A Morphology-based Analysis of Biliary Ductules After Kasai Procedure and a Review of the Literature.

OBJECTIVE: To explore the distribution of biliary ductules in biliary remnants of patients with biliary atresia and to investigate the relationship between the ductules and the prognosis after Kasai portoenterostomy. PATIENTS AND METHODS: From October 01, 2015 to September 30, 2017, 46 patients who were diagnosed as type 3 biliary atresia were enrolled in this study. Continuous sections of biliary remnants were stained with cytokerantin 19 antibody. The number, area, and distribution of micro-biliary ductules of each section were recorded. According to the number of ductules in the most proximal section (n ≥ 20 or n < 20), patients were divided into two groups (A or B) and followed up for 1-3 y, including cholangitis, jaundice clearance, and survival with native liver. RESULTS: Four patients had no micro-biliary ductules. In 17 patients with ductules, the numbers at bilateral parts were similar (P > 0.05), while the ductules in the middle part were significantly less than bilateral parts (P < 0.05). Starting from 2 mm from the proximal end of remnants, the number of ductules significantly and gradually decreased (P < 0.05). The total area of ductules in Group A was significantly increased compared to that in Group B (P < 0.05). Patients in Group A had significantly higher jaundice clearance rate and better survival rate with native liver than patients in Group B (both, P < 0.05). Patients had significantly higher incidence of cholangitis in Group A compared to Group B (P < 0.05). CONCLUSIONS: The number/area of ductules yielded by technical precision is closely related to effective bile drainage, jaundice clearance, and first onset of cholangitis in patients after Kasai procedure.

Optimal time for single-stage pull-through colectomy in infants with short-segment Hirschsprung disease.

OBJECTIVE: Short-segment Hirschsprung disease (HSCR) is the predominant type of HSCR that affects approximately 75% of patients. Whether single-stage endorectal pull-through (ERPT) surgery is appropriate for neonatal patients with HSCR has not been definitively determined. This retrospective cohort study concerning infants with short-segment HSCR investigated the optimal age for single-stage ERPT surgery, regardless of the operative approach. METHODS: The 198 patients were stratified by operative age ≤ 3 or > 3 months (groups A or B, respectively, n = 62 and 136, respectively). Diagnoses of short-segment HSCR were conducted by preoperative contrast enema and rectal suction biopsy with acetylcholinesterase immunohistochemical staining. The perioperative clinical course for all patients was reviewed and the accuracy rate of the preoperative diagnoses and postoperative short- and midterm outcomes were assessed. RESULTS: The rates of diagnostic accuracy, according to the results of the preoperative contrast enema or rectal suction biopsy, were lower in group A (67.2 and 93.5%, respectively) than in group B (81.4 and 94.9%, respectively). In groups A and B, 49 (79.1%) and 108 (79.4%) infants, respectively, completed follow-up examinations. The short-term outcomes were postoperative HSCR-associated enterocolitis, adhesive bowel obstruction, anastomosis leakage, and anal stenosis during the first 12 months after surgery. The midterm outcomes were incontinence and constipation at ~24 months after surgery. Compared with group B, group A experienced more incidences of anastomotic leakage in the short-term and more soiling in the midterm. In groups A and B, the rates of constipation recurrence were nil and 1.9%, respectively. CONCLUSION: Infants with HSCR ≤3 months old at the time of single-stage ERPT surgery showed lower rates of accurate and conclusive diagnostic results and poorer postoperative outcomes. Waiting to perform this surgery until infants are older might be more beneficial.

GDNF and NT-3 induce progenitor bone mesenchymal stem cell differentiation into neurons in fetal gut culture medium.

