Objective To search for a good method for generation of hematopoietic stem cells (HSC) by embryonic stem cells (ESC),and to investigate the potential of HSC derived from ESC to reconstruct hematopoiesis in vivo.Methods Using a three-step method to induce a mice ESC line, E14.1-into HSC.And identifying HSC by flow cytometry analysis cell markers with CD34 +/Sca-1+, then HSC (1×109L-1)from ESC differentiation were injected into severe combined immunodefieency (SCID) mice for observing teratoma formation.To validate function of HSC by colonngenic cells assay and to reconstitute the hematopoiesis in lethally irradiated mice.Results Combining to use more hematopoietic stimulating factor to availably promote the El4.1 cell into embryoid body (EBs) with abundant hematopoietic progenitors.EBs were induced after 14 day to fast differentiate for HSC with peak percentages of CD34+/Sca-1+ cells reached to (13.72±2.07)%.To harvested ceils from EBs by day 14 for second -step HSC differentiation, percent of CD34+/Sca-1+ cells rise to(24.62±2.50) % after day 16 induction.Cloning forming units (CFU) analysis showed that more Erythro -myeloid cloning generation was observed at this period.Cells obtained in the second step are subsequently plated on monolayer of mice bone marrow stromal cells, in the presence of TPO, FLt3 ligand and superoatant of mice fetal liver stromal cells, cultured for additional 15 days, followed fast expansion of CD34+/Sca-1+ to maximally (58.64±4.20 ) % with more CFU-E, CFU-GM and CFU-GEMM population.Wright-Giemsa stain showed that its had the character of hematopoietic progenitors.Cells from the third-step were injected into SCID mice, but no teratomas were recovered in 2 mices after 6 weeks.Positive selection of CD34+/Sca-1+ cells by magnetic sorting from the third - step differentiation were transplanted into 7 lethally irradiated female mice while predominant hematopoietic reconstitution were observed in 10 days after transplantation, with 71.4% successful engraftment rate.And 3 recipients showed that the cell population of the peripheral blood leukocytes,red cells, hemoglobin approached normal index at 40 days after transplant, hut followed relative'slow renew in platelet count.Survival rate of transplant group is 43.0%, compared to 100% mortality in control mice.Karyotyping assays confirmed female mice with XY.Conclusions The results showed that the three-step differentiation and the culture conditions described here good support differentiation of ESC into HSC.HSC derived from ESC were safe without teratomas formation in body, and its can reconstruct hematopoiesis.

Objective To explore whether human umbilical venous endothelial cells could be used as feeder layer to support the growth of embryonic stem cells (ESC) and keep ESC undifferentiated.Methods The venous vessels of umbilical cord obtained from healthy puerperal were perfused with collagenase.The isolated endothelial cells went through primary culture and passages for expansion.Factor Ⅷ antigens determination was implemented.Endothelial cells with good growth and 3 or above passages were treated with mitomycin-C(10 mg/L) and prepared as feeder layer,on which E14.1 ESC was transplanted for subculturing to observe the morphological characterization and determine ESC alkaline psphatase (AKP) activity and the expression of stem cell marker Oct-4.Severe combined immune deficiency(SCID) mouse in vivo terotoma formation experiment was performed to identify its pluripotent properties.Results Human umbilical vein-derived endothelial cells grew well in culturing in vitro and regenerate in large numbers.The endothelial cells maintained normal cellular morphological and biological characterization after 10 passages.The cells stopped proliferating after being treated with mitomycin-C,but its activity and morphological properties were well-maintained with 24 hours,which was a fundamental property of serving as feeder layer.E14.1 ESC remained undifferentiated in human umbilical venous endothelial cells after 3-8 passages,the cells grew in colony and showed high expression of AKP and stem cell Oct-4.In vivo pluripotency experiment showed that 6 weeks after being transplanted to SCID mice E14.1 ESC of 6 and 10 passages in endothelial cells both could form teratoma containing 3 layers of tissue cells.Conclusions Human umbilical venous endothelial cell serve as a convenient feeder layer cell with rich sources.It can effectively support ESC growth and heterogenous and prevent the heterogeneous protein pollution and pathogenic microorganisms caused by animal cell feeder layers,thus solve the problem of biological safety of ESC clinical application.

