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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.
Subject(s)
Animals , Amino Acid Sequence , Base Sequence , Cell Line , Cloning, Molecular , Methods , Endothelial Cells , Metabolism , Indoleamine-Pyrrole 2,3,-Dioxygenase , Genetics , Metabolism , Molecular Sequence Data , Sequence Alignment , SwineABSTRACT
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.
ABSTRACT
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.
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BACKGROUND: It has been reported that in China, human bone marrow mesenchymal stem ceils are mostly harvested from adults. Studies on bone marrow mesenchymal stem cells in children are few.OBJECTIVE: To isolate and expand bone marrow mesenchymal stem cells from children, and to analyze the biological characteristics of bone marrow mesenchymal stem cells and their potential of differentiating into osteoblasts, adipocytes and neural like cells.DESIGN: Observational comparative study.SETTING: Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: Experiments were performed at the Laboratory of Department of Orthopaedics of Wuhan Tongji Hospital from March to September 2006. Bone marrow mesenchymal stem cells were collected from one boy patient and two girl patients aged 5-8 years, who received pelvis osteotomy for dysplasia of the hip joint. The experimental procedures were approved by the Hospital Ethics Committee and family members of all children patients singed the informed consent.Dexamethasone, vitamin C, β-sodium glycerophosphate, 3-1sobutyl-1-methylxanthine, insulin, indometacin and butylated hydroxyanisole were bought from Sigma Company. Dimethyl sulphoxide was purchased from Amersco Company.METHODS: Bone marrow mesenchymal stem cells were cultured from mononuclear cells isolated over a Percoll gradient.Bone marrow mesenchymal stem cells were observed under an inverted phase contrast microscope. Bone marrow mesenchymal stem cells could differentiate into osteoblasts, adipocytes and neural like cells with osteoblast inductor (β-sodium glycerophosphate, dexamethasone, vitamin C), lipoblast inductor (dexamethasone, 3-isobutyl-1-methylxanthine,bovine insulin, indometacin) and serum-free medium inductor (dimethyl sulphoxide, butylated hydroxyanisole) respectively.Osteoblast marker (alkaline phosphatase, osteocalcin mRNA, calcium node), adipocyte marker (lipid droplet, PPAR γ-2mRNA) and neural ceil-like marker (nissl body, neuron specific enolase, neurofilament protein) were respectively determined by the immunohistochemical method, polymerase chain reaction and immunocytochemical method.MAIN OUTCOME MEASURES: ①Appearance and proliferation of bone marrow mesenchymal stem ceils from children,and ②determination results of osteoblast, adipocyte and neural cell markers.RESULTS: ①Children bone marrow mesenchymal stem cells could easily adhere to the wall, appeared fusiform, had high reproductive activity and arranged vortically after fusing. ②Appearance of bone marrow mesenchymal stem cells changed after receiving inductor. Osteoblast marker, adipocyte marker and neural cell-like marker were positive after chemical staining, polumerase chain reaction and immunocyte staining.CONCLUSION: Children bone marrow mesenchymai stem cells show stable proliferation, passage and multi-direction differentiation towards osteoblasts, adipocytes and neural like cells.
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[Objective]To study the effects of verapamil on the immediate-early gene(IEGs)expression of bone marrow mesenchymal stimulated by mechanical strain,and deduce the effects of calcium ion channel on the early responses of children bone marrow mesenchymal stem cells(MSCs)to mechanical strain in vitro.[Method]MSCs were isolated and cultured.The 3-6th generational MSCs were stimulated in different time by mechanical and(or)verapamil(20?mol/l).Flow cytometry was applied to measure intracellular free Ca2+ at once.Early-response genes /proteins(c-fos,c-jun and c-myc)were examined by RT-PCR and immunocytochemical stain.[Result]The application of mechanical strain increased intracellular c-fos,c-jun and c-myc mRNA /protein levels.The resulting effect is partially inhibited before MSCs were preincubated with verapamil(P
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<p><b>OBJECTIVE</b>To summarize the experience of heart-shaped anastomosis operation in patients with Hirschsprung's disease.</p><p><b>METHODS</b>Hirschsprung's disease treated by heart-shaped anastomosis, improvement of surgery procedure, and complications were reviewed retrospectively.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>Heart-shaped anastomosis procedure can effectively reduce the complications ceased by Hirschsprung's disease operation and is superior to other procedures.</p>
Subject(s)
Child , Child, Preschool , Female , Humans , Infant , Infant, Newborn , Male , Follow-Up Studies , Gastroenterostomy , Methods , Hirschsprung Disease , General Surgery , Postoperative Complications , Retrospective StudiesABSTRACT
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.