With the increasing use of bone marrow mesenchymal stem cells (BMSCs) in cell therapies, factors regulating BMSC differentiation have become the interest of current research. In this study, we investigated the effects of glial cell-derived neurotrophic factor (GDNF) and neurotrophin-3 (NT-3) on the course of BMSC differentiation. BMSCs were isolated from rat bone marrow and transfected with GDNF and NT-3 genes. Compared to mock-transfected BMSCs, GDNF and NT-3 induced BMSC differentiation to reveal neuron-like characteristics, i.e., the positive expression of neuronal marker MAP-2 and astrocyte marker GFAP, as detected by immunofluorescence assays. Semi-quantitative polymerase chain reaction (PCR) and western blot analyses showed that the increase of expression of GDNF and NT-3 in BMSCs also simultaneously elevated the mRNA expression of NSE, nestin, and MAP-2. Furthermore, the cell patch-clamp test demonstrated that the overexpression of GDNF and NT-3 in BMSCs enhanced voltage-activated potassium currents, implying that BMSCs possess great potential as a cell-based therapeutic candidate to treat neurological diseases.

Mesenchymal Stem Cells Attenuates Hirschsprung Disease-Associated Enterocolitis by Reducing M1 Macrophages Infiltration via COX-2 Dependewhant Mechanism.

OBJECTIVE AND DESIGN: Hirschsprung disease-associated enterocolitis (HAEC) is a common life-threatening complication of Hirschsprung disease (HSCR). We aimed to investigate the effectiveness, long-term safety and the underlying mechanisms of Mesenchymal stem cells (MSCs) based therapy for HAEC. MATERIAL OR SUBJECTS: Specimens from HSCR and HAEC patients were used to assess the inflammatory condition. Ednrb knock-out mice was used as HAEC model. MSCs was intraperitoneally transplanted into HAEC mice. The therapy effects, long-term outcome, safety and toxicity and the mechanism of MSCs on the treatment of HAEC were explored in vivo and in vitro. RESULTS: Intestinal M1 macrophages infiltration and severe inflammation condition were observed in HAEC. After the injection of MSCs, HAEC mice showed significant amelioration of the inflammatory injury and inhibition of M1 macrophages infiltration. The expression levels of pro-inflammatory cytokines (TNF-α and IFN-γ) were decreased and anti-inflammatory cytokines (IL-10 and TGF-ß) were increased. In addition, we found that effective MSCs homing to the inflamed colon tissue occurred without long-term toxicity response. However, COX-2 inhibitor could diminish the therapeutic effects of MSCs. Using MSCs and macrophages co-culture system, we identified that MSCs could alleviate HAEC by inhibiting M1 macrophages activation through COX-2-dependent MAPK/ERK signaling pathway. CONCLUSIONS: MSCs ameliorate HAEC by reducing M1 macrophages polarization via COX-2 mediated MAPK/ERK signaling pathway, thus providing novel insights and potentially promising strategy for the treatment or prevention of HAEC.

Subtotal colectomy with a single-incision laparoscopic surgery technique in children with long-segment Hirschsprung disease and allied disorders.

BACKGROUND: Over the last 15 years, the laparoscopic-assisted endorectal pull-through procedure has become the standard treatment for Hirschsprung disease in many centers around the world. Recently, single-incision laparoscopic techniques have drawn more attention. We describe a single-incision laparoscopic surgery (SILS) subtotal colectomy to treat long-segment Hirschsprung disease (LSHD) and Hirschsprung disease allied disorder (HAD) in children. METHODS: A total of 22 patients who underwent SILS subtotal colectomy, including three patients with a failed first surgery, were included in this retrospective study. For SILS, a 1-cm skin incision was first made below the umbilical margin and a 5-mm trocar was placed into the abdomen after incising the peritoneum. Two 5-mm trocars were then placed on both sides of the umbilicus. Subsequently, based upon preoperative examination and biopsy results, we performed subtotal colectomy. The affected colon was mobilized successively beyond the peritoneum using high-frequency cutting and sealing devices, followed by a pull-through procedure and colon-anal anastomosis. RESULTS: The average operative time was 206.39 min. No case needed conversion from SILS to either conventional laparoscopy or open surgery. Of the 22 patients, 15 were diagnosed as LSHD, while 6 cases were diagnosed with intestinal neuronal dysplasia and one was diagnosed with hypoganglionosis. There were no intra-operative complications. One child had incision dehiscence on postoperative day three. During the follow-up over 12 months, all patients were noted to have excellent cosmetic outcomes, and enterocolitis was observed in four children. CONCLUSIONS: Subtotal colectomy with the SILS technique can be safely performed in LSHD or HAD patients in the pediatric population without major complications.

