Objective To investigate the relationship between the promoter of IL-12B gene polymorphism and the susceptibility and clinical features of chronic gastritis and duodenal ulcer with or without Helicobacter pylori(Hp) infection in children and adolescent.Methods Mucosal biopsies were obtained from 132 children and adolescent (patient group),including 100 children with chronic gastritis and 32 children with duodenal ulcer,undergoing an upper gastrointestinal endoscopy for dyspeptic symptoms.Biopsy specimens were stained with hematoxilin and eosin (HE),and gastritis was graded according to the Sydney system.Serology,urease test and histology were taken to assess Hp status.Genomic DNA was obtained from peripheral blood or gastric biopsies of patients and 102 healthy children as normal control group.The promoter of IL-12B +1188A/G gene polymorphism was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing.The genotype distributions and allele frequencies were compared between the study group and the normal control group,and the association of genotypes with clinicopathological features was studied.IL-12B mRNA level expressions in gastric mucosa were confirmed by reverse transcription PCR biopsy-based tests.Results The genotype distributions and allele frequencies of IL-12B +1188A/G gene polymorphisms were similar in gastric upper gastrointestinal diseases and healthy subjects.The IL-12B +1188A/G gene polymorphisms were not associated with Hp status.IL-12B+1188A/G gene polymorphisms did not affect IL-12B mRNA level expressions and were not associated with the degree of antrum chronic inflammation.Conclusions These data suggest that IL-12B+1188A/G gene polymorphisms are not associated with susceptibility to chronic gastritis and duodenal ulcer in children and adolescent.

Objective To construct the bioartificial liver by bone mesenchymal stem cell (BMSC) and alginate scaffold. Method Alginate scaffold was used as the cell carrier for the cultivation of BMSC and the differentiation from BMSC into hepatic like cells was induced by the cell factors of HGF, EGF and FGF-4 in the scaffold in vitro. The compatibility of the cells and the scaffold was observed by microscopy and the function of the differentiatd cells was tested. The gene of AFP and ALB was detected by RT-PCR. The secretion of ALB and the urea synthesis of the cells were tested by ALB kit and urea kit respectively. The glycogen synthesis and the CK-18 was tested by the glycogen stanning method and the immunofluorescence test. Results BMSC was able to attach, grow and proliferate well in the alginate scaffold, the well compatibility was observed by microscopy. ALB and urea were detected in the cultivating medium, the gene of ALB and AFP was identified by RT-PCR. The glycogen synthesis ability and the expression of CK-18 were induced during the differentiation. Conclusion The three dimensional atginate scaffold exhibited well compatibility with BMSC, BMSC could be differentiated into the hepatic like cell in the scaffold. BMSC and the alginate scaffold could be used to construct the bioartificial liver for the hepatic tissue engineering.

BACKGROUND: There is no accepted treatment for liver fibrosis recently. Bone marrow meaenchymal stern cells (BMSCs) used in the treatment of liver fibrosis has been reported as an effectively treatment, but the mechanism is unclear.OBJECTIVE: To study the regulation of hepatic stellate cells mediated by human BMSCs in vitro.DESIGN, TIME AND SETTING: The cytological in vitro study was performed at the Center for Stem Cells and Tissue Engineering of Sun Yat-sen University and the Central Laboratory of Third Affiliated Hospital of Sun Yat-sen University from June to December 2008.MATERIALS: Human bone marrow masenchymal stem cells were collected from normal youth volunteers; Human hepatic stellate cells and normal liver call line L-O2 were supplied by the Animal Experimental Center of Sun Yat-sen University.METHODS: The purified human BMSCs and hepatic stellate calls were set up in Transwell co-culture system. The incubation density was 2×104cells/well. L-O2 was set up instead of human BMSCs as negative control. Hepatic stellate cells cultured alone served as blank control group. The culture was performed for 72 hours.MAIN OUTCOME MEASURES: Morphology of hepatic stellate cells and results of immunocytochemical staining. Apoptosis of hepatic stellte calls was determined by flow cytometry. Western blot were used to assay the expression of α-actin.RESULTS: Activated hepatic stellate cells presented fiat and thin shape under an inverted microscope. Fat drop was lack in cytoplasm, a -actin located in hepatic stellate calls, with the presence of high tension fibers. Compared with the L-O2 + hepatic stellate cell and hepatic stellate call groups, the apoptotic rate of hepatic stellate cells was significantly increased in the BMSC + hepatic stellate cell group (P < 0.05). α -actin expression was significantly down-regulated.CONCLUSION: Human BMSCs can inhibit activation of hepatic stellate ceils and promote them apoptosis, which may be the anti-hepatic fibrosis mechanism of BMSCs.