Subject(s)
Child , Child, Preschool , Female , Humans , Infant , Infant, Newborn , Male , Colon , Heme Oxygenase (Decyclizing) , Genetics , Hirschsprung Disease , Genetics , RNA, Messenger , GeneticsABSTRACT
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.
Subject(s)
Colon/enzymology , Heme Oxygenase (Decyclizing)/biosynthesis , Heme Oxygenase (Decyclizing)/genetics , Hirschsprung Disease/enzymology , Hirschsprung Disease/etiology , Hirschsprung Disease/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/geneticsABSTRACT
Objective To evaluate the efficacy of laparoscopic first-stage Fowler-Stephens for high cryptorchidism. Methods Seven children with high cryptorchidism (10 testes) were enrolled in this study. After detecting the testicles under a laparoscope, we dissected the spermatic cord vessels. Then, the spermatic duct was separated, and the gubernaculum testis was retained. Afterwards, the first-stage orchidopexy was performed laparoscopically. Results Nine of the 10 testes were successfully placed into the scrotum by first-stage Fowler-Stephens. In one patient who had bilateral cryptorchidism, the left testis was placed into the scrotum at the second stage operation. The seven patients were followed up for 6-24 months (mean, 14 months). None of them had retraction or atrophy of the testis. Conclusions Laparoscopic first-stage Fowler-Stephens is effective for high cryptorchidism. The procedure is worth being widely used.
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Objective To analyze the relationship between polymorphisms of EDNRB gene and Hubei provincial patients of Han ethnicity with sporadic Hirschsprung disease(sHD). Methods Peripheral blood samples from 104 patients with sHD and 84 parents of 42 patients, and 120 normal children(as controls) were collected. PCR-SSCP and direct DNA sequencing were used to detect mutations and polymorphisms of exon-4 in EDNRB gene. The differences of allele frequencies and genotype distribution in polymorphic sites were further analyzed between the three groups. Allele frequencies of SNPs in forty-two sHD trios were analyzed by transmission disequilibrium test(TDT), and the association between phenotype of HD and SNPs was analyzed. Results No mutant site was detected and one polymorphic site of c831 G→A(L277L) was observed in Hubei provincial patients of Han ethnicity with sHD. The allele frequency of A(68% vs 53%) and genotype frequency of AA(49% vs 30%) were significantly higher in sHD group than that in control group(P
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Objective To evaluate the long-term results of partial internal sphincterectomy for the treatment of internal anal sphincter achalasia in childhood. Methods The clinical,radiographic,manometrical and histochemical data of 6 cases were reviewed retrospectively. All patients had received partial internal sphincterectomy and were followed-up for 2 to 8 years. Results All patients presented with severe constipation with or without soiling. No stenosis zone of intestine could be noted in 3 patients by barium enema examination. The rectoanal inhibition reflex on rectal balloon inflation was absent in all patients. The normal acetylcholinesterase activity on rectal biopsies was demonstrated by histochemical staining. Ganglion cells within internal anal sphincter was noted in all cases. On follow-up,all patients regained regular bowel habits and are not on any laxatives. Conclusion The long term results of partial internal sphincterectomy for the treatment of internal anal sphincter achalasia in childhood are satisfactory.