Differentiation of GDNF and NT-3 dual gene-modified rat bone marrow mesenchymal stem cells into enteric neuron-like cells.

Bone marrow mesenchymal stem cells (BMSCs) have been shown to be multipotent cells that possess high self-replicating capacity. The purpose of our study was to investigate the feasibility of using enteric neuron-like cells obtained by in vitro induction and differentiated from rat BMSCs for the treatment of Hirschsprung's disease (HD). Glial cell-derived neurotrophic factor (GDNF) and neurotrophin-3 (NT-3) are neurotrophic factors that play important roles in neuronal development, differentiation, survival and function. Meanwhile, GDNF mutations are a major cause of HD. In this study, BMSCs were transfected with eukaryotic expression plasmids co-expressing GDNF and NT-3, and the transfected cells displayed neuron-like changes after differentiation induced by fetal gut culture medium (FGCM). Immunofluorescence assay showed positive expression of the neuronal marker NSE and the enteric neuronal markers PGP9.5, VIP and nNOS. Reverse transcription-polymerase chain reaction (RT-PCR) revealed the expression of GDNF and NT-3 in transfected BMSCs. The present study indicates that genetically modified BMSCs co-expressing GDNF and NT-3 are able to differentiate into enteric neuronal cells and express enteric nerve markers when induced by FGCM. This study provides an experimental basis for gene therapy to treat enteric nervous system-related disorders, such as HD.

Molecular cloning and characterization of porcine indoleamine 2, 3-dioxygenase and its expression in various tissues.

In order to confirm the existence of indoleamine 2, 3-dioxygenase (IDO) gene in swine, and to clone the novel gene followed by the molecule structure properties and expression pattern analysis, the porcine mRNA sequences homologous to human IDO were obtained from GenBank database by bioinformatics method. By using RT-PCR, the IDO gene was cloned from porcine endothelial cell line and the accuracy of the nucleic acid sequence was confirmed, and the expression pattern of the gene was detected. The three-dimensional structure model of porcine IDO was built referring to the tertiary structure of human IDO using biological sequence analysis software and database. The results showed that the porcine IDO was identified by sequencing. The nucleotide sequences were confirmed as a novel gene after submitted to Genbank. Porcine IDO was expressed in the lung, thymus, epididymis and anterior chamber with a basic level, however in peripheral blood mononuclear cells (PBMCs) the IDO gene was highly expressed. The three-dimensional structure model of porcine IDO was similar to that of human IDO. It was suggested that identification of the structure information of porcine IDO is essential to further investigate the immunologic function of the gene. Study of IDO on NK cells-mediated xenograft rejection will be a novel therapeutic target for the development of xenotransplantation.

Treatment of postoperative intestinal dysfunction of hirschsprung's disease based on the principle of "anorectal balance".

Purpose: Radical surgery is the most effective treatment for Hirschsprung's disease. However, some children still have symptoms of intestinal dysfunction such as constipation, abdominal distension, and recurrent enterocolitis after operation. The purpose of this study was to evaluate treatment outcomes of postoperative intestinal dysfunction in children with Hirschsprung's disease by using the principle of "anorectal balance". Methods: The clinical data of postoperative intestinal dysfunction in children with Hirschsprung's disease in the single treatment group from July 2019 to July 2021 were retrospectively analyzed. All the enrolled children underwent botulinum toxin injection (2.5 U/kg); 3 to 6 months later, the injection was performed again; the children who had received more than two botulinum toxin injections underwent the internal sphincter myectomy. Anorectal manometry was performed routinely after operation, and abdominal distension and defecation were recorded. Results: A total of thirty children with postoperative intestinal dysfunction underwent radical surgery for Hirschsprung's disease were included in this study. Symptoms of constipation, abdominal distension and enterocolitis were improved after botulinum toxin injections in most children compared to before surgery (P < 0.01). After re-injection of botulinum toxin in twelve children, the frequency of defecation increased, the anal resting pressure decreased, and the clinical symptoms were relieved again (P < 0.05). Eleven children underwent internal sphincter myectomy, and the symptoms of constipation, abdominal distension and enterocolitis were significantly improved after the operation (P < 0.01). Conclusion: Botulinum toxin injection and internal sphincter myectomy based on the principle of "anorectal balance" can effectively reduce the resting pressure of the anus and relieve intestinal dysfunction, and have satisfactory clinical effect.