Objective To explore the change of apoptosis factor Caspase-3 and Bcl-2 in the injured segment of rat with spinal cord injury after inhibiting lentivirus expression of inflammation factor TNF-α. To study the relationship between Caspase-3, Bcl-2, Bax and TNF-α in spinal cord injury. Mthods Spinal cord contusion model was prepared by Allen method. The relation between tumor necrosis factor alpha and Bcl-2, was predicted by the method of GeneMANIA bioinformatics. The RNA which was packaged by lentivirus constructed the RNA interference model of tumour necrosis factor alpha. After interference of tumor necrosis factor alpha, we used the method of QRT-PCR to assays the mRNA expression of Caspase-3 and Bcl-2 in spinal cord and detect of the localization of Caspase-3 and Bcl-2 by immunohistochemistry. Statistical analysis with SPSS17.0. Results SD rats had paraplegia and urinate retentaion because of spinal cord injury. The result of QRT-PCR showed that in the seventh day after SCC, the expression of Caspase-3 reduced significantly (P < 0.05) and Bcl-2 did not change significantly (P > 0.05). Immunohistochemistry experiment results showed that Caspase-3 Bcl-2 and Bax immunoreactive cells were observed in the neurons and glial cells of both white matter and gray matter in the spinal cord. The results were the same with QRT-PCR.. Conclusion TNF-α in rats after SCC can effectively regulate the ratio of Bcl-2 and Bax , and then regulate the expression of Caspase-3 , which may affect the function of apoptosis and function recovery after spinal cord injury.

Objective To explore the characteristics of proinsulin secretion in latent autoimmune diabetes in adults (LADA). Methods Fasting and 2 h sera in oral glucose tolerance test from 36 LADA patients, 37 type 2 diabetic patients and 43 healthy controls were collected to test glucose, proinsulin (PI) and C-peptide (CP) by radioimmune assay. Glutamie acid decarboxylase antibodies (GAD-Ab) were determined by radioligand assay.Results (1) Fasting proinsulin (FPI) and 2 h proinsulin (PPI) level in LADA patients were lower than those in type 2 diabetic patients (P<0.05), being both significantly inereasad compared with healthy controls (P<0.05 or P<0.01); The ratios of FPI/FCP and PPI/PCP (%) in LADA were beth significantly higer than those of type 2 diabetes mellitus and controls (P<0.05 or P<0.01); (2) LADA type-1 (GAD-Abe>0.3) patients showed lower PI levels(P<0.05 or P<0.01) and higher PI/CP ratio (all P<0.05) than LADA type-2 (0.05≤GAD-Ab<0.3); Meanwhile, there were no significant differences in above parameters between LADA type-2 and type 2 diabetes meUitus (P>0.05). (3) GAD-Ab index was negatively correlated with FPI and PPI in LADA group (r=-0.236 and-0.268, both P<0.05), and positively correlated with PPI/PCP (r=0.254, P=0.030).Meanwhile BMI was positively correlated with FPI, PPI and PI/CP in type 2 diabetes mellitus (all P<0.01). No factor entered the multiple regression analysis for predieting the hyperproinsulinemia and dispropriately elevated proinsulin levels in LADA patients. (4) According to the 99.5 th percentile of proinsulinemia in the healthy controls, which is defined as the cutoff point dispropriately elevated proinsulin levels, the proportion of subjects with fasting dispropriately elevated proinsulin levels (FPI/FCP) were 77.8%, 62.2% and 2.3% in LADA, type 2 diabetes meUitus and controls respectively, and PPI/PCP 83.3%, 51.4% and 2.3% respectively. Conclusion LADA patients, as well as type 2 diabetic patients, all showed hyperproinsulinemia and disproportionately elevated proiasulin levels that were one of characteristics of defective β-cell function. Moreover, disproportionately elevated nproinsulin level is more evident in LADA patients than that in type 2 diabetics and this may be related to humoral immunity.

Objective To evaluate the clinical efficacy and safety of Holmium laser in the treat ment of calyceal stricture and atresia through antegrade percutaneous nephrostomy. Methods Ante grade percutaneous nephrostomy was performed in 68 patients with calyceal stricture and atresiathrough a rigid 8/9.8 F ureteroscope. The stricture and atresia was incised in a linear fashion by theHolmium laser with a 550 mm fiber. After completion of the incision,a double J ureteral stent wasplaced for 6-8 weeks and nephrostomy tube was kept for 7 days thereafter. Patients were then fol lowed up with IVU and/or ultrasound at 3-6 month intervals. Results The mean operative timewas 90 min,ranged from 80 to 120 min. The mean postoperative hospital stay was 8 d(7-9d). Hy dronephrosis was significantly improved in 38 cases in an average follow up of 9 months (4-26 months). Repeated laser incision was performed to 4 treatment failures and all turned out to be suc cessful. Conclusions The Holmium laser treatment through antegrade percutaneous nephrostomyfor calyceal stricture and atresia has characteristics of minimal invasion,short hospital stay,good effi cacy in short term and repeated cases. This procedure to be used as the first choice for patients withgood renal function and mild hydronephrosis,especially accompanied with renal calculus.

