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BACKGROUND:At present, a lot of research about culture methods for umbilical cord mesenchymal stem cels, but not for the waste of primary system. OBJECTIVE:To explore the best culture method of human umbilical cord mesenchymal stem celsin vitro. METHODS:Human umbilical cord mesenchymal stem cels were prepared by tissue explants method, recorded as initial culture group. The centrifugal fluid and tissue of the primary culture flask were centrifuged and divided into three groups for secondary culture: tissue group, mixed group and pure liquid group. Cel morphology, time for cel acquisition, and yield of primary cels in the four groups were observed; the cel growth curve was analyzed by MTT assay; and cel cycle and phenotype were detected by flow cytometry. RESULTS AND CONCLUSION: The average time for cel acquisition in the initial culture group, tissue group, mixed group and pure liquid group were (15.00±0.45), (7.0±0.3), (8.00±0.25) and (8.00±0.25) days, respectively. The number of cels at first generation was (4.0±0.5)×105, (9.0±0.55)×105, (15.0±0.2)×105 and (7.0±0.33)×105 markers of the four groups had no significant differences. The human umbilical cord mesenchymal stem cels can be obtained rapidly and largely through the secondary culture to the primary culture system. T75 culture bottle, respectively. Under the inverted microscope, cels in the four groups were fusiform-like adherent cels, which were in paralel or circinate arrangement. Growth curve, proliferative activity, surface markers of the four groups had no significant differences. The human umbilical cord mesenchymal stem cells can be obtained rapidly and largely through the secondary culture to the primary culture system.
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Objective To explore the role of apelin-13 in regulating stem cell differentiation into vascular net. Meth?ods Mesenchymal stem cells were isolated from human umbilical Wharton’s jelly using tissue adherence method.Their immunophenotypes were detected by flow cytometry . Passage 3 of WJ-MSCs (Wharton’s jelly-mesenchymal stem cells) were inoculated in 4 flasks, denoted as A1, A2, A3, A4 group. TwentyμL of apelin-13 at concentrations of 0, 1×10-6, 10 × 10-6 and 100 ×10-6 mol/L were added to A1, A2, A3 and A4 respectively each day. After being induced for 7 days, cell mor?phology and viability were observed under inverted microscope. Von Willebrand factor (vWF) was examined by immunofluo?rescence and CD31 was identified by flow cytometry. Upon incubating with three dimensional culture medium of hydrogel, those cultured A1, A2, A3 and A4 were renumbered as S1, S2, S3, S4. Again, twentyμL of apelin-13 at concentrations of 0, 1×10-6, 10 ×10-6and 100 ×10-6 mol/L were used to treat S1, S2, S3 and S4 respectively. After 7 days, cell morphology, via?bility and vas-like networks were observed with inverted microscope. Results Our study showed that WJ-MSCs can be in?duced by apelin 13 to differentiate into endothelial cells lineage indicated by positive of vWF staining. Moreover, CD31 expres?sion increases significantly upon apelin-13 addition in a dosage dependent manner. The endothelial cells line formed vas like networks when cultured with three-dimensional medium containing hydrogel. Conclusion This study demonstrated that ape?lin-13 could promote human umbilical cord-MSCs to differentiate into endothelium lineage then to form vascular networks.
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Objective To construct a recombinant plasmid pUbi-apelin-FLAG-pSV40-EGFP and package with lentivirus to co-express enhanced green fluorescent protein (EGFP) and apelin, and to investigate optimal multiple of infec-tion (MOI) to transfect human umbilical cord mesenchymal stem cells (hUCMSCs) and expression of target gene. Methods The apelin gene was chemically synthesized and amplified by polymerase chain reaction (PCR), and which was inserted into linear plasmid vector. The gene fragment and linear plasmid vector were connected by In-Fusion technology after enzyme di-gestion and transformed into competent DH5αcells. The positive clones of lentiviral expression vector were obtained after screening and followed by sequencing. The lentiviral vector was used to transfect 293T cells and package virus, and then the virus titers were determined. HUCMSCs were transfected with lentivirus vector in vitro via different values of MOI. The trans-fection efficiency was obtained according to the optimal MOI. The expression of target gene was detected by RT-PCR and Western blot assay. Results A 284 bp target gene fragment with the restriction sites was obtained by PCR and connected to the lentiviral vector. The positive clones of lentiviral expression vector were corresponded to the expected result. The lentivi-ral particles were successfully packaged. HUCMSCs could be transfected by the lentivirus vector with high efficiency. The mRNA and protein levels of target gene were stably up-regulated within 2 weeks. Conclusion The lentivirus vector pUbi-apelin-FLAG-pSV40-EGFP can transfect apelin gene into hUCMSCs with high efficiency. The infected cells can express high levels of apelin gene in two weeks.
