BACKGROUND:In vitro isolation and purity technique of stem cells mostly depends on the identification of cell surface marker,such as monoclonal antibody adherent spreading method,flow cell sorting method and immunomagnetic beads sorting method,but the operation was complicated and the price was high.OBJECTIVE:To observe the biological characteristics of human amniotic fluid-derived embryonic mesenchymal stem cells,which were isolated and cultured using the two-step method.DESIGN,TIME AND SETTING:The opening study was conducted at the Stem Cell Research Room of Xinjiang Medical University from March 2008 to March 2009.MATERIALS:Totally 10 amniotic fluid specimens were obtained from pregnant women who underwent prenatal diagnosis following 16-22 weeks of gestation or voluntarily induced abortion.With ultrasonic guidance,amniocentesis was performed to collect 20-40 mL amniotic fluid.METHODS:Human amniotic fluid-derived embryonic mesenchymal stem cells were isolated and cultured using the two-step method.Amniotic fluid was first centrifuged and incubated till spindle-shape cells were seen,with the presence of flbroblast-tike cell colonies.Supematant was moved to a new 25 cm~2 culture flask for further culture till spindle-shape fibroblast-like mesenchymal stem cell colonies.When 70% confluence,cells were digested,and incubated in α-MEM,supplemented with basic fibroblast growth factor,served as the first passage.MAIN OUTCOME MEASURES:Morphological changes in human amniotic fluid-derived embryonic mesenchymal stem cells of primary culture and subculture were measured.Karyotype,cycle,growth curve and colony formation ability of human amniotic fluid-derived embryonic mesenchymal stem cells were measured.Surface antigen and cytokine were examined using flow cytometry,immunofluorescence and RT-PCR.RESULTS:Human amniotic fluid-derived embryonic mesenchymal stem cells were successfully isolated and subcultured.During metaphase,primarily cultured amniotic fluid cells presented scattered spindle cells and flbroblast-like mesenchymal stem cell colonies every 7 days.Passaged cells completely adhered in 12 hours.Following 1 or 2 days of latent period,cells proliferated rapidly.About 90% confluence was observed following 6 or 7 days of culture.Cell arranged regularly,showing whirlpool-shape,radiated shape.Cells were spindle-shape,with unclear boundary.Chromosome karyotype of human amniotic fluid-derived embryonic mesenchymal stem cells was normal diploid.Growth curve showed "S" shape,but the two-step method reached a peak at (6.1±0.5) days,which was significantly rapid compared with the one-step method (7.2±0.6) days (P=0.035).Flow cytometry analyses showed that P3 cells at S phase took up (14±2.3)% using the two-step method,which was more than the one-step method (9.0±1.4)% (P=0.031).Low-density human amniotic fluid-derived embryonic mesenchymal stem cells were incubated for 7 days prior to cells formed scattered cell colonies.However,colony forming efficiency using the two-step method (15.0±2.3)% were significantly more than the one-step method (10.0±1.8)% (P=0.021).Flow cytometry results showed that human amniotic fluid-derived embryonic mesenchymal stem cells expressed CD44,CD29 and CD105,but were negatively for CD45,CD34,HLA-DR.Immunofluorescence suggested that Oct-4-positive cells were observed in amniotic fluid.However,the proportion of Oct-4-positive cells using two-step method (1.2±0.3)% was significantly greater than the one-step method (0.9±0.2)% (P=0.041).RT-PCR suggested that human amniotic fluid-derived embryonic mesenchymal stem cells obtained using the two methods expressed Oct-4.CONCLUSION:Human multipotent mesenchymal stem cells are present in human amniotic fluid.The two-step culture protocol could be a kind of high performance and simple protocol which may not interfere with the normal prenatal diagnosis procedure.

