AIM: To investigate whether a pathological micro-environmental culture system consisting of cholestatic sera induces embryonic stem cells (ESC) to differentiate into hepatocyte-like cells in vitro, and select hepatic stem cells from differentiating embryonic stem cells. METHODS: Mouse ESC, E14 cell line, were cultured in Dulbecco's modified Eagle's medium containing 106 U/L recombinant mouse leukemia inhibitory factor (rmLIF) and 10% FCS. After embryonic bodies formed by the hanging drop culture method, they were exposed to fibroblast growth factor-4 (FGF-4) and hepatocyte growth factor (HGF) for one week, and then placed to a pathological micro-environmental culture system consisting of 5% cholestatic sera and cultured for 2 weeks. Morphological examination, immunocytochemical staining of albumin and CK8/18 were carried out, and mRNA level of albumin and transthyretin were detected by RT-PCR. Glycogen storage and urea synthesis of the cells were tested with PAS staining and colorimetric assay, respectively. RESULTS: The proliferation of cells was inhibited at the early stage when cultured in a pathological micro-environmental culture system consisting of 5% cholestatic sera, but 2 weeks later, a large number of epithelial-like cell colonies were observed, which exhibited hepatocellular phenotype, expressing albumin and CK8/18, transcribing mRNA of albumin and transthyretin and synthesizing glycogen and urea. CONCLUSION: A pathological micro- environmental culture system consisting of 5% cholestatic sera could not only induce embryonic stem cells to differentiate into hepatocyte-like cells, but select hepatic stem cells from differentiating embryonic stem cells initially induced by FGF-4 and HGF in vitro as well.

Objective To investigate the role and intracellular signal transduction mechanism in the injury of renal tubular cells induced by postasphyxial-serum in neonate.Methods Human renal proximal tubular cell(HK-2 cell) was used as target cell. The experiment was designed as:control group, asphyxia group ,and pyrrolidine dithiocarbamate (PDTC)blocking group. The attacking concentration of serum was 20%, and the apoptosis rate of HK-2 cells was detected by flow cytometer.Results Compared with controls[(13.3?1.70)%],after being stimulated with postasphyxial-serum, the apoptosis rate of HK-2 cells of asphyxia group [(46.73?3.68)%] and PDTC blocking group [(31.19?2.79)%]were significantly increased(P

Objective To investigate the role of postasphyxial-serum of neonate in inducing injury of human renal proximal tubular cells(HK-2 cells).Methods HK-2 cells were used as target cell.The neonatal different concentration postasphyxial-serum of 1,3,7 days after asphyxia were used as attacking means.The experimental groups were divided into 15 groups:the 2.5%,5.0%,10.0%,(20.0%) attacking concertration groups of 1,3,7 day after asphyxia and control group of each concertration.The culture medium and concertration of the control group and the experimental groups were the same.The changes of morphology were observed under inverted microscope,the cell viability was measured by 3-(4,5-dimethyl-2-thiazoly1)-2,5-diphenyl-2H-tetrazolium bromide(MTT) method and the leakage rate of lactate dehydrogenase(LDH) was determined by biochemical methods.Results Compared with control group,the changes in morphology of HK-2 were most serious and obvious,the cell viability were obviously decreased(all P

