BACKGROUND: Stem cells are relatively primitive cells possessing the capabilities of self-renewal, high proliferation and multi-potential differentiation in vivo under certain conditions. Pancreatic stem cells and umbilical cord blood mesenchymal stem cells (MSCs) may serve therapeutic purpose clinically, but they are still difficult to culture in vitro at present.OBJECTIVE: To explore the method for isolation, purification and culture of pancreatic stem cells and umbilical cord blood MSCs in vitro and observe their morphological changes during culture in vitro.DESIGN: Completely randomized experiment with repeated measurement.SETTING: Stem Cell Research Center, Teaching and Research Division of Physiology, Medical School of Zhengzhou University.MATERIALS: This experiment was conducted in the Stem Cell Research Center, Teaching and Research Division of Physiology, Medical College of Zhengzhou University, between April 2004 and January 2005. Ten to fifteen newborn SD rats (1-3 days) were selected for culture in vitro of pancreatic stem cells, and fresh umbilical cord blood was collected from healthy woman (24-35 years old, with informed consent) at full-term delivery for culture in vitro of umbilical blood SMCs.METHODS: The abdomen of the newborn SD rat was opened under aseptic condition to obtain the pancreas, which was cut into small tissue blocks and digested with type-V collagenase for islet isolation. The isolated islets were purified in continuous roller-bottle culture. Umbilical cord blood was freshly collected for isolating the monocytes by means of density gradient centrifugation in lymphocyte separation medium (with density of 1.077 g/cm3). The islet cells and umbilical cord blood monocytes were cultured in the incubator at 37 ℃ with 5% CO2. The morphological changes of the cells were observed at designed time points and flow cytometry was used to determine the expression of cell surface molecules.MAIN OUTCOME MEASURES: The isolation and culture of pancreatic stem cells and umbilical cord blood MSCs, and their morphological changes during culture in vitro.RESULTS: During culture in vitro, the fusiform islet progenitor cells showed adherent polar growth and continuous proliferation, which covered the whole bottom of the flask after 12-14 days and could be subcultured for passages. However round cells appeared after removal of the growth factor and serum in the culture medium. The monocytes isolated from the umbilical cord blood grew initially into numerous hematopoietic cell clones, most of which proved to be granulocyte clones by Switzerland staining. Seven days later, flat flask wall-adhering epithelial cells and long fusiform fibroblasts were observed mixed with a number of osteoclasts. As the cell culture was prolonged, the cell number increased steadily.CONCLUSION: Pancreatic stem cells and umbilical cord blood SMCs can be cultured in vitro for further experiments.

Objective To assess the diagnostic value of MSCT in congenital bridging bronchus (BB).Methods Three-dimensional (3D) reconstructed CT images including MPR,MinIP,MIP,VR were respectively analyzed in 12 patients with congenital bridging bronchus on postprocessing workstation.Results Six of 12 BB patients were type Ⅰ bridging bronchus which originated from the left main bronchus at the level of fourth and fifth thoracic vertebral body,while the right bronchus was originated from the normal carina at the level of second and third thoracic vertebral body.The average angle of normal carina was about 59.2°,and the average angle of pseudocarina where BB originated from the left main bronchus was about 108.8°.The left main bronchus proximal to the origin of BB showed significant stenosis,with 1-2 mm width and 17 mm length in the involved segment.Six cases were type Ⅱ bridging bronchus,the right bronchus was absent in normal carina,BB originated at the level of fourth and fifth thoracic vertebral body,the average angle of pseudocarina was about 131°,the whole right lung was supplied by BB.The lower portion of trachea had stenosis in all 6 patients with 2-3 mm width and 30 mm length in the involved segment The lower portion of the trachea was found moving to the left in 4 patients and the left pulmonary artery sling was found in 2 patients.Conclusion MSCT can afford a definite diagnosis of BB by showing the morphology of trachea,bronchus,carina and relationship with surrounding organs with 3D reconstructions.

