Objective To further approach the effect of miR-33 to melanoma cells line B16F10 proliferation and apoptosis.Methods Constructing targeted miR-33 over-expression mimics and inhibitor,the same B16F10 cells were divided into five groups,control group,miR-33 mimics group,mimics control group,miR-33 inhibitor group,inhibitor control group,then gene transfer technology was used to transfer corresponding gene into B16F10 cells.The effect of miR-33 on B16F10 cell' s proliferation and apoptosis were analysed.Results The relative miR-33 gene expression of miR-33 mimics group (1773.3±245.83) was higher than that of control group,which had statistical significance (P ＜ 0.01).The gene expression of miR-33 inhibitor group (0.6973±0.1958) was lower than those of control group and inhibitor control group.The cell growth rate of miR-33 mimics group was lower than those of control group and the trend after transfection 48 h (1.1875±0.0502) and 72 h (1.7500±0.0933) was significant (P ＜ 0.05).The cell apoptotic ratio of miR-33 mimics group [(1.8050±0.2050) ％] was higher than that of control group [(1.13000±0.1414) ％] (P ＜ 0.05).Compared with control group the cell proportion ofG1 period in miR-33 mimics group [(62.7000±1.7321) ％]increased,the cell proportion of S period [(23.4000±2.5044) ％] decreased,both of them had statistical significance (both P ＜ 0.05).Conclusion miR-33 over-expression can restrain the proliferation of B16F10 cells line,promote B16F10 cells' apoptosis.

OBJECTIVETo analyze the influence of retinoic acid (RA) on the undifferentiated state and EB formation abilities of human embryonic stem cells.

METHODSThe biological characteristics of H9 ESCs after RA treatment were characterized by real-time PCR, MTS proliferation assay and immunofluorescence staining. The expression of three germ layers markers, osteogenic differentiation markers and adipogenic differentiation markers in H9-differentiated embryoid bodies (EBs) with RA treatment were quantified by real time PCR.

RESULTSThe proliferation of H9 ESCs in the early logarithmic growth phase was accelerated by RA treatment. In addition, RA induced differentiation of H9 ESC coupled with morphology changes, decreased expression of undifferentiated markers Oct4, Nanog, Sox2 and OCT4 mRNA binding protein Lin28 at mRNA level, and reduced expression of Oct4 at protein level. RA induced formation of cavities in EBs. Real time PCR results showed that the expressions of ectodermal markers: NeuroD1, Noggin; mesodermal markers: Brachyury, Twist and endodermal markers: AFP, GATA-4 were significantly increased (P < 0.05), especially for AFP (P < 0.01), by RA treatment in a dose-dependent manner. In addition, the expression of adipogenic differentiation marker C/EBPalpha was increased while the osteogenic differentiation marker OPN was decreased in EBs after RA treatment for 5 days.

CONCLUSIONSHigh concentrations of RA induced the loss of stemness in H9 ESCs and excessive differentiation in EBs, and damaged the balance between osteogenic and adipogenic differentiation during early EB differentiation, which may be relevant to the congenital malformations.

Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Embryonic Stem Cells ; cytology ; drug effects ; Humans ; Tretinoin ; pharmacology

Objective To evaluate a surgical approach and anastomosis for the treatment of carcinoma of the gastric cardia. Methods Transabdominal intramediastinal esophagogastric anastomosis covered by sero-muscular flap of gastricwall for cardial carcinoma in 187 cases. Results The method reached the satisfactory surgical result in terms of tumor free cut edge on esophagus end of the resected samples. And the morbidity rate was 5.8%. Conclusion The technique enables lymphadenectomy within the lower mediastinum and a sufficiently long enough resection of esophagus. Transabdominal incision of the crus dextrum of the diaphragm makes a clear operative field for the purpose of radical operation for carcinoma of the gastric cardia. The anastomosis effectively prevents anastomotic leakage. This procedure is indicated for cardial carcinoma cases in which the esophaged involvement is within 2cm.

ObjectiveTo summarize the application experience of laparoscopic totally extraperitoneal prosthetic(TEP) for inguinal hernia in grass-root hospitals.MethodsThe clinical data of 63 cases of inguinal hernia treated by TEP were retrospectively analyzed,and operation scheme suited to the actual conditions of grass-root hospitals was summed up.ResultsAmong the 63 cases,5 cases were treated by transabdominal preperitoneal repair of inguinal hernia(TAPP) to complete the operation because of relatively serious peritoneal rupture.No case was converted to open surgery.11 cases were subjected to continuous epidural anesthesia and the other cases to endotracheal anesthesia.17 cases gained satisfactory result withoutstapling fixation.Among them,a few people had postoperative complications:3 cases of pneumoscrotum,1 case of hematoma of scrotum and 2 cases of early groin pain.No case recurred.ConclusionTEP is a safe and effective operation method in grass-root hospital for inguinal hernia repair.A reasonable selection can be taken according to the individual condition of patients including operation,anaesthesia,mesh style and stapling fixation,to ensure the operation quality,to reduce the medical wst and to reduce the economic burden of the patients.

