Preliminary Study On The Anting Neyronal Differentiation Of Retinoic Acid Related Gene HA117

PART ONE THE EFFECT OF HA117 ON THE NEUTONAL DIFFERENTIAYION OF HUCMSCObjective: HA117 is a multidrug resistance gene expressed in HL-60 cells treated with all-trans retinoic acid. Previous bioinformatics analyses suggested a role for HA117 during neural development. Therefore, we investigated the effects of HA117 on the neuronal differentiation of human umbilical cord-derived mesenchymal stem cells(h UCMSCs).Methods: We obtained h UCMSCs and verified their identity and differentiation potential through flow cytometric analysis of cell surface markers and in vitro differentiation into adipocytes. We then transduced cells with lentiviruses expressing HA117 and induced differentiation by culturing the cells in neuronal differentiation media. We then assessed the efficiency of differentiation by measuring the expression levels of the neuronal markers β-tubulin III, neuron-specific enolase(NSE), and nestin by quantitative PCR(q PCR), Western blot, and immunofluorescence.Results: The transduction of h UCMSCs with HA117-expressing lentivirus was highly efficient(68.9%). We found that the overexpression of HA117 resulted in significantly reduced levels of β-tubulin III, NSE, and nestin compared with GFP-expressing or untransduced cells.Conclusions: Our findings suggest that HA117 inhibits the differentiation of h UCMSCs into neuron-like cells.PART TWO EXPRESSION CHARACTERISTICS AND SIGNIFICANCE OF HA117 AND SHH SIGNALING PATHWAY IN HIRSCHPRUNG’S DISEASEObjective:To research the relationship between HA117 and SHH signaling pathway( SHH, Ptch1,Gli1) and HSCR by studying their expression in different colon segments of patients with Hirschsprung’s disease(HSCR).Methods:Colon specimens were collected from 30 patients dignosed with HSCR during 1operation in Children’s Hospital of Chongqing Medical University from2014 to 2015 and grouped into three segments: proximal anastomosis, dilated segment and stenotic segment.The m RNA levels of HA117,SHH,Smo,Ptch1,Gli1 were evaluated by using quantitative real—time PCR. Protein levels of SHH,Ptch1,Gli1 were tested by Western Blot and immunohistochemistry.Results:The m RNA levels of HA117 in stenotic segment(1.52±0.17)was significantly higher than proximal anastomosis(0.28±0.04)(P<0.05). While The m RNA levels of SHH, Ptch1,Gli1 in stenotic segment(0.53±0.11,0.48±0.11,0.62±0.13) were significantly lower than proximal anastomosis(1.23±0.26,1.20±0.22,1.71±0.46)(P<0.05). Protein levels of SHH,Ptch1,Gli1 were consistent with the m RNA levels(P<0.05). Immunohistochemistry staining of SHH,Ptch1,Gli1 in stenotic segment were significantly less intense than which in proximal anastomosis.Conclusions:HA117 and SHH signaling pathway may have intimate connection with the development of enteric nervous system(ENS),which may be one of the important mechanisms that activing the pathogenesis occurrence of HSCR.