BACKGROUND:Currently,there is not a standard method for in vitro culture and large scale amplification of umbilical cord blood mesenchymal stem cells(UCB-MSCs).OBJECTIVE:To investigate the isolation,purification and culture of UCB-MSCs in vitro,and to detect its surface marker variation.METHODS:The monocytes were harvested from UCB using 1.077 g/cm3 lymphocytes separating solution and density gradient centrifugation,followed by incubation in an incubator containing 5%CO2 at 37℃.The cell morphological changes were observed at different time points and the expression of surface marker was detected using flow cytometry.RESULTS AND CONCLUSION:The monocytes isolated from the UCB grew initially into numerous hematopoietic cell clones,most of which were granulocyte/macrophage colony-forming units and burst forming unit-erithroid,increasing by(37.1±2.3)and (10.4±1.7),respectively.Switzerland staining showed most of them were granulocyte clones(80,1±85.2)%,next was erythroid clones(14.2±1.8)%.At 7 days after culture,some shuttle fibroblast-like cells and fiat osteogenic-like cell spread the whole plastic well.At 14 days after culture,flow cytometry showed CD38+ cells accounted for 1.64%,and CD34+/CD38+ cells accounted for 1,71%,and CD34+/CD38- were 0.55%.PI+ and Annexin-V+ cells accounted for 0.05% and 0.18% respectively.At 21 days after culture,CD38+,CD34+/CD38+ and CD34+/CD38- cells were 74.32%,1.61%,and 0.24%.The results reveled that UCB-MSCs can be isolated and cultured in vitro.

Objective To develop a double-antibody sandwich ELISA for determining the concentration of nucleoprotein (NP) of rabies virus in various products of rabies vaccine.Methods The purified rabies antibodies from a rabbit were used to coat microwell plates. Horseradish peroxidase-conjugated anti-NP monoclonal antibody was used to probe the NP bound to coated antibodies.The assay was used to quantitatively determine the concentration of NP of rabies virus.Results The results showed that the coefficient of linear correlation was higher than 0.97.The optimal linear range was 0.000625~0.01 IU/ml and the detection limit was 0.000625 IU/ml. The recovery rate was 102~109% and the coefficient of variation was only 7.2%~9.4%.No cross reactions were observed with bovine serum,bovine serum albumin,ovalbumin,refined solution of influenza vaccine,encephalitis B vaccine,and hepatitis A vaccine.Conclusions The results indicated that the assay is specific,sensitive,accurate,reproducible,and stable,and could be suitable for quantitative determination of different rabies vaccine's processes products.

Objective To investigate the clinical characteristics of bronchial tumors in 3 children to improve the diagnosis of pediatric bronchial tumor.Methods Three cases of children bronchial malignant tumors diagnosed by rigid bronchoscopy were analyzed retrospectively.Results The 3 children were males,aged from 6 to 10 years old, and presented with cough, sputum, and fever symptoms for 1 day to 3 months.Chest CT scan and airway remodeling examinations indicated the children ~ main bronchi were blocked and then the diagnoses of bronchial foreign bodies were made.However bronchial tumors were found in the 3 children by rigid bronchoscopy and were determined as mucoepidermoid carcinoma, large cell lung carcinoma with rhabdoid phenotype, and inflammatory myofibroblastic tumor, respectively.Conclusions Children with bronchial tumor often present with cough, wheezing and other respiratory symptoms that are not specific to bronchial tumor.When a child complaint of repeated cough and wheezing symptoms with unknown cause,not only bronchial foreign body and also bronchial tumor should be considered.