BACKGROUND:Human amniotic membrane-derived mesenchymal stem cells (AMSCs) are considered to be one kind of adult stem cells that can be easily obtained in large quantities without using an invasive method. Because of their low immunogenicity, anti-inflammatory properties, multipotency of differentiation and without ethical issue, human amniotic membrane-derived mesenchymal stem cells have been proposed as a good candidate to be used in celltherapy and regenerative medicine. However, the biological properties and the differentiation capacity of human amniotic membrane-derived mesenchymal stem cells are stil poorly characterized. OBJECTIVE:To establish a practical method for isolation and purification of human amniotic membrane-derived mesenchymal stem cells, and to study the biological characteristics and dopaminergic neural-like celldifferentiation potential of the human amniotic membrane-derived mesenchymal stem cells. METHODS:Human amniotic membrane-derived mesenchymal stem cells were disassociated and isolated from the amniotic membrane by trypsin and col agenase based enzymic digestion, and purified by percol mediated density gradient centrifugation. Expressions of surface antigens and transcription factors of the human amniotic membrane-derived mesenchymal stem cells were determined by flow cytometry and western blot assays. Based on the osteogenic and adipogenic induction, the multipotent differentiation capability of human amniotic membrane-derived mesenchymal stem cells was determined. Induction of neural celldifferentiation of human amniotic membrane-derived mesenchymal stem cells was conducted in Neurabasal conditioning medium with ATRA supplement. Neural cellassociated bio-markers were determined by immunofluoresence staining and confocal microscope. RESULTS AND CONCLUSION:In this study, we performed a practical method to isolate and purify human amniotic membrane-derived mesenchymal stem cells and amniotic epithelial cells simultaneously, with high cells yield. We demonstrated a group of constitutive expressions of neural antigens and embryonic associated transcription factor proteins (OCT-4, SOX-2 and KLF4) in fresh isolated human amniotic membrane-derived mesenchymal stem cells as wel as in human amniotic membrane-derived mesenchymal stem cells after in vitro passage, which suggested that the human amniotic membrane-derived mesenchymal stem cells not only possessed intrinsic tendency to neural celldifferentiation, but also maintained their stem cellcharacteristics after in vitro passage. We stimulated the human amniotic membrane-derived mesenchymal stem cells in the neurobasal-A and B27 based conditioning medium to induce neural celldifferentiation. The induced human amniotic membrane-derived mesenchymal stem cells displayed an up-regulation of expression in panel of neural and dopaminergic associate molecules (β-tubulin III, neuron-specific nuclear protein, tyrosine hydroxylase, glial fibril ary acidic protein, myelin basic protein and nestin) by flow cytometry and immunofluorescence staining, which demonstrated the multipotent differentiation capability and dopaminergic neuron-like differentiation potential of the human amniotic membrane-derived mesenchymal stem cells.

Objective To explore the impact of nucleos(t)ide analogue antiviral therapy on anxiety and depression of patients with chronic hepatitis B (CHB),and analyze the related factors.Methods Before nucleos(t)ide analogue antiviral therapy,1 year and 2 years after antiviral therapy,120 CHB patients were investigated with self-rating anxiety scale (SAS) and self-rating depression scale (SDS).The demography data of patients were collected.Serum levels of alanine transaminase (ALT) and hepatitis B virus (HBV) DNA and other biochemical indicators were measured regularly.Results Before nucleos(t)ide analogue antiviral therapy,1 year and 2 years after antiviral therapy,both the mean scores of SAS and SDS became lower gradually (F=12.661 and 22.395,respectively;both P＜0.01).The percentage of patients with SAS and SDS scores more than 50 were 5.8％,4.2％,1.7％ and 13.3％,7.5％,5.0％,respectively.After 2 years of therapy,the anxiety improvement rate of the patients obtained HBV DNA＜1000 copy/mL was 69.0％,while those with HBV DNA≥1000 copy/mL was 22.2％ (x2 =22.325,P＜0.01).Meanwhile,after 2 years of therapy,the depression improvement rate of the patients obtained HBV DNA＜1000 copy/mL was 77.4％,while those with HBV DNA≥1000 copy/mL was 22.2％ (x2 =32.179,P＜0.01).Multiple factors Logistic regression analysis indicated that the odds ratios (OR) of improvement of anxiety and depression in patients with HBV DNA＜1000 copy/mL were 7.751 (95％ CI:3.026-19.853) and 15.069(95％ CI:5.309-42.770),respectively,compared with those with HBV DNA≥1000 copy/mL; and OR of improvement of depression in patients with ALT≤40 U/L waa 4.103 (95％ CI: 1.376 - 12.238).Conclusions Nucleos(t) ide analogue antiviral therapy could improve the anxiety and depression of CHB patients.The HBV DNA negativity is the independent impact factor of improvement of anxiety and depression in CHB the patients.

