni post hoc, n = 3. Knockdown, infected with Doc2a-lentivirus; negative control, Lentivirus Luciferase Reporter negative control; blank control, cultured with an equal medium. |
Comparison of neuron polarity among different neural sphere groups. (A–C) Nerve fiber phenotypes of neural spheres in different groups. (D–F) Partial enlarged drawing of (A–C) separately. (G–I) IF (Tuj1) showed the nerve fibers and neurites of neural spheres. Red arrow, nerve fiber; yellow arrowhead, neurite. (J) Comparison of average number of nerve fiber connections. (K) Comparison of average number of neurite outgrowth per neural sphere. **, p < 0.01; ***, p < 0.001, one-way ANOVA with Bonferroni post hoc, n = 3. Knockdown, infected with Doc2a-lentivirus; negative control, Lentivirus Luciferase Reporter negative control; blank control, cultured with an equal medium. Abbreviations: IF, immunofluorescence; DAPI, 4’,6-diamidino-2-phenylindole. |
Investigation of Doc2a down-regulation promoting hyperplastic nerve fibers in zebrafish intestinal tract. (A–D) Comparison of enteric nerve fibers (Tubulin) in different groups. (E–H) Comparison of enteric neurons (HuC/D) in different groups. Red arrow, enteric nerve fiber; yellow arrowhead, enteric neuron. (I) Diagram of zebrafish. (J) Comparison of average number of enteric neurons in zone 1 and 2. (K) Comparison of length proportion of intestinal tract with hyperplastic nerve fibers in zone 1 and 2. #, p > 0.05; ***, p < 0.001, one-way ANOVA with Bonferroni post hoc, n = 3. Abbreviations: MO, Morpholino; ret, ret proto-oncogene. PHENOTYPE:
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Expression relationship between DOC2A and UNC13B. (A,B) Change in UNC13B after DOC2A being knocked down in N-2a cell lines and neural spheres. (C–J) Expression levels (IHC) of DOC2A and UNC13B in different grades of HSCR patient colons. **, p < 0.01; ***, p < 0.001, one-way ANOVA with Bonferroni post hoc, n = 3. (K,L), protein interaction (CO-IP) between DOC2A and UNC13B. Abbreviations: UNC13B, unc-13 homolog B; IHC, immunohistochemistry; HE, hematoxylin-eosin staining. |