Objective To identify Brucella species by means of bacteriological and polymerase chain reaction(PCR)methods,and to understand the drug susceptibility by in vitro susceptibility test of these strains to eight antimicrobial drugs.Methods The isolated Brucella strains were identified by standard method with conventional positive serum experiment,monophase specificity serum(A,M and R) agglutination experiment and brucella phage splitting experiment(Tb and BK2).Reference strains were set as control group.Molecular typing was performed by polymerase chain reaction(PCR)targeting Brucella surface protein 31(BCSP-31)and(abortus melitensis ovis suis,AMOS)-PCR assay which is able to distinguish among B.abortus,B.melitensis,B.ovis and B.porcine.Microdilution broth method was used to determine the minimum inhibitory concentrations(MIC)of 8 antibiotics to 27 Brucella strains isolated from blood culture,including azithromucin,ciprofloxacin,levofloxacin,doxycycline,rifampicin, gentamicin,acheomycin,and streptomycin.Results Twenty-seven strains were identified as B. melitensis,of which 21 were B.melitensis biovar 3 isolates,6 were B.melitensis biovar 1.The BCSP-31-PCR confirmed that all 27 isolates were Brucella.spp.AMOS-PCR assay confirmed that all isolates were B.melitensis.All isolates were susceptible to ciprofloxacin,levofloxacin,doxycycline,gentamicin, acheomycin,and streptomycin.Doxycycline was the most effective antibiotic(MIC900.064 mg/L),while rifampicin was moderately active to 3 isolates(MIC 2 mg/L).Conclusions Brucella isolates are susceptible in vitro to the antibiotics recommended by world health organization.Regular evaluation of antibiotic susceptibilities of Brucella strains is helpful for epidemiological investigation and antibiotic resistance monitor.

Objective To get knowledge of the molecular epidemiological characteristics of human derived Brucella isolated in Hohhot,and to provide experimental basis in guiding prevention and treatment of Brucella infection.Methods Twenty-seven Brucella isolates derived from patients in Affiliated Hospital of Inner Mongolian Medical University from 2013 to 2015 were identified by routine bacteriological methods and molecular methods.Multiple-locus variable number tandem repeats analysis (MLVA-16) was used to detect molecular typing and do cluster analysis.Sixteen virulent genes were detected and analyzed by polymerase chain reaction (PCR).Results Twenty-seven Brucella isolates were identified as Brucella melitensis (B.melitensis) by routine bacteriological methods and PCR.Out of them,six isolates were B.melitensis biovar 1,and twenty-one isolates were B.melitensis biovar 3.MLVA-16 analysis showed that seven genotypes were obtained from nine Brucella isolates,which showed significant difference in variable number of tandem repeats,which suggested that they originated from sporadic outbreak.Moreover,two isolates were clustered into the same clade,which suggested they were epidemiologically correlated and may be derived from the same origin.Sixteen virulent genes were detected in all of the twenty-seven isolates.Conclusions Brucella isolates from patients in Hohhot are mainly B.melitensis biovar 3 and B.melitensis biovar 1,and the distribution profile of multiple virulence genes is similar.Some isolates have showed epidemic correlation,and the epidemic mechanism should be further explored.

Objective This study aims to analyze the independent risk factors of acute respiratory dysfunction (ARD) in children with airway foreign body and to assess possible prevention and treatment option in the future.Methods Clinical data of 456 cases of children with airway foreign body were retrospectively collected and analyzed by cluster sampling,including 246 males and 210 females,who received operation in our hospital between July,2009 and December,2012,aged 0.5-11 years old,onset to treatment time was 0.15-14 days.Chnical characteristics including age,gender,past medical history,time of onset,temperature,location of the foreign body,category of foreign bodies,complicated by pneumonia,complicated by subcutaneous and mediastinal emphysema were gathered.Temperature,respiratory rate,heart rate,cyanosis,transcutaneous oxygen saturation or arterial blood analysis were assayed before operation.Risk factors with statistical significance were screened with univariate logistic regression analysis,independent risk factors of ARD were determined with multivariate logistic regression analysis.Results Acute respiratory dysfunction occurred in 78 (17.1％) patients.The foreign bodies in 455cases were successfully removed brocboscopically in the first time.One case received chest surgery for foreign body removal.Total of 452 cases were successfully extubated and ventilator weaned 4-6 h after brochoscopy.In 2 cases,the ventilator was weaned 2-4 d after brochoscopy in ARD gup,and 2 cases with severe pneumonia died.Age,location of the foreign body,temperature,complicated by pneumonia,complicated by subcutaneous and mediastinal emphysema did not show significant difference between acute respiratory dysfunction group and non-acute respiratory dysfunction group (P ＜ 0.05).Multivariate logistic regression analysis showed location of the foreign body and complicated by pneumonia,complicated by subcutaneous and mediastinal emphysema were independent risk factors for ARD.Conclusion Early judgement of the risk factors of acute respiratory dysfunction in children with airway foreign body can provide a reference for the operation and perioperation period treatment.