Objective To observe the change of six cytokines in mice infected with Echinococcus multilocularis as part of the study on immunological mechanism in the infection. Methods Mice were infected by abdominal inoculation of echinococcus protoscoleces. The change of serum level of the cytokines IL-2、IFN-?、TNF-?、IL-4、 IL-5 and IL-10 was determined by ELISA during the infection which lasted for 260 d. Results Compared with uninfected control, the levels of the cytokines all significantly increased in the 260 d. The level of IL-2 reached a peak after 80 d post-infection (p.i.), then decreased quickly after 140 d p.i., High level of TNF-? was detected after 40 d, compared to uninfected control, reached a peak at 100 d p.i., and decreased quickly after 140 d. The level of IFN-? reached a peak after 80 d p.i., and decreased slowly after 140 d p.i., The levels of IL-4, IL-5 and IL-10 remained lower before 80 d, and increased sharply after 100 days. The levels of IL-4 and IL-10 reached peaks at 100 d p.i., and that of IL-5 at 140 d p.i. Conclusion The data suggest that the induction of Th2 antibody-mediated immunity (AMI) with a parallel expansion of Th1 cell-mediated inflammatory (CMI) responses are important mechanism of the host in defending against the metacestodes. Th1 CMI plays an important role at the early stage of infection, and Th2 AMI is important in the later stage of infection.

Described in the paper are contents and characteristics of building smart healthcare system at a research hospital,and practices of the hospital in exploring smart healthcare.Specific measures include staged building of an e-hospital emphasizing information security; closed-loop transformation with mobile and barcode technology for higher efficiency and patient safety;optimizing the service flow with smart interconnection to create an orderly outpatient environment; implementing healthcare regulations with information systems to ensure a homogeneous medical environment;and integrating the entire medical system to promote coordinated development of regional healthcare service. These measures help upgrade the information service effectiveness and quality of care of the hospital.

Objective: To explore the inhibition effects of ectogenic wild type p53 cDNA(Ad wtp53) on colorectal carcinoma cell lines with different p53 gene status and search for the role of wild type p53 tumor suppressor gene in occurrenc and progress of malignant tumor. Methods: MTT process was taken to choose optimal transfection titre. Three kinds of cell lines(p53 gene deletion, mutation and nomal) were transferred by Ad wtp53 in optimal titre. The inhibition effects of these cell lines were observed and compared. Results: The best titre is 1000 MOI and p53 gene deletion cell line (THC 8908) shew the highest sensitivity. G 1 S transition period blocking effects occurred in all cell lines and G 2 M phase regulation effects were not coincidence in three colorectal cell lines. Conclusions: Recombinant adenovirus mediated wild type p53 gene observably inhibited colorectal carcinoma cell lines growth and proliferation, blocked cell cycle in G 0 /G 1 phase and displayed obvious different actions on G 2 M phase among cell lines with different p53 status.

ObjectiveTo investigate the changes in serum levels of M30, M65, and interleukin-17 (IL-17) and their clinical significance in patients with acute pancreatitis. MethodsA total of 126 patients with acute pancreatitis who were admitted to our hospital from December 2009 to December 2013 were selected, and according to clinical diagnosis, they were divided into mild acute pancreatitis group (82 patients) and severe acute pancreatitis group (44 patients). A total of 107 healthy subjects who underwent physical examination during the same period of time were enrolled as the control group. On days 1, 2, and 4, the serum levels of M30, M65, and IL-17 were measured, and M30/M65 ratio was calculated. Comparison of coutinous data between multiple groups was made by ANOVA and pairwise comparison between any two groups was made by SNK-q test, comparison between two groups was made by independent-sample t test, while comparison of categorical data by chisquare test. ResultsOn days 1, 2, and 4, the severe acute pancreatitis group and mild acute pancreatitis group had significantly higher serum levels of M30, M65, and IL-17 (P＜0.05), and a significantly lower M30/M65 ratio (P＜0001), as compared with the healthy controls. On days 1, 2, and 4, the severe acute pancreatitis group had significantly higher serum levels of M30 and M65 than the mild acute pancreatitis group (P＜0.001); on day 1, the severe acute pancreatitis group had a significantly lower M30/M65 ratio than the mild acute pancreatitis group (P＜0.001); the serum level of IL-17 showed no significant difference between the two groups (P＞0.05). M65 and IL-17 had high sensitivity and specificity in the diagnosis of acute pancreatitis. ConclusionThe serum levels of M65 and IL-17 and M30/M65 ratio at 24 hours after the attack of acute pancreatitis can be used as the serological biomarkers for early evaluation of the severity of acute pancreatitis.