Colonoscopic Diagnosis of Postoperative Gastrointestinal Bleeding in Patients With Hirschsprung's Disease.

Aim: Postoperative lower gastrointestinal bleeding in children with Hirschsprung's Disease (HSCR) is a non-specific symptom, which may be caused by various etiologies. Our current study aims to utilize colonoscopy to diagnose the causes of postoperative hematochezia and to analyze its feasibility, accuracy, and safety. Methods: Twenty-four patients with HSCR with postoperative lower gastrointestinal bleeding or occult blood in the stool were enrolled in this study. The postoperative onset duration, age at examination, accompanied anomalies were recorded. After bowel preparation, all patients underwent colonoscopy. According to visual findings, mucosal biopsy was performed, followed by pathological diagnosis. Further treatment was determined according to the visual findings and pathological diagnosis. All patients were followed up for 6 months including therapeutic outcomes and recurrence of symptoms. Results: The mean onset duration was (221.3 ± 216.8) days postoperatively (ranging from 25 to 768 days). The mean age at examination was (41.0 ± 29.4) months. There was no significant difference in the onset days among each group (all, p > 0.05). Based on visual and pathological findings, there were 11 cases of HSCR associated enterocolitis (HAEC), 4 cases of anastomotic leakage, 7 cases of anastomotic inflammation, 1 case of juvenile polyp, and 1 case of inflammatory pseudopolyp. Intraluminal saline irrigation, thrombin treatment or colorectal polyp electrocision was performed according to intraoperative diagnosis. Patients with HEAC and anastomotic inflammation underwent antibiotics therapy and colorectal irrigation. Patients with leakage underwent reoperation. The highest incidence of accompanied symptoms of diarrhea existed in HEAC group (p = 0.02) and fever in leakage group (p = 0.02), respectively. No perforation or aggravated bleeding occurs in any patients. All patients gained uneventful recovery during follow-up period. Conclusions: Colonoscopy is a safe, accurate and timely examination for HSCR patients with postoperative lower gastrointestinal bleeding. The visual findings and biopsy may provide accurate diagnosis and guide treatment for this subset of patients.

Expression of NF-kappaB in rotavirus-induced damage to the liver and biliary tract in neonatal mice.

BACKGROUND: Biliary atresia, the etiology of which still remains unclear, occurs exclusively in newborns and most are infected with rotavirus. In this study, we aimed to investigate the histopathological patterns of different kinds of rotavirus in the liver and biliary tract of neonatal mice and the expression of NF-kappaB in the liver and biliary tract of infected mice. METHODS: Twenty-three adult mice (8 were male and 15 female) were divided into 8 breeding pairs, and each pair (1 male and 2 females) was housed in a cage in a laminar flow hood. Newborn mice, 24-48 hours old were randomly divided into A, B and C groups. The A and B groups were respectively inoculated with MMU18006 and SA11 rotavirus through the intraperitoneal route, while group C as blank control was only inoculated with culture medium. The liver was dissected after 5, 10, 15, 21 and 28 days; the weight of each mouse and the histopathological patterns in the liver were recorded. The expression of NF-kappaB in the liver and intrahepatic bile ducts was detected by immunohistochemical staining and the expression intensity was analyzed with a GT-2 imaging instrument. RESULTS: The average increase in weight of infected mice was significantly slower than that of the normal control, while the growth rate of group A (injected with MMU18006 rotavirus) was slower than that of group B (SA11 rotavirus). In infected mice, the acute and chronic inflammation of liver and intra- and extra-hepatic bile ducts was more significant in group A. Stenosis was found in most intrahepatic bile ducts, and sporadically in extrahepatic bile ducts. The expression of NF-kappaB in infected mice was dramatically higher than that of the normal control, while the expression in group A was higher than in group B. CONCLUSIONS: Significant damage to the liver and biliary tract of neonatal mice can be induced by inoculating MMU18006 rotavirus through the intraperitoneal route, which is very similar to the pathology of biliary atresia in the newborn human. Similar inoculation with SA11 rotavirus can only result in moderate impairment that disappears quickly. The difference of pathogenicity between the two rotaviruses may depend on their differing capacities to increase the expression of NF-kappaB in the liver and biliary tract.