BACKGROUND: Recently, little attention has been paid to how to induce and identify the functions of differentiated cells in the methods for embryonic stem (ES) cells differentiation into hepatocytes. Whether the differentiated cells express functional characteristics of hepatocytes should be one of the markers to identify the hepatic differentiation of ES cells.OBJECTIVE: To direct mouse embryonic stem cells in vitro differentiation into functional hepatocytes by introduction of murine cholestatic serum in hepatocyte growth factor (HGF)-induced system.DESIGN: A controlled observation and in vitro cytological trial.SETTING: Department of Hepatobiliary Surgery, the Second Affiliated Hospital of Sun Yat-sen University.MATERIALS: The experiment was carried out in the Medical Research Center of the Second Affiliated Hospital of Sun Yat-sen University from October 2004 to February 2007. The mouse E14 ES cell line was kindly provided by the Stem Cell and Tissue Engineering Center of Sun Yat-sen University. Twenty male SD rats, aged 2 weeks, were purchased from the Experimental Animal Center of Sun Yat-sen University. All animal experimental procedures were abided by the rules of animal ethnics.METHODS: The SD rats were undergone common bile duct ligation to induce cholestasis. Ten days after the operation, the whole blood of rats was collected to prepare cholestatic serum. The ES cells were cultured using hanging-drop method for 5-7 days to develop embryonic bodies (EBs). The dissociated EBs cells were then induced hepatic differentiation with spontaneous system, HGF (20 μg/L) system and cholestatic serum (5%) plus HGF (20 μg/L) system, respectively.MAIN OUTCOME MEASURES: The cellular morphologic changes were observed using transverse microscopy dynamically. (2) The cell staining for albumin, α-fetoprotein, CK18/19, glycogen, indocyanine green (ICG) and fluorescein diacetate (FDA) was done after 4 weeks differentiation. (3) The hepatocyte-specific metabolic functions of synthesizing albumin, triacylglycerol and urea nitrogen were assayed at 3 days interval.RESULTS: (1) The differentiation of ES cells cultured in spontaneous system was uncontrolled and the cells could grow into a wide range of three-germ cells. The HGF could promote ES cells differentiation into endoderm and mesoderm (myocardium). But the differentiated cells only expressed low levels of hepatic specific functions in these two induced systems. (2) Under cholestatic serum plus HGF system, the ES cells could differentiate into polygonal cells with very uniform morphology which were positive in glycogen, ICG and FDA staining and showed higher capabilities of synthesizing albumin, triacylglycerol and urea nitrogen than the differentiated cells in the other systems (P＜0.05-0.01).CONCLUSION: The cholestatic serum, a mimic pathological microenvironment in vitro, could effectively promote ES cells-derived hepatocytes induced by HGF to express high level of liver-specific metabolism functions.

BACKGROUND: It is recently found that some kinds of antibiotics can aggravate the obstruction of neuromuscular junction(NM J) transmission,exacerbate myasthenia gravis (MG). Hitherto, there are few reports about the effect of antibiotics on transitive function on animal models. Along with the appearance of new antibiotics, the effects of the antibiotics on NMJ transitive function need to be further observed.OBJECTIVE: To investigate the effect of aminoglycoside antibiotics, fluoroquinolone antibiotics and cephalosporin antibiotics on the transitive function of NMJ in MG, and to provide an experimental basis for using those antibiotics securely in clinic and for selecting those antibiotics to treat MG properly.DESIGN: Randomized controlled study based on experimental animals.SETTING: Department of nosocomial infection, neurology and pharmacy in a university hospital.MATERIALS: The experiment was conducted at the Neurological Institute of Tongji Medical College, Huazhong University of Science and Technology from March 2002 to January 2003. Totally 150 healthy female C57BL/6mice, 6 - 8 weeks old, weighting 18 - 20 g, were divided randomly into 4groups: normal group( n = 10), MG group( n = 10), saline group( n = 10)and antibiotics group( n = 120) . Mice in antibiotics group were divided randomly again into gentamicin group, etimicin group, ciprofloxacin group,fleroxacin group, cefuroxime group and cephradine group, with 20 mice in each group.INTERVENTIONS: C57BL/6 mice were immunized with the acetylcholine receptor(AChR) protein in complete Fruend' s adjuvant(CFA) to establish experimental autoimmune myasthenia gravis(EAMG) . Mice in saline group were injected normal saline and mice in antibiotics group were injected antibiotics(10 mg/kg), lasted 14 days. Mice in MG group were without any treatments. On the 7th day after the last immunization and the 14th day after the antibiotics treatments, MG scores was evaluated, repetitive nerve stimulation(RNS) and the levels of acetylcholine receptor antibody(AChRab)were tested at the same time.RESULTS; The mean symptom scores on the 14th day after the antibiotics treatment with gentamicin, etimicin, ciprofloxacin and fleroxacin were higher than that in MG group, and there was no significant difference in the mean symptom scores among cefuroxime group, cephradine group and MG group. The decrement percent of RNS in gentamicin group [ (21.22 ± 4.63)% ], etimicin group[ (19.08 ±4. 25)% ], ciprofloxacin group[ (22.25 ±4.95)% ] and fleroxacin group[ (21.71 ±4.99)% ] were higher than that in MG group[(15.75 ±2.22)% ], but no difference was found in the attenuation rate among cefuroxime group[(15.25 ±2. 87)% ],cephradine group[ ( 15.25 ± 3.30)% ] and MG group. The levels of AChRab in gentamicin, etimicin, ciprofloxacin and fleroxacin groups were also higher than that in MG group, but no difference was found among cefuroxime group, cephradine group and MG group.CONCLUSOIN: Aminoglycoside and fluoroquinolone antibiotics can aggravate the obstruction of NMJ transmission, and cephalosporin antibiotics have no obvious effect on the obstruction of NMJ transmission function in MG.