Objective To explore hemodynamic parameter changes in pulmonary arterial hypertension(PAH)treated by transplantation of bone marrow mesenchymal stem cells(MMSCs)in the experimental rats.Methods MMSCs cells were collected from bone marrow of Sprague-Dawleye(SD)rat's femoral and tibial bones,cultured and passaged in vitro,then stained by Hoechst 33342 fluorescence dye stuff.Ninety healthy male SD rats were randomly divided into 3 groups(n=30):normal control group(group N),MMSCs transplanted group(group M),PAH model group(group H).The rats in the two latter groups were given a single subcutaneous crotaline(50 mg/kg)to establish the model of PAH.The rats of group N were injected respectively a single subcutaneous 9 g/L saline water(6 mL/kg).After 21 days,5?109 L-1 MMSCs cultured in 1 mL phosphate-buffered saline were infused into the rats respectively in group M by sublingual vein and 1 mL L-DMEM was given in group H.The indexalso of right ventricle systolic pressure(RVSP),right ventricle hypertrophy index,arterial blood gas analysis and the changes of small pulmonary blood vessel were observed after 28 days.Results The administration of MMSCs 28 days after PAH nearly completely prevented the increase in RVSP with PAH alone [(32.20?2.32)mmHg vs(48.30?1.56)mmHg P

The global incidence rate of nonalcoholic steatohepatitis (NASH) continues to rise. The pathogenesis of NASH is complex, and there is no effective clinical treatment. Previous study has shown that DEAD box protein 5 (DDX5) can significantly alleviate the NASH process in mice. This study screened the natural product library of the research group and found that the active compound hypercalin B (HB) in Hypericum beanii N. Robson, a traditional Chinese medicine, can upregulate the expression of DDX5 protein in a dose-dependent manner. In this study, an in vitro model of NASH stimulated by palmitic acid (PA) and an animal model of NASH induced by the methionine- and choline-deficient diet (MCD) were constructed. Different concentrations of HB were used to investigate the effect and mechanism of HB in alleviating NASH progression. All animal experiments in this paper were approved by the Ethics Committee of China Pharmaceutical University (NO: 2021-02-003). In vitro model results showed that HB significantly reduced the intracellular lipid deposition induced by free fatty acid (FFA). Animal experiments showed that HB improved liver injury by significantly reducing lipid accumulation in the liver of NASH mice, and reducing serum aspartate transaminase (AST) and alanine transaminase (ALT) levels. Moreover, HB could inhibit liver inflammation by reducing the mRNA levels of liver pro-inflammatory cytokines including interleukin 6 (IL-6), interleukin 1β (IL-1β), and tumor necrosis factor α (TNFα). Further research showed that HB could reduce the phosphorylation level of the mechanical target of rapamycin (mTOR) and reduce the expression of sterol regulatory element binding protein 1 (SREBP1) and fatty acid synthase (FASN), thereby improving lipid metabolism and alleviating NASH progression, and the effects of HB against NASH were dependent on DDX5. In conclusion, HB can improve lipid metabolism and inhibit inflammatory activation by suppressing mTORC1 pathway via upregulating DDX5 protein, and showed promising anti-NASH activity in vitro and in vivo.

OBJECTIVETo observe the effect of salidroside on tic behavior and in vivo dopamine DA) and serotonin (5-HT) levels in Tourette syndrome (TS) model rats.

METHODSForty rats were randomly divided into the blank control group, the TS model group, the haloperidol-treated group (0.5 mg/kg x d(-1)), and the salidroside-treated group (50 mg/kg x d(-1)), 10 in each group. TS rat model was induced by imino-dipropio-nitrile (IDPN). Peritoneal injection of haloperidol and salidroside was started from the 4th day of modeling in the haloperidol-treated group and the salidroside-treated group respectively. Normal saline was peritoneally injected to rats in the blank control group and the TS model group respectively. Stereotyped behavior was scored, and changes of DA and 5-HT levels in blood and striatum were measured before modeling, after modeling, and after intervention.