Heart-shaped anastomosis for Hirschsprung's disease: Operative technique and long-term follow-up.

AIM: To study the long-term therapeutic effect of "heart-shaped" anastomosis for Hirschsprung's disease. METHODS: From January 1986 to October 1997, we performed one-stage "heart-shaped" anastomosis for 193 patients with Hirschsprung's disease (HD). One hundred and fifty-two patients were followed up patients (follow-up rate 79%). The operative outcome and postoperative complications were retrospectively analyzed. RESULTS: Early complications included urine retention in 2 patients, enteritis in 10, anastomotic stricture in 1, and intestinal obstruction in 2. No infection of abdominal cavity or wound and anastomotic leakage or death occurred in any patients. Late complications were present in 22 cases, including adhesive intestinal obstruction in 2, longer anal in 5, incision hernia in 2, enteritis in 6, occasional stool stains in 7 and 6 related with improper diet. No constipation or incontinence occurred in any patient. CONCLUSION: The early and late postoperative complication rates were 7.8% and 11.4% respectively in our "heart-shaped anastomosis" procedure. "Heart-shaped" anastomosis procedure for Hirschsprung's disease provides a better therapeutic effect compared to classic procedures.

Interleukin-10 modified dendritic cells induce allo-hyporesponsiveness and prolong small intestine allograft survival.

AIM: To investigate whether IL-10-transduced dendritic cells (DCs) could induce tolerogenicity and prolong allograft survival in rat intestinal transplantation. METHODS: Spleen-derived DCs were prepared and genetically modified by hIL-10 gene. The level of IL-10 expression was quantitated by ELISA. DC function was assessed by MTT in mixed leukocyte reaction. Allogeneic T-cell apoptosis was examined by flow cytometric analysis. Seven days before heterotopic intestinal transplantation, 2 X 10(6) donor-derived IL-10-DC were injected intravenously, then transplantation was performed between SD donor and Wistar recipient. RESULTS: Compared with untransduced DC, IL-10-DC could suppress allogeneic mixed leukocyte reaction (MLR). The inhibitory effect was the most striking with the stimulator/effector (S/E) ratio of 1:10. The inhibition rate was 33.25%, 41.19% (P<0.01) and 22.92% with the S/E ratio of 1:1, 1:10 and 1:50 respectively. At 48 hours and 72 hours by flow cytometry counting, apoptotic T cells responded to IL-10-DC in MLR were 13.8% and 30.1%, while untransduced group did not undergo significant apoptosis (P<0.05). IL-10-DC pretreated recipients had a moderate survival prolongation with a mean allograft survival of 19.8 days (P<0.01), compared with 7.3+/-2.4 days in control group and 8.3+/-2.9 days in untransduced DC group. Rejection occurred in the control group within three days. The difference between untreated DC group and control group was not significant. CONCLUSION: IL-10-DC can induce allogenic T-cell hyporesponsiveness in vitro and apoptosis may be involved in it. IL-10-DC pretreatment can prolong intestinal allograft survival in the recipient.

Enzymes activity of intestinal grafts after liver small bowel transplantation in rats.

OBJECTIVES: To investigate the activity alterations of enzymes in intestine grafts after liver/small bowel transplantation in rats and the relations of these changes to immune rejection of grafts. METHODS: A model of liver/small bowel transplantation (LSBT) was established in closed colony SD and Wistar rats. The activity of enzymes including triphosphatase (ATPase), alkalinophosphatase (AKP), acytelcholinesterase (AchE), oxidesynthase (NOS) and monoamine oxidase (MAO) in bowel grafts was studied histochemically at regular postoperative intervals. RESULTS: The activity of enzymes in the wall of the grafts disappeared eventually in isolated small bowel transplantation (SBT) rats. In contrast, the activity in LSBT rats remained and recovered postoperatively. CONCLUSIONS: The rejection in grafted intestine could be prevented or delayed in LSBT rats. The changes in the activity of enzymes and neurons might be used to detect the rejection and function of the graft.