Adult ; Burkitt Lymphoma ; metabolism ; pathology ; CD3 Complex ; metabolism ; DNA Nucleotidylexotransferase ; metabolism ; Diagnosis, Differential ; Female ; Granulosa Cell Tumor ; metabolism ; pathology ; Humans ; Ki-67 Antigen ; metabolism ; Leukocyte Common Antigens ; metabolism ; Ovarian Neoplasms ; metabolism ; pathology ; surgery ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; metabolism ; pathology ; surgery ; Sarcoma, Myeloid ; metabolism ; pathology

Objective To explore the causes of dengue fever resurgence in Guangxi, and to analyze the risk factors of dengue fever. Methods The descriptive epidemiological analysis was conduced based on the dengue fever data reported from 2006 to 2015, and the surveillance results of aedes and antibody levels in health population from 2013 to 2015 in Guangxi. Results Before 2013, dengue fever was imported from foreign country in Guangxi, accounting for 95.35％(42/45), and 75.71％of the imported cases was imported from Southeast Asia. The local outbreak of dengue fever was happened in 2014, accounted for 94.02％(849/903) of the total number of 10 years. From onset to diagnosis, Guangxi dengue fever cases need 0-70 d (median time interval is 6 d). Cases were reported year-round, but the peak season for the onset of dengue fever was from September to November, accounting for 96.46％of all cases (871/903). The number of cases reported in Nanning was the most (83.37％), followed by Wuzhou city (7.44％) and Guilin city (4.81％), and all the three cities had dengue fever outbreaks. The cases were mainly commercial service staff (27.80％) and houseworkers and unemployed people (18.16％). Results of aedes monitoring showed that the density of aedes was high in Guangxi. In more than 50％ of the monitoring seasons the breteau index (BI) stayed greater than 20. However, the antibody positive rate was only 3％ in the healthy residents of Guangxi. Conclusion The risk of dengue fever is high in Guangxi. Therefore, it is essential to emphasizing idea of prevention and control, strengthening immigration surveillance, improving diagnosis ability, enhancing public health education, and expanding monitoring range.

OBJECTIVETo study the effect of melatonin (MLT) in in vitro apoptosis of hepatocarcinoma cells and its mechanism.

METHODSThe apoptotic cells, bcl-2 and bax were detected through immunocytochemical method (ICC) and Tolt-mediated x-duTP nick end labeling (TUNEL). Computer image analysis system was used to quantify the expression of bcl-2 and bax by detecting the absorbance value of positive products. Apoptosis index (AI) was used to quantify the number of apoptotic cells.

RESULTSIn vitro, AI increase was both concentration- and time-dependent through TUNEL. During the same duration, AI of medium dose group was higher than that of low dose and control group (P < 0.05); AI of high dose, medium dose and 5-Fu group were higher than those of low dose and control group (P < 0.01), however, there was no significant difference between the low dose and control group (P > 0.05). At the same dose, in high dose, medium dose and 5-Fu group, the change of AI showed significant difference from 24 to 36 hours (P < 0.05). The expression of bcl-2 was down-regulated as the MLT increased, and there was significant difference between the low dose and control group (P < 0.01). But, the expression of bax was up-regulated as the dose of MLT increased, showing significant difference between the high dose and control groups (P < 0.01). As time went on, the expression of bcl-2 was decreased and in every group, with the change in absorbance value of bcl-2 significantly different from 24 to 36 hours (P < 0.05), whereas that of bax remained almost unchanged. The ratio of bax/bcl-2 was increased with the increase in the concentration of MLT.

CONCLUSIONMelatonin may induce apoptosis in the hepatocarcinoma cells which is concentration- and time-dependent, in which bcl-2 and bax are involved.

Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; drug therapy ; pathology ; Cell Line, Tumor ; Dose-Response Relationship, Drug ; Humans ; Liver Neoplasms ; drug therapy ; pathology ; Melatonin ; pharmacology ; Proto-Oncogene Proteins ; analysis ; Proto-Oncogene Proteins c-bcl-2 ; analysis ; Time Factors ; bcl-2-Associated X Protein

On-line analysis and control are critical for the optimization of product yields in animal cell culture. The close monitor of viable cell number helps to gain a better insight into the metabolism and to refine culture strategy. In this study, we use the oxygen uptake rate (OUR) to estimate the number of viable cell and the OUR-based feed-back control strategy for nutrients feeding to improve the efficiency of cell culture. A hybridoma cell line (HAb18) was cultured in fed-batch and perfusion model using serum free medium in 5L CelliGen Plus bioreactor (NBS Co., American) and 5L Biostat B bioreactor (Braun Co., Germany). The system and the method for online monitoring OUR in bioreactors, based on the dynamic measurement of dissolved oxygen (DO), were developed. The method of on-line cell concentration estimation was established based on the relationship between the growth of the hybridoma and the uptake rate of oxygen. This method was then used to determine OUR and the concentrations of cell, antibody, glucose, lactate, glutamine and ammonia in the bioreactors at given times. The relationship between OUR and nutrients metabolism was studied and OUR-based feed-back control strategy, which used the state deltaOUR = 0 as the regulation point, was established and used to control the rates of nutrients or medium feeding rate in the perfusion culture. The results showed that there was close relationship between OUR, concentration of live cells, productivity of antibody and consumption of glutamine. The sudden decrease in OUR may be caused by glutamine depletion, and with different delay times, the viable cell concentration and antibody productivity also decreased. The further analysis revealed the linear relationship between OUR and the density of live cells in the exponential growth phase as qOUR = (0.103 +/- 0.028) x 10(-12) mol/cell/h. These findings can be applied to the on-line detection of live cell density. Our study also indicated that by adjusting the perfusion rate with OUR-based feed-back control strategy, it is feasible to continuously increase in viable cell density and antibody concentration in the perfusion culture.
Bioreactors ; Cell Culture Techniques ; methods ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Hybridomas ; metabolism ; Oxygen ; metabolism

