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.

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.

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.

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

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.