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Objective To investigate the optimal condition of lentivirus,which was recombined with marker gene of enhanced green fluorescent protein (Lentivirus-EGFP) transfect human umbilical cord wharton’s jelly-derived mesenchy-mal stem cells (HUWMSCs) and the effect of transfection on the proliferation in HUWMSCs. Methods HUWMSCs were transfected with EGFP by lentivirus vector in vitro via different multiplicity of transfection (MOI) in four different transfec-tion methods (A, B, C and D). The fluorescence expression and the transfection efficiency in different methods were analyzed by both fluorescent microscope and flow cytometry. The proliferation rates of infected HUWMSCs was evaluated by MTT method. Results The transfection efficiency was 10.6%-87.3%after 4 days in all experimental groups, which showed the dose-effect relationship with MOI. Polybrene (5 mg/L) could significantly increase the transfection efficiency (P＜0.05). Re-sults of MTT assay showed that there were significant differences in the proliferation rates of infected HUWMSCs between different transfection methods (P ＜ 0.001). There was better cell proliferation in method A (MOI=10) group and method B (MOI=10) group than that of other groups. Conclusion Method B (MOI=10) is the optimal transfection method in this exper-iment. HUWMSCs could be transfected by lentivirus-EGFP with high efficiency and could stably express transfected gene within 2 weeks, which is a safe and effective gene transfer vector.
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BACKGROUND: Radiofrequency catheter ablation (RFCA) in dog triggers myocardial nerve sprouting and sympathetic hyperinnevation. It is possible that RFCA in humans has the same effect. Nerve growth factor (NGF) is a neurons nurture that supports the survival and differentiation of sympathetic neurons and enhances target innervation. Therefore, it is hypothetic that RFCA can increases plasma NGF concentration in humans.OBJECTIVE: To test the hypothesis that RFCA increases plasma NGF concentration in humans.DESIGN: Self-control experiment.SETTING: Department of Cardiology, Tangdu Hospital, the Fourth Military Medical University of Chinese PLA.PARTICIPANTS: Forty-three patients were selected from the Cardiological Department of Tangdu Hospital, the Fourth Military Medical University of Chinese PLA from January to June 2005, including atrioventricular nodal reentrant tachycardia (AVNRT) (n=18), right-sided accessory pathways (RSAP) (n=13) and left-sided accessory pathways (LSAP) (n=12), 20 males and 23 females, ages 28-65 years, all agreed to participate in the study voluntarily.METHODS: Blood samples were obtained from the peripheral veins before ablation and at 6 hours, 1, 3, 5, 7 days after ablation. The plasma concentration of NGF was determined with enzyme-linked immunosorbent assay (ELISA).MAIN OUTCOME MEASURES: The plasma concentration of NGF was determined with ELISA before RFCA and at 6 hours, 1, 3, 5, 7 days after RFCA in each patient.RESULTS: Total 43 patients who were referred for ablation therapy for AVNRT, RSAP and LSAP were involved in the result analysis without loss. Plasma NGF increased at 6 hours after RFCA. Increased NGF continued to 7 days in the RFCA treated patients. The plasma NGF concentrations at 6 hours, 1, 3, 5, 7 days after RFCA in AVNRT, RSAP and LSAP ablations treated patients were (29.72±7.04), (30.94±5.68),(31.39 ±4.92), (31.06 ±4.56), (29.11 ±4.59), (31.77 ±6.25), (30.69 ±5.10),(31.46±4.96), (30.15±4.01), (30.43±3.14), (31.42±6.75), (31.00±5.20),(32.08±4.62), (30.67±3.71), (29.27±2.75) μg/L, respectively, and all more than that before RFCA [(14.89±2.84), (15.00±2.71), (15.51±2.75) μg/L, P ＜ 0.01]. However, there were no significant differences in the NGF levels at 6 hours, 1, 3, 5 and 7 days after RFCA (P ＞ 0.05). The plasma NGF concentration was not significant different among AVNRT, RSAP and LSAP ablation patients at any given time (P ＞ 0.05). The number of RFCA applications, the procedure time and the total energy have no correlation with NGF concentration at any given time instance.CONCLUSION: RFCA increases plasma NGF concentration in humans and lasts for at least 7 days. The number of RFCA applications, the procedure time and the total energy have no correlation with NGF concentration at any given time instance.
