1.The Experimental Study On NESCs And ICC Intramural Co-Transplantation2.the Relatlonship Between IL-6/Stat3Expression Status And Wilms Tumor Progression

Background:Hirschsprung’s disease (HSCR) is a serious congenital malformation. It was confirmed that the deficiency or reduced intramural ganglia cells and interstitial cells of Caja (ICC) are the main pathological changes. Currently, the etiology and pathogenesis of this disease have not been fully stated. Although previous studies have showed some genetic factors responsible for human HSCR, other events such as environmental factors have been proved also involved in the incidence of the disease.Studies have shown that enteric nervous system (ENS) was an independent neural regulation system contributed to the alimentary motility. In the process of human development, neural crest cells which contribute to the migration and differentiation of enteric neural cells disabled to move into primitive digestive tube or failed differentiated into neural cells can cause neurological movement disorders of digestive tract. Advances in molecular and stem cell biology have provided new avenues for therapy of ENS disorders. It has been suggested that HSCR could be treated by stem or progenitor cell transplantation to generate a neo-ENS to restore function to the aganglionic bowel. Neuroepithelial stem cells (NESCs), recognized as the most primitive neural stem cells existing in early neural tube wall had the ability of self-renew and differentiated into neurons, astrocytes and oligodendrocytes cells. Moreover, considering the favorableness of isolation, culture, amplification, low immunogenicity and neural crest cell differentiated potential, NESCs have been recognized as the ideal donor for cell transplantation in neurological movement disorders of digestive tube.Interstitial cells of Cajal (ICC) ICC originated from mesodermal mesenchymal cells are the pacemakers in gastrointestinal (GI) muscles which could generate and propagate slow waves and mediate neuromuscular neurotransmission. Evidences showed that absence or reduction of ICC muscles could result in gastrointestinal motility disorders. For now, numbers of researches have separated out the ICC in the alimentary tract through the combination of enzymatic hydrolysis by density gradient centrifugation technology, which facilitated further researched on cell function in vitro.Considering the pathogenesis of HSCR, the ideal treatment is to compensate the missing ganglion cells and ICC, that is, cell transplantation therapy. Since1978, Sato first reported benzalkonium chloride (BAC) could selective destroyed the ENS of rat and successfully established HSCR rat model, the method is widely used for the study of this disease. However, the influence of BAC on ICC is still indistinct. Based on the previous studies, this research aimed to product rat models of HSCR using BAC and observe the effect of BAC on ganglion cells in the colon as well as ICC. Meanwhile, we transplanted NESCs together with ICC to the HSCR rat models to observe the treatment effect of the transplanted cells in intestine micro-environment and evaluate the effect of ICC on neural stem cells after co-transplanted into aganglionic bowel.Methods:NESCs and ICC were isolated from neural tube of embryonic rat (embryonic day11.5) and colon of neonatal rats respectively. Immunofluorescence histochemistry method was used to identify the extracted cells in vitro.0.5%benyalkonium chloride (BAC) was used to damage enteric nervous system to build the rat models of HSCR. After co-transplantation into the BAC-induced rat aganglionic colon, the status of survival and differentiation of the implanted cells were assessed8weeks post-transplantation using immunofluorescence, transmission electron microscope (TME) and western blotting. Colonic motility was assessed by measuring the response of muscle strips to electrical field stimulation (EFS) and the changes of intraluminal pressure responding to inflating stimulation.Results:The isolated NESCs which exhibited the Nestin immunoreactivity could differentiate into neurons and astro-cytes with MAP2and GFAP immunoreactivity respectively. The isolated ICC displayed c-Kit immunoreactivity and could interconnected with adjacent ICC by the multiple processes extended from the cell body. Four weeks after surgery, histological examinations and TEM showed that ganglion cells and interstitial cells of Cajal (ICC) significantly reduced or completely disappeared at the segment of BAC treated sit. Eight weeks after transplantation, the results showed the transplanted cells survived well. The BrdU labeled NESCs could differentiate into PGP9.5positive neurons and GFAP positive glial cells. The DAPI marked ICC with c-Kit immunoreactivity survived well and form connections with the differentiated NESCs. The co-transplantation group showed more differentiated PGP9.5-positive neurons compared with transplantation of NESCs alone (P<0.05). Western Blotting detection showed the neuronal Nitric oxide synthase (nNOS) and choline acetyl transferase (ChAT) expression were markedly elevated in the co-transplantation goup (P<0.05). Moreover, the EFS-induced reaction of the colon and contractile response to inflation after co-transplantation were successfully evoked.Conclusions:Our data indicate that reconstitution of elements in the intestinal environment such as ICC enhances the differentiation of NESCs. This may aid development of a stem cell-based treatment for HSCR. Background:Wilms’tumor (WT) is one of the most common malignant tumors in children with an incidence of approximately1in10000. The prognosis of the patients has greatly improved by the advancement of combination therapy. Nevertheless, about10%patients with WT have poor survival suffering from metastasis or recurrence. Although previous studies have showed some aberrant molecular events responsible for tumor progression, the biological factors governing Wilms’ tumor invasion and metastasis remain largely unknown. Research has suggested that deregulated inflammation is associated with most tumors. Tumorigenesis and progression associated with inflammation are known to be influenced by multiple growth factors and cytokines including interleukin-6(IL-6). The IL-6signaling pathway is activated via binding to its receptor IL-6R resulting in downstream signal transmission through various signal pathways including JAK2/STAT3signal pathway and ultimately leading to induction of target genes that control cell proliferation, tumor invasion and metastasis. This study aimed to investigate the expression profiles of IL-6and STAT3in Wilms’ tumor (WT) and their relationship with disease progression.Methods:Immunohistochemistry was used to examine IL-6and STAT3expression status in58primary tumors and18invasive/metastatic ones.Results:Positive expression rate of IL-6/STAT3was39.7%(23/58)/29.3%(17/58) in primary WT tissues, while61.1%(11/18)/33.3%(6/18) in associated invasive/metastatic tissues. The expression rate of IL-6and STAT3was higher in primary WT tumors of invasive/metastatic group than that of non-invasive/metastatic group (P=0.033; P=0.012). The expression rate of IL-6and STAT3was also associated with histopathological type (P=0.013; P=0.027). There was a positive correlation between IL-6and STAT3expression in76WT tissues (P＜0.001, r=0.444). The expression of IL-6/STAT3between primary WT and matched invasive/metastatic tissues was concordance (P=0.727; P=0.99). IL-6expression status and histopathological type were associated with disease-free survival (DFS) and overall survival (OS)(P=0.025; P=0.037), while STAT3was only correlated with DFS (P=0.004).Conclusions:IL-6and STAT3expression in WT might be correlated with progression and predict unfavorable prognosis, highlighting a new therapy target for invasive or metastatic WTs.