RESULTSCompared with the blank control group, the score of the tic behavior was elevated (P < 0.01) , levels of DA and 5-HT in plasma and striatum were reduced in the model group (P < 0.01, P < 0.05). Compared with the same group after modeling, the tic behavior score decreased and plasma DA levels increased in the two treated groups after intervention (P < 0.01). 5-HT content increased in the salidroside-treated group (P < 0.01). Compared with the model group after intervention, the tic behavior score was significantly reduced (P < 0.01), and DA levels in plasma and striatum were elevated (P < 0.01, P < 0.05) in the salidroside-treated group and the haloperidol-treated group. Compared with the haloperidol-treated group, the tic behavior score increased (P < 0.01), DA levels in plasma and striatum were lowered (P < 0.01, P < 0.05), the 5-HT level increased in plasma and striatum (P < 0.01, P < 0.05) in the salidroside-treated group.

CONCLUSIONSIn the salidroside-treated group, the tic behavior was significantly reduced, and DA levels in plasma and striatum were elevated. Its mechanism might be related to regulating activities of dopamine neurons in striatum.

Animals ; Corpus Striatum ; Dopamine ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Glucosides ; pharmacology ; therapeutic use ; Haloperidol ; Phenols ; pharmacology ; therapeutic use ; Rats ; Serotonin ; Stereotyped Behavior ; Tics ; drug therapy ; Tourette Syndrome ; drug therapy

Objective To investigate whether the bilateral nipple discharge with the background of hypothyroidism and pituitary tumor causes intraductal neoplasm. Methods Clinical data of 222 bilateral nipple discharge patients with hypothyroidism or pituitary tumor undergoing mammary ductoseopy (MDS) examination were analyzed. Those diagnosed as intraductal papilloma received surgery and pathological exam. 158 cases with inflammatory comedomastitis diagnosed by MDS received milk duct washing with gentamycin, dexamethasone and corresponding medical treatment. Results Among the 222 cases, 158 cases(71.17%) were diagnosed as inflammatory comedomastitis by MDS, 64 cases (28.83%) were diagnosed as intraduetal papilloma (64/222) by MDS and received operation,59 cases were pathologically diagnosed as intraductal papilloma,the other 5 cases were pathologically diagnosed as mammary duct ectasia. The pathological coincidence was 92.2% and no breast cancer was observed. 68.10% of inflammatory comedomastitis cases suffered from breast pain. There were no significant difference in the disease distribution of the ethnic groups. During pre-menopause period, the incidence of period neoplasia is higher in pituitary tumor than hypothyroidism; However, during the post-menopause the situation was vice versa. The color of niplle discharge is mostly milk-like in patients with pituitary tumor, while serous in patients with hypothyroidism. 71.19% of duct papilloma locate at the branch of the main duct. Conclusion The bilateral nipple discharge with hypothyroidism or pituitary are usually concomitant with intraduetal lesions. Mammary ductosopy is helpful to discover intraduetal neoplasia.

Objective To investigate the pathogenesis of the expression and activation of signal transducers and activator of transcription(STAT) 6 in patients with ulcerative colitis(UC).Methods Thirty patients with active UC(diagnosed with colonoscopy with pathologicaly conformation),who were not treated with steroids or immunomodulators,and 30 age-matched healthy controls were studied.The expressions of phosphorylated STAT6 and non-phosphorylated STAT6 in nucleus and cytoplasm were deterimined by Western blot.DNA binding activity of STAT6 was tested by electrophoresis mobility shift assay(EMSA).Results The expression of phosphorylated STAT6 was higher in nucleus than that in cytoplasm in mild UC patients,but the expression was higher in cytoplasm than that in nucleus in moderate to sever UC patients (P