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Objectives:To examine the effect of chloride blocker (NPPB and tamoxifen)on volume sensitive chloride channels in mouse cardiac ventricular myocytes. Methods:Isolated mouse cardiac ventricular myocytes were subjected to whole cell patch clamp to record the hypotonicity activated chloride currents. Results:When the myocytes were exposed to hypotonic solution, an obvious whole cell currents were activated. The currents were inhibited by extracellular NPPB reversibly and significantly. The specific blocker for volume sensitive chloride channel , tamoxifen (50 ?mol/L), could apparently block the activity of this channel in a voltage dependent manner. Conclusions:Mouse cardiac ventricular myocytes process volume sensitive chloride channel which is sensitive to NPPB and tamoxifen.
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Objective To investigate the possibility, effect and safety of intracoronary autologous moronuclear bone marrow cell (MBMC) transplantation in patients with ischemia heart failure (IHF). Methods 41 patients with IHF were enrolled in this prospective nonrandomized study. 14 patients were transplanted with autologous MBMC via a balloon catheter placed into the infarct-related artery during balloon dilatation by highpressure infusion, which was performed 6-8 times for 2 minutes each. 13 patients were transplanted via selective the infarct-related arteries by highpressure infusion. Results There were no major periprocedural complications. Two patients had limited premature ventricular contractions during cell infusion forseveral seconds. Two patients felt cold after 15-30 minutes infusing cell and got better several minutes later. There were no new onset of arrhythmias found on 48 h ECG monitoring. After 3 months of follow up, the symptoms and cardiac function were significantly improved in the transplantation group. FDG-PET analysis revealed a significant increase in myocardial metabolism (23.94?7.28)% (P=0.015). Plasma BNP lever decreased significatly at 3 days and 7 days after transplantation than before transplantation (P
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Objective To investigate the possibility、 efficacy、 safety of the repair of infarcted myocardium and improvement of cardiac function by intracoronary autologous mononuclear bone marrow cell (MBMC) transplantation in patients with AMI. Methods Thirty-eight patients with AMI were enrolled in this prospective study and divided into PCI+cell transplantation group and PCI only group. Baseline and follow up evaluations included complete clinical and laboratory evaluations, 2D echocardiogram, positron emission tomography (PET), 48 ECG monitoring. MBMC were harvested, isolated, washed, and resuspended in saline for infusion. 21 patients were transplanted with autologous MBMC via a balloon catheter placed into the infarct-related artery during balloon dilatation by highpressure infusion 14.6 days after AMI, which was performed 6-8 times for 2 minutes each and 7 patients were transplanted via non-selective infarct-related arteries by highpressure infusion. Results Surgery was safe. 3 patients had limited premature ventricular contractions during cell infusion for several seconds. 3 patients felt cold after 15-30 minutes infusing cell. After 3 months of fellow-up, there were viable myocardium in the infarct defected regions in 13 cases after MBMCs transplantation determined by PET, which occupied [(40.08?8.82%), P
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Objective To discuss the influence of several physico-chemical factors existed in the surrounding environment on the endothelium function of pilots,submarine sailors and ground crew,so as to provide a basis for preventing the cardiovascular disease.Methods 48 submarine sailors(SM group),22 pilots(PI group)and 17 ground crew(control group),all of them were healthy males,were chosen as the subjects.The dynamic features of 3H-L-Arg in the platelets transportation were checked with isotope labeling technique.The yielded nitrite(NO-2),a product of NO metabolism,and the activity of NO synthase(NOS)in platelets were detected.Results In SM group,the ability of L-Arg transportation in platelets was much lower than that of both control group and PI group.The maximum transport velocity(Vmax)of SM group was lower by 31.4% than that of control group(P
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AIM: To elucidate the cellular source of adrenomedullin (AM) in myocardial tissue, the effects of 5 substances on secretion of AM from cultured rat fibroblasts and myocytes were examined in this study. METHODS: Myocardial fibroblast and myocyte isolation and culture; AM was measured by radioimmunoassay; AM receptors were assayed by radioactive analysis method. RESULTS:Tumor necrosis factor ? (TNF?), lipopolysaccharide (LPS) and basic fibroblast growth factor (bFGF) all could stimulate AM synthesis and secretion in myocardial Fb and myocyte , and the amount of AM secreted by Fb was greater than that by myocytes, whereas transforming growth factor-?(TGF?),and interferon-?(IFN?) suppressed in it in myocardial Fb and myocytes. Myocardial Fb has two kinds of AM binding sites, but myocyte has only one high affinity- binding site.CONCLUSIOIN:Myocardial fibroblasts could synthesize and secrete AM, and there are AM receptors in both myocardial fibroblasts and myocytes.