An experimental study on the expression of heme oxygenase-2 mRNA in Hirschsprung's disease.

In order to investigate the relationship between the expression of heme oxygenase-2 (HO-2) mRNA and the pathogenesis of Hirschsprung's disease (HD), total ribonucleic acid (RNA) was extracted in the aganglionic and ganglionic segments of colon respectively from 15 cases of HD. The single-stranded cDNA of HO-2 was synthesized and further amplified by reverse transcription-polymerase chain reaction (RT-PCR). The expression of HO-2 mRNA was normal in ganglionic segments, but absent in aganglionic segments. It is concluded that the absence of HO-2 mRNA expression may be an important mechanism responsible for HD.

[Long-term follow-up of Hirschsprung's disease patients treated by heart-shaped anastomosis operation].

OBJECTIVE: To summarize the experience of heart-shaped anastomosis operation in patients with Hirschsprung's disease. METHODS: Hirschsprung's disease treated by heart-shaped anastomosis, improvement of surgery procedure, and complications were reviewed retrospectively. RESULTS: Of 193 cases, 152 completed follow-up. Early complications included urine retention (2 cases), enteritis (10), anastomosis stricture (1), and intestinal obstruction (2). Late complications (22 cases) included adhesive intestinal obstruction (2), constipation (5), incision hernia (2), enteritis (6), and occasionally stool stains (7). Neither infection in celiac, pelvic cavity and wound nor incontinence or death occurred in all patients. CONCLUSION: Heart-shaped anastomosis procedure can effectively reduce the complications ceased by Hirschsprung's disease operation and is superior to other procedures.

Effects of verapamil on the immediate-early gene expression of bone marrow mesenchymal stem cells stimulated by mechanical strain in vitro.

BACKGROUND: To study the effects of verapamil on the immediate-early genes (IEGs) expression of bone marrow mesenchymal stem cells (MSCs) stimulated by cyclic mechanical strain, in order to deduce the role of calcium ion channel in the cell signaling responses of MSCs to mechanical strain. MATERIAL AND METHODS: MSCs were isolated and cultured, and the passage of 3-6 MSCs were stimulated by mechanical strain and pretreated with or without verapamil. After that, flow cytometry was used to measure the fluorescence intensity of intracellular Ca(2+) immediately. The expression of early-response genes/proteins (c-fos, c-jun and c-myc) were examined by RT-PCR, immunohistochemistry and Western blot. RESULTS: Intracellular Ca(2+) concentration of MSCs significantly changed when stimulated by cyclic strain, and the expression of c-fos, c-jun and c-myc remarkably increased in both mRNA and protein levels, while verapamil pre-treatment partially inhibited these effects (P<0.01). CONCLUSIONS: The changes of the intracellular calcium concentration of MSCs induced by mechanical strain, dependent on the regulation of calcium channel activation, might play a role in the early response of MSCs to cyclic strain.

NOTES for surgical treatment of long-segment hirschsprung's disease: report of three cases.

PURPOSE: The aim of this study was to evaluate the feasibility of natural orifice translumenal endoscopic surgery (NOTES(®); American Society for Gastrointestinal Endoscopy [Oak Brook, IL] and Society of American Gastrointestinal and Endoscopic Surgeons [Los Angeles, CA]) for the surgical management of long-segment Hirschsprung's disease. PATIENTS AND METHODS: Three children with long-segment Hirschsprung's disease were enrolled in this study. In all three cases the transition zone was proximal to the splenic flexure, with too long a segment of distal aganglionic colon to perform an isolated transanal pull-through. Our procedure was as follows. A rectal mucosectomy was performed starting 0.5 cm proximal to the dentate line and extending proximally to the level of the intraperitoneal rectum. Three cannulas were inserted through the muscular sleeve into the abdominal cavity. The mesocolon, lateral peritoneum, and greater omentum were ligated and divided in order to mobilize the colon. After mobilization, the aganglionic distal bowel segment was pulled through the anus and resected. Finally the colo-anal anastomosis was created. RESULTS: All three operations were successfully performed without intraoperative complications. No additional ports or conversion to an open procedure was required. The operative times were 242, 195, and 174 minutes, respectively. All three children were discharged without complication with follow-up for at least 1 year. One year after the procedure the 3 patients were stooling one to three times per day, with no fecal soiling or constipation. CONCLUSIONS: This NOTES procedure may be a safe and feasible option for the surgical treatment of long-segment Hirschsprung's disease.