The relationship of connexin43 (Cx43) and bystander effect in ovarian tumor cells in herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) gene therapy in vitro was explored and the effect of all-trans retinoic acid (RA) on the expression of Cx43 and bystander effect investigated. The Cx43 expression was detected by flowcytometry, Western blot, and immunofluorescence in two ovarian tumor cell lines OVCAR3, CaOV3 before and after RA treatment. Bystander effect was determined by the cells growth inhibitory rate with methyl thiazolyl tetrazolium. Following exposure to ganciclovir, there was much greater bystander killing in OVCAR3 than that in CaOV3 (P<0.05). The expression of Cx43 was detected in OVCAR3 by flowcytometry and Western blot, but it could not be detected in CaOV3. The expression of Cx43 in both cell lines could be induced by RA. Immunofluoresence staining showed that Cx43 protein of OVCAR3 was located on membrane surface, whereas CaOV3 in cytoplasm. RA could not change the location of Cx43 protein in both cell lines. There is relationship between Cx43 expression and HSV-TK/GCV bystander effect. HSV-TK/GCV bystander effect can be enhanced by RA in ovarian cancer.
Antiviral Agents ; pharmacology ; Bystander Effect ; Cell Line, Tumor ; Connexin 43 ; biosynthesis ; genetics ; Female ; Ganciclovir ; pharmacology ; Genes, Transgenic, Suicide ; Genetic Therapy ; Humans ; Ovarian Neoplasms ; metabolism ; therapy ; Pregnancy ; Simplexvirus ; genetics ; Thymidine Kinase ; genetics ; Tretinoin ; pharmacology

Objective:To analyze the clinical characteristics, treatment, and prognosis of ectopic bronchogenic cysts in children.Methods:A retrospective analysis was conducted on the data including the clinical characteristics, auxiliary examination and treatment of 21 children with ectopic bronchogenic cysts diagnosed pathologically at Children′s Hospital Affiliated to Zhengzhou University from July 2015 to December 2023. There were 16 males and 5 females, with a male-female ratio of 3.2∶1, and the age ranged from 4 days to 8 years old (median age 2 years and 8 months).Results:Among the 21 cases of ectopic bronchogenic cysts, 11 cases were found in the pharynx, with symptoms including dyspnea (4 cases), snoring during sleep (3 cases), and choking on milk(4 cases).Ten cases were found in the head, neck or anterior chest, 5 of these cases had infection history, and 5 showed progressive mass growth.Imaging and endoscopy showed 9 patients underwent preoperative color ultrasonography revealed cystic masses with well-defined boundaries. CT examination was performed on 13 patients, which showed round or nearly round masses with homogeneous density, smooth margins, and regular cyst walls. CT attenuation values ranged from 2 to 52 Hounsfield Units (HU). Four cystic lesions were assessed via MRI, 3 cases demonstrated long T1 and long T2 signals, while 1 case had a slight short T1 and long T2 signal, with high signal intensity on fat-suppressed images. Eleven cases of pharyngopharyngeal cysts were examined by electronic nasopharyngoscopy. The cysts appeared as spherical or ovoid masses with smooth surfaces, close to or slightly light in color with the surrounding tissue, with one cyst presenting with a bluish blue in the oropharynx. All 11 pharyngeal cysts were excised using low-temperature plasma under general anesthesia and intubation assisted by a nasal endoscope. The cysts were pulled and excised as completely as possible.Ten cases of neck and anterior chest cysts were completely excised. Postoperative histopathology confirmed bronchogenic cyst. Twenty-one children were followed up postoperatively for 4 months to 7 years without recurrence, except for 1 patient who was lost to follow-up.Conclusions:Ectopic bronchogenic cysts are uncommon and lack of typical imaging and clinical features.Combination of ultrasonography, CT and MRI is recommended for cases occuered in neck and anterior chest, while electronic nasopharyngoscopy complements pharyngeal evaluations. Surgical intervention is the preferred treatment choice for this disease.