Background To optimize the culture method of human retinal microvascular endothelial cells is very important for the study of retinal angiogenesis disease.Human retinal microvascular endothelial cells have been successfully cultured in previous studies,but further improvement of the culture method to harvest higher yields and purity cells is still needed.Objective This study was to design a modified method to isolate and purify human retinal microvascular endothelial cells much easily and quickly,and to compare the expression of specific markers of vascular endothelial cells,factor Ⅷ and CD31/CD34 in the cells.Methods The use of human donor eyeballs was approved by the Ethic Commission of Zhongshan Ophthalmic Center of Sun Yat-sen University.The retina tissue from healthy donor was isolated and digested by the two-step digestion method with 2％ trypsin and 0.133％ collagenase Ⅳ.Human retinal microvascular endothelial cells were collected and plated in 60 mm dishes coated by 0.1％ fibronectin and cultured in endothelial cell-specialized medium supplemented with 10％ fetal bovine serum,0.3 mg/L β-endothelial cell growth factor (ECGF) and 100 ng/L sodium heparin.During the culturing,the growth situation of the cells was monitored by morphological observation,and immunohistochemical staining was performed to probe vascular endothelial cell-specific membrane protein CD31,CD34 and factor Ⅷ for identification of the cell purity.Results Human retinal microvascular endothelial cells were isolated successfully from the retina by the twostep digestion method.The primary cultured cells adhered to well 72 hours later and achieved confluence with the typical cobblestone appearance 9 to 10 days after cultured.The cells exhibited the blue nuclei and reddish cytoplasm by regular haematoxylin and eosin stain and showed a strong positive response for CD31,CD34 and factor Ⅷ by immunohistochemistry.The positive dye of CD31 and CD34 was lower than Ⅷ factor in both endothelial cells.Conclusions Modified culture method of human retinal microvascular endothelial cells can improve cell culture result and purify target cells.

Objective To investigate a new method of improving design of the skin flap pedicled with descending hranch of lateral femoral circumflex artery,in order to increase the accuracy of preoperative Doppler location.Methods Six fresh cadavers underwent a whole body,intra-arterial injection of a lead ox- ide and gelatine preparation.Observe the perforators of anterolateral thigh by dissection,measured their diam- eter;course,branches and location for there were showed up by angiography and photography.A specific pro- gram,3D-doctor,was used for the detection of the regions of interest in angisome and its 3D-reconstructive.In addition to the average area supplied by a single perforator within that territory was calculated from digital an- giograms using Scion Image.Results There were 16 perforators that the diameter≥0.5 ram,20%were septocutaneous,80%were musculocutaneous.Their average external diameter was 0.8 ram.The average ped- icle length from the deep fascia was(3.15?1.43)ram;the perforators from descending branch of lateral cir- cumflex femoral artery walk length average about 2.63 cm in superficial fascia.The area of each perforator supplied blood was average 45.61 cm~2.Conclusion Modified lead oxid-gelatine technique provides high quality angiograms for the study of cutaneous artery and perforator falp.Based on our data,the maximum di- mension of the anterolateral thigh perforator flap can be 30 cm?20 cm in with only a single dominant perfora- tor.The perforator flaps deviced by perforators from the anterolateral thigh are transplanted to the lower limbs and the other part of the body.

BackgroundHuman retinal gliocytes play an important role in proliferative diseases,which are the basis of in vitro studies.Researchers have cultured human retinal gliocytes in the past.In our study,we found that the cells we cultured presented a unique shape different from those by other researchers.ObjectiveThis study was to design to produce a new culture and purification method for retinal gliocyte in vitro.Methods Retinal tissue was isolated from human eyeballs and digested using the two-step digestion method (2％ pancreatin and 0.133％collagenase Ⅵ) to harvest the retinal glio cytes.The cells were collected and cultured in endothelial cell-targeted nutrient culture containing 10％ fetal calf serum and supplemented with β-endothelial cell growth factor (ECGF) and sodium heparin,and the culture dishes were coated with fibronectin(FN) to promote the attachment of retinal gliocyte.During the culturing process,the gliocytes were identified by the observation of morphological characteristic and regular histological examination.The identification of the cells also was performed by immunochemistry targeting glial fibrillary acidic protein (GFAP),Vimentin,neuron specific enolase ( NSE ),S-100,CD34,and Ⅷ factor.Results Retinal gliocytes were isolated successfully from the human retina by the two-step digestion method.Primary cultured cells attached after 72 hours and achieved confluency between day 9 and 10 that were aligned petaliform in shape.Regular histological examination after H&E staining showed blue cell nuclei and light red cytoplasm.The target cells presented with strong responses for GFAP and Vimentin and no response for NSE,S-100,CD34 and Ⅷ factor.ConclusionsLarge amount of purified human retinal gliocytes can be obtained by two-step digestion and cultured in endothelial cells-targeted culture medium supplemented with β-ECGF and sodium heparin in plates coated with FN.The cultured cells expressed markers for retinal gliocytes.However,specific features of these cells remain to be further elucidated.