Objective To validate genetic suppression of metastastic capability of highly metastastic melanoma cells by endostatin transfection.Method pcDNA3.1-Endo eukaryotic expression vector contained insulin signal peptide sequence was transfected into highly metastatic mice melanoma cell strain B 16.The expression of endostain was detected by RT-PCR and Western blot experiment,melanoma cells were determined with adhere experiment,in vitro invasion and migration experiment and pulmonary metastasis experiment on C57BL/6 mice.Result Endostatin can obviously inhibit the capability of adherence,in vitro invasion and migration and pulmonary metastasis of melanoma cells.Among them,adhere inhibition ratio was 67.3%,in vitro invasion inhibition ratio was 48.4%,cell migration inhibition ratiowas 52.1%and pulmonary metastasis inhibition ratio was 67.3%.Conclusion Endostatin transfection can obviously inhibit the highly metastatie capability of melanoma cells.

BACKGROUND:Umbilical cord mesenchymal stem cells are plentifully and conveniently obtained with a high proliferative ability, and have opened up a new way to treat patients with liver failure as they can differentiate into hepatocyte-like cells.OBJECTIVE:To observe the safety and efficacy in the treatment of chronic liver failure by transplanting umbilical cord mesenchymal stem cells.METHODS:Using parallel contrast method, 50 patients with chronic liver failure were divided into two groups, namely a stem cell group and a control group, containing 25 patients in each group. For the first group, transplantation of umbilical cord mesenchymal stem cells, (1.4-2.3)×106/kg, 100 mL, was given on the basis of medical comprehensive treatment,while for the second group only simple medical comprehensive treatment was given. The injection was done every 15 days, totally three times. Liver functions, prothrombin activity, Model for End-Stage Liver Disease (MELD) score, clinical symptoms, survival and side effects of the patients were observed before and 2, 4, 12 and 24 weeks after the treatment.RESULTS AND CONCLUSION:Compared with the control group, the albumin level and prothrombin activity were significantly increased in the stem cell group 12 and 24 weeks after treatment (P < 0.05), while the MELD score was significantly decreased in the stem cell group at 4, 12 and 24 weeks after treatment (P < 0.05). There was no significant difference in alanine aminotransferase and total bilirubin levels between the two groups (P > 0.05). Four weeks after treatment, clinical symptoms of the stem cell group improved significantly in comparison with the control group (P < 0.05).During the 24-week follow-up, the survival rate in the stem cell group was significantly higher than that in the control group (P < 0.05). Additionally, there were no adverse reactions and liver cancer associated with the stem cell therapy.Results show that it is safe and effective to treat patients with chronic liver failure through the transplantation of umbilical cord mesenchymal stem cells, and the cell transplantation can significantly improve patients' survival rate.

AIM To compare liver-protective and anti-inflammatory effects of extracts from root,stem,leaf,and flower of Gentiana rigescens.METHODS The mouse model for the immunological liver injury induced by Concanavalin A,and mouse ear swelling model for inflammation caused by dimethylbenzene were used for the comparison of the liver-protective or anti-inflammatory effects of four parts individually.RESULTS Four aqueous extracts of Gentiana rigescens showed the dose-dependent decrease in the activity of ALT and AST in serum and liver index,and alleviation of hepatic tissue injury induced by Concanavalin A in mice.The effects of the extracts from the leaf and root were better than those from the stem and flower.These extracts presented dose-dependent inhibition against the ear swelling caused by dimethylbenzene in mice.The effects of the extracts from the leaf and stem were better than those from the flower and root.CONCLUSION Extracts from the root and leaf of G.rigescens have liver-protective effect,and parts from the stem and leaf have anti-inflammatory effect.

Objective To evaluate the efficacy of confocal laser endomicroscopy(CLE)for the assessment of the grading of gastric intestinal metaplasia(GIM)in vivo.Methods Patients with known GIM underwent CLE(Pentax EC-3870K).The presence of GIM was indentified immediately by the endoscopist during the procedure.The updated Sydney Classifycation System as reference,GIM was subdivided as mild,moderate and severe according to the area of intestinal metaplasia glands and the number of goblet cells.The histological evaluation remained the gold standard for the final diagnosis of GIM.The presence of the dysplasia and the expression of the Ki67 were examined.Results A total of 151 GIM positive areas were found in 58 patients with mild in 92,moderate in 34.and severe in 25 by CLE. One hundred and forty-six GIM areas were examined histopathologically with positive rate of 96.7%(mild in 82,moderate in 36 and severe in 28).The sensitivity and specificity of CLE were 90.2%and 73.9%in diagnosis of mild GIM,69.4 oA and 92.2%in moderate GIM,71.4% and 95.9%in severe GIM.The kappa coefficient of CLE criteria and the histopathological grading for mild,moderate and severe GIM were 0.65,0.63 and 0.70,respectively.The more severe the GIM,the higher the ratio of incomplete GIM,the ratio of dysplasia and the stronger expression of Ki67.Conclusions CLE may offer an instant and reliable diagnosis for GIM with high accuracy.It is helpful in grading of GIM.