Objective To investigate the prevalence and risk factors of mild cognitive impairment (MCI) among the old people in Chongqing, China. Methods From October to November, 2015, 1850 persons more than 60-year-old in Chongqing were cluster sampled. They were investigated with self-made general situation questionnaire, Mini-Mental State Examination (MMSE), Instrumental Activity of Daily Living Scale (IADL) and Geriatric Depression Scale (GDS) through face-to-face interview. Enumeration data were tested withχ2 test and multiple Logistic regression. Results MCI prevalence was 11.73%in the old people in Chongqing, and various with the residential re-gions, ages, marital status, literacy, career, smoking, drinking, seafood-eating, egg-eating, exercising, intensive labor, low intensity of labor, housework, outdoor activities, Mahjong/chess/cards playing, TV-watching/radio-listening/newspaper-reading, social activities, obesity, hy-pertension, diabetes, stroke, hyperglycemia, cerebrovascular insufficiency and depression (χ2>4.092, P<0.05). According to multiple Logis-tic regression, living in rural areas, aging, celibacy, obesity, hypertension and depression were the risk factors of MCI;while middle school and above years of schooling, mental workers, smoking cessation, frequent seafood-eating and egg-eating, exercising, daily intensive labor and low intensity of labor, daily and sometimes housework, daily and weekly outdoor activities at least once, daily Mahjong/chess/cards playing and daily TV-watching/radio-listening/newspaper-reading were the preventing factors. Conclusion The morbidity of MCI is high in the old people in Chongqing. It is necessary to take effective intervention in view of the related factors of MCI as early as possible, to delay or prevent the development of MCI to dementia.

AIM: To investigate the mechanism of the AP-1 signal transduction pathway inhibited by JIP in nasopharyngeal carcinoma cells. METHODS: AP-1 activity was triggered by Dox-induced LMP1 expression in Tet-on-LMP1-HNE 2 cells (L7). The retention of phospho-JNK in the cytoplasm caused by JIP was examined with immunofluroscence assay. RESULTS: 24 h after transfection of L7 cells with the JIP expression plasmid, the translocation of activated JNK was inhibited, which resulted in the retention of phospho-JNK in the cytoplasm and down-regulation of the AP-1 activity. CONCLUSION: JIP down-regulates the activity of AP-1 through the inhibition of the translocation of JNK.

OBJECTIVESalvia miltorrhiza Bunge (SMB) is a traditional Chinese herb, which is considered to promote blood flow and remove blood stasis. This study examined whether SMB can alleviate injury induced by hypoxia/reoxygenation (H/R) in human kidney proximal tubular cells-2 (HK-2 cells).

METHODSThere were 3 experimental groups: control, H/R injury and SMB-treated H/R injury. H/R injury of HK-2 cells was induced by first covering the cells with and then removing liquid paraffin wax. Different concentrations of compound SMB solution (0.05%, 0.10%, 0.15% or 0.20%) were administered to the SMB-treated H/R injury group before the hypoxic injury. After 4, 12 and 24 hrs of hypoxia and 4, 12, 24 and 48 hrs of reoxygenation, morphologic changes of HK-2 cells were observed under an inverted microscope. Cell viability was measured by the MTT method. Lactate dehydrogenase (LDH) activity in the culture supernatants was assayed using biochemical methods; TNF-alpha levels were determined by radioimmunoassay (RIA).