[Transumbilical laparoscopic pull-through for children with hypoganglionosis].

OBJECTIVE: To investigate the surgical outcomes after on transumbilical laparoscopic pull-through procedure for pediatric hypoganglionosis(HYP). METHODS: Twelve children with HYP had received transumbilical laparoscopic pull-through procedure from June 2009 to June 2010. Specially designed curved and elongated laparoscopic instruments were used during the procedures. All the patients were followed up over 10 months. Data were collected and analyzed. The diagnosis of hypoganglionsis was pathologically confirmed. RESULTS: No conversions to laparotomy or traditional laparoscopic surgery were required and there were no damages to the abdominal blood vessels, intestine, ductus deferens, or ureters. The average duration of operation was 140 min. The mean intraoperative blood loss was 45 ml. The mean length of specimen was 40 cm. Postoperatively there were no complications such as anastomotic leak, anastomotic stricture, constipation, seepage, or fecal in continence. The average hospital stay after surgery was 9 days. During 10 to 22 months of follow-up(median 16 months), no postoperative recurrence was noticed. No obvious scar was seen 1 months after surgery. CONCLUSION: It is safe and effective for children with hypoganglionosis to undergo transumbilical laparoscopic pull-through procedure.

A novel method for establishment and characterization of extrahepatic bile duct epithelial cells from mice.

Culture of extrahepatic bile duct epithelial cells is a useful model to investigate physiology of extrahepatic bile duct epithelia and hepatobiliary disease mechanisms. The aim of this work was to establish and characterize a primary murine extrahepatic bile duct epithelial cell culture. Epithelial cells were isolated from extrahepatic bile ducts of BALB/c mice that were intraperitoneally injected with newborn bovine serum to induce the proliferation of extrahepatic bile ducts' epithelial cells and cultured on rat tail type I collagen-coated plastic culture flask containing DMEM/HamF12 with 10% FBS and 10 ng/ml epidermal growth factor at 37°C in an incubator with 5% humidified CO(2). The cells showed typical morphologic characteristics of epithelial phenotypes with cobblestone appearance in monolayer within 5-6 d after culture; they were positive against anticytokeratin-19 immunostaining. Transmission electron microscopy showed typical bile duct epithelia with microvilli on the cytomembrane, Golgi complex, massive mitochondria, and rough endoplasmic reticulum in the cytoplasmic. The growth curve of the epithelial cells was determined by a MTT assay which showed a normal sigmoidal growth curve. This culture technique might be a reliable method for isolation, purification, and primary culture of extrahepatic bile duct epithelial cells that can serve as a model for in vitro studies on the pathophysiology of hepatobiliary diseases as well as pharmacological and toxicological targets relevant to hepatobiliary diseases.

[Distribution of mast cells and its role in the pathogenesis of Hirschsprung disease].

OBJECTIVE: To investigate the distribution of mast cells (MC) in colon tissue of Hirschsprung disease (HD) and explore the role of mast cells in the pathogenesis of HD. METHODS: Forty-one cases of HD (male 23, female 18), age from 2 months to 15 years, and eight age-matched normal cases were enrolled in this study. The distribution of MC in all layers of colon was examined by immunohistochemistry with mouse antihuman mast cell tryptase monoclonal antibody. RESULTS: The count of MC in all layers of colon aganglionic segments of HD was significantly higher as compared with colon ganglionic segments of HD and normal controls (21.47+/-3.59 vs 3.18+/-0.87, 2.75+/-0.51). The average optical density values(A) of MC in aganglionic and ganglionic segments significantly decreased as compared to normal control (0.38+/-0.10,0.31+/-0.11 vs 0.51+/-0.08). CONCLUSION: Mast cells may play an important role in the pathogenesis of HD.