Cyclin-Dependent Kinase 4 ; genetics ; Exons ; genetics ; Humans ; Magnetic Resonance Imaging ; Mutation ; genetics ; Polymerase Chain Reaction ; Polymorphism, Genetic ; genetics ; Pulmonary Artery ; metabolism ; pathology ; Sarcoma ; genetics

Hybridoma cells were cultured by continuous perfusion in Fibra-Cel of 5L packed-bed bioreactor for 22 days in low serum or serum-free media. The corresponded amino acids were fed and serum concentration was decreased by analyzing glucose concentration, oxygen uptake rate, secretary antibody amount and amino acids concentration in culture supernatant. Comparing with continuous perfusion culture that amino acids were not fed, antibody amount of production was increased about 2-3 times. The inoculated cell density was 2.5 x 10(5) cells/mL, while the final cell density was 8.79 x 10(8) cells/mL. Antibody production was reached 295 mg/L/d at average level, and the highest level was reached 532 mg/L/d. These results provided a primary mode of enlarge culture for monoclonal antibody industralization.
Animals ; Antibodies, Monoclonal ; biosynthesis ; Cell Division ; Cells, Cultured ; Glucose ; pharmacology ; Hybridomas ; metabolism ; Mice ; Mice, Inbred BALB C ; Oxygen Consumption ; Perfusion

OBJECTIVETo investigate the mechanisms of apoptosis induced in human hepatoma cell line SMMC7721 by plasmid pVHN constructed with Newcastle disease virus (NDV) HN gene.

METHODSTwenty-four h after transfection with liposome-plasmid pVHN complexes in vitro, the mortality rate of SMMC7721 cells was determined by MTT staining and flow cytometry (FCM) with PI staining. The alteration of mitochondrial trans-membrane potential of the cells was detected by FCM with rhodamine 123 staining. Cell genomic DNA was detected by agarose electrophoresis. The activation of caspase-3 was assayed by its substrate color reaction.

RESULTSSignificant apoptosis was induced by transfection with plasmid pVHN into the cells for 24 h and the mortality rate was 50.0% (the mortality rate of control group was 5.2%). Genomic DNA was fragmented and mitochondrial trans-membrane potential was decreased, but caspase-3 activity increased.

CONCLUSIONSignificant apoptosis in SMMC7721 cells can be induced by NDV HN gene. Apoptosis may be resulted from the decrease of mitochondrial trans-membrane potential and activation of Caspase-3.

Apoptosis ; physiology ; Cancer Vaccines ; immunology ; Carcinoma, Hepatocellular ; pathology ; Caspase 3 ; metabolism ; Cell Line, Tumor ; HN Protein ; genetics ; Humans ; Liver Neoplasms ; pathology ; Newcastle disease virus ; genetics ; Transfection ; Vaccines, DNA ; immunology

OBJECTIVETo study the different features of hyperplasia in castrated and uncastrated mice after testosterone (T) treatment.

METHODSForty-eight BALB/c mice were randomly divided into 6 groups of 8 in each: castrated (A), uncastrated (B) , castrated + low T (C), uncastrated + low T (D), castrated + high T (E), uncastrated + high T (F). Groups C and D were treated with testosterone solution at the dose of 12.5 mg/(kg d) and Groups E and F at 125 mg/(kg d) for 20 consecutive days, while Groups A and B received saline only. All the mice were sacrificed on the 21st day, their ventral and dorsal prostate glands weighed and their pathological features studied.

RESULTSAtrophic prostates were observed in Group A, but normal in Group B; prostatic hyperplasia was found in both Group C and D, but more obvious in the latter (P <0.05); and a slightly higher degree of hyperplasia was noted in Groups E and F than in C and D. There was an increase in serum T and vascular endothelial growth factor (VEGF) concentration and a decrease in serum estrogen (E2) concentration in the testosterone treated groups.

CONCLUSIONBoth castrated and uncastrated mice develop prostate hyperplasia after short-term testosterone treatment, although in different degrees and with different features, which may help further the studies on the association of castration and androgen with prostate diseases.

Animals ; Hyperplasia ; Male ; Mice ; Mice, Inbred BALB C ; Orchiectomy ; Prostate ; pathology ; Prostatic Hyperplasia ; drug therapy ; pathology ; Testosterone ; therapeutic use