RESULTSThe number of HK-2 cells was significantly reduced in the H/R injury group after hypoxia, and reached a nadir 24 hrs after hypoxia treatment. Various concentrations of SMB-treated groups showed significantly greater number of HK-2 cells than the H/R injury group. SMB solution (0.10%) produced the best effect. The levels of LDH and TNF-alpha in the H/R injury group were significantly increased, and reached a peak between 24 hrs of hypoxia and 4 hrs of reoxygenation when compared to the control group. Pre-treating with 0.10% SMB resulted in significantly lower levels of LDH and TNF-alpha than in the untreated H/R injury group at various time points of H/R.

CONCLUSIONSSMB has protective effects against H/R injury of HK-2 cells, possibly through inhibition of inflammatory cytokines.

Calcium ; metabolism ; Cell Hypoxia ; Cells, Cultured ; Cytoprotection ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Kidney Tubules, Proximal ; drug effects ; pathology ; L-Lactate Dehydrogenase ; metabolism ; Plant Extracts ; pharmacology ; Salvia miltiorrhiza ; Tumor Necrosis Factor-alpha ; analysis

OBJECTIVETo observe the biological changes of primary human nasopharyngeal epithelial cells in the early stage of immortalization.

METHODSThe morphological changes of nasopharyngeal epithelial cells were observed by phase contrast microscopy, and the activity profile of senescence-associated beta-galactosidase (SA-beta-Gal) was detected by SA-beta-Gal staining. The expression of p16(INK4a) protein was tested by immunochemical assay, and the life span in vitro of nasopharyngeal epithelial cells was calculated as population doublings. In addition, the expression of Epstein-Barr (EB) virus latent membrane protein 1 (LMP1) was also detected by immunofluorescence staining.

RESULTSMorphologically, cells treated with EB virus and 12-o-tetradecanoyl-phorbol-13-acetate (TPA) formed multi-layer foci, and their cellular life span in vitro was extended (about 155 days of culture). A low percentage of cells (about 4.8%) expressed SA-beta-Gal activity at late primary culture, and did not always express p16(INK4a) protein in the progression of culture.

CONCLUSIONSNasopharyngeal epithelial cells treated with EB virus in cooperation with TPA can pass through the stage of senescence and enter the early stage of immortalization. Some changes of phenotype occur in these cells. Our results provide data for further studying the mechanism of immortalization and the establishment of a human nasopharyngeal epithelial cell line.

Cell Transformation, Viral ; Cellular Senescence ; Cyclin-Dependent Kinase Inhibitor p16 ; analysis ; Epithelial Cells ; physiology ; virology ; Herpesvirus 4, Human ; physiology ; Humans ; Nasopharynx ; cytology ; virology ; Tetradecanoylphorbol Acetate ; pharmacology

Objective: To explore the application of asymmetrical sculpture in the ear reconstruction with autologous cartilage through comparing the difference of the balanced sculpture and asymmetrical sculpture. Methods: We used the method of retrospective survey and classified the patients who had undergone ear reconstruction because of microtia into two groups. Group A included the patients who had undergone operation with the application of balanced sculpture method. Group B included the patients who had undergone operation with the application of unsymmetrical sculpture method. We picked out 35 patients containing 19 patients of group A and 16 patients of group B according to our grouping criteria and exclusion criteria. The number of cases with complications was recorded, such as collapse of framework, exposure of cartilage, necrosis of skin flap and reconstructed ear infection. The clinical outcomes of two methods in refining the delicate structures of the reconstructed ear were evaluated (that is the definition, shape, size, color and projection). The time for sculpture was compared. Then we analysed the results and evaluated the effectiveness, safety and feasibility of asymmetrical sculpture′s application in the ear reconstruction. Results: Group A had 2 cases which suffered skin flap necrosis. One of them had cartilage exposure and then had secondary infection leading to local cartilage necrosis and absorption, but after active treatment and local skin flap transplantation it was cured. Group B had no skin flap necrosis, cartilage extrusion, framework deformation and distortion, framework collapse and necrosis or absorption. Group B had a higher score in definition, shape, size, color and projection, and the difference was significant in definition, shape, color and projection. In our clinical practice, the sculpture time in both group was similarly about 40 minutes after the surgery operator had skillfully mastered both techniques. Conclusions Both methods are safe and feasible. The method of asymmetrical sculpture does not require additional operative time, and the method of asymmetrical sculpture is a better one compared with the method of balanced sculpture in refining the reconstructed ear.