Animals ; Cell Culture Techniques ; Cell Differentiation ; Cells, Cultured ; Embryonic Stem Cells ; cytology ; Female ; Hematopoiesis ; Hematopoietic Stem Cells ; cytology ; Male ; Mice

Objective To evaluate effects of trichostain A (TSA) on cell proliferation, cell cycles, apoptosis and invasiveness of multiple myeloma cell line U266; as well as active changes of methylation regulating proteins including DNA methyl-transferase(DNMTs), methyl-binding domain (MBD) proteins: MBD2 and MeCP2 after treated with TSA. Methods U266 cells were treated with different concentrations of TSA for 12, 24, 48 and 60 h. The proliferation activity of U266 cells was detected by MTT and the IC50 of 24 h was calculated. After U266 cells were treated with IC50, cell cycles were check out by dying with PI. mRNA of matrix metalloproteinase-2(MMP-2), bc1-2, bcl-xl and methylation regulating proteins (DNMTs, MBD2 and MeCP2) were detected by real-time PCR. FCM and Western blotting were used to measure expressions of MMP-2 and MBD2. Results MTT results revealed TSA inhibited proliferation of U266 cells in a dose-and time-dependent manner and the IC50 of 24 h was 0.07 μmol/L FCM analysis showed that TSA could arrest the cell cycle in G0/G1 and the proliferation index (PI) in U266 cells [(49.90 0.39)%]were significantly different after exposed to TSA (0.7 μmnol/L for 24h compared with that in the control cells[(55.78 0.49)%](P <0.01). After treated by TSA, the 2-△△Ct of MMP-2, bcl-2 and bcl-xl were 0.71 0.06, 5.04 0.92 and 2.95 0.35, respectively. There were great changes on mRNA of DNMT, MBD2 and MeCP2. TSA could reverse the transcription of DNMT, MBD2 and MeCP2. Conclusion TSA can arrest the U266 cell cycle in GVG, to prevent its proliferation and promote apoptosis, which maybe greatly connect with the changes of the methylation regulating proteins.

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 the effect of treatment of children's stress hyperglycemia with glucose-insulin-potassium.Methods Thirty children with stress hyperglycemia were randomly divided into two groups,15 cases in each group.Patients in the treated group were admi-nistered with glucose-insulin-potassium,whereas those in the control group,glucose were transfused at the speed

Objective To induce mice bone marrow(BM) mono-nuclear cells into dendritic cells(DC) in vitro,and then produce function vaccine of anti-myeloma by bone marrow derived dendritic cells(BM-DC).Methods Mono-nuclear cells separated from mice bone marrow were cultured in tissue culture plastics which supplemented granulocyte-macrophage clonystimulating factor(GM-CSF),IL-4.During the 8 days,there were numerous of mature DCs outgrown,and then fused with SP 2/0 myeloma cells in logarithmic growth phase cultured with 8-azaguaine by polyethylene(PEG).The resulting purified DC/myeloma hybrids were generated by selecting with hypoxanthine-aminopterin-thymidine(HAT).The specific anti-tumor activity was examined in vitro.Results Mono-nuclear cells derived from BM cultured by supplement propriate cytokines milieu in tissue culture plastics,and then exposed to lipopolysaccharide(LPS).There were plenty of mature DCs appear as early as day 8,and exhibit dramatic veils of cytoplasm and extensive dendrites in their surfaces,high express CD11c(83.19%),major histocompatibility complex(MHC)-Ⅱ(95.25%),CD86(89.24%),their capacity to stimulate primary T cell responses in mixed leukocyte reaction(MLR) would be shown strongly.Hybrids could grow well through PEG fusion and HAT selection.These hybrids could stimulate naive T cells into cytotoxic T-lymphocyte(CTLs) directed against SP 2/0 myeloma,and exhibit strong specific killed ability.Conclusions BM-DCs is fused with tumor cells ensure the tumor associated antigens could be processed and presented effectively,and stimulate specific anti-tumor immunity so that prevent or cure this kind of tumor.So it's feasible in practice to produce anti-tumor vaccine by BM-DCs.

Although elevated prolactin levels have been shown to inhibit penile erection, the relationship between prolactin and erection of the penile tip or base has not been extensively researched. We therefore investigated the prolactin's effects on erection of the penile tip and base, with a cross-sectional study of 135 patients with erectile dysfunction, based on scores of ≤21 on the International Index of Erectile Function-5. All patients were tested for nocturnal penile tumescence, blood pressure, serum glucose, total cholesterol, triglyceride, high-density lipoprotein, low-density lipoprotein, luteinizing hormone, follicle-stimulating hormone, prolactin, estradiol, testosterone, and progesterone. Univariate and multivariate analyses were used to assess the associations between prolactin levels and erection at the penile tip and base. We found no obvious relationship between erection time at penile tip and prolactin levels, but observed a negative correlation between base erection time and prolactin level (hazard ratio: -2.68; 95% confidence interval [CI]: -5.13 - 0.22). With increasing prolactin concentration, multivariate analysis showed obvious reduction in base erection time among patients with normal Rigiscan results (hazard ratio: -3.10; 95% CI: -7.96-1.77; P < 0.05). Our data indicate that prolactin inhibits penile erection, particularly at the penile base. In addition, when the effective erection time of the penile base lasts longer than 10 min, prolactin has a more obvious inhibitory effect on penile base erection.

ObjectiveTo explore the effect of earlier rehabilitation on activities of daily living(ADL) of patients with spinal cord injury(SCI).Methods50 SCI patients received earlier rehabilitation and improvement of ADL of patients was evaluated.ResultsAfter two months treatment,the scores of Barthel index,functional independence measure(FIM) grade of patients increased significantly compared with that of before treatment(P<0.05) and ADL improved.ConclusionEarlier rehabilitation can improve ADL of SCI patients.

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.