Objective: To study the comprehensive utilization of Herba Ephedrae resources during the extraction process of ephedrin, that is the extracting of porphyrin compound.Methods: The process conditions for preparing sodium copper porphyrin were optimized by the two preparation processes and twice orthogonal tests. Results: Herba Ephedrae was powdered, steeped and boiled in alcohol, saponified, then extracted with xylene. The ephedrin was extracted from the upper layer of xylene extraction solution and sodium copper porphyrin was obtained from the lower layer by copper substituton, acidulation and alkalization. Conclusion: The optimum preparation process for producing sodium copper porphrin of Herba Ephedrae which is superior to porphyrin in the stability and colour is ascertained.

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.

Aim To estimate the affinity between C085 and Hsp90 and the inhibitory effects of C085 on the activity of Hsp90 ATPase. Methods The fluores-cence spectrum experiment was applied to examine the affinity between different C085 concentrations and Hsp90 , NHsp90 , MHsp90 , CHsp90; fluorescence in-tensities were recorded in the range of 290-510 nm at 293 K, 303 K and 310 K, respectively;a colorimetric assay for inorganic phosphate based on the formation of a phosphomolybdate complex and subsequent reaction with malachite green was used to examine the inhibitory effects of C085 on the activity of Hsp90 ATPase. Re-sults The dissociation constant KD value of C085 was (11. 163 ± 0. 316 ) μmol · L-1 . The interaction be-tween C085 and Hsp90 was driven mainly by electro-static interaction. C085 showed strongest affinity with CHsp90. When the concentration of ATP was 1 mmol· L-1 ,the inhibition of Hsp90 ATPase activity of C085 with the IC50 value was 6. 04μmol·L-1 . Conclusions The interaction mechanism between C085 and Hsp90 can be analyzed by fluorescence spectrum. C085 shows strong inhibition ATPase activity of Hsp90 .

Objective: To obtain ephedrine with porphyrin copper sodium from Herba Ephedrae. Methods: Application of ethanol to obtain ephedrine and porphyrin copper sodium from ephedra. The spoinfy reaction of porphyrin will take place, the integral ephedrine will turn into free ephedrine (dissolved in ether), when the liquor of ethanol of ethanol of ephedra are heated with NaOH. Using dimethylponyle to extract, the liquor will into two phases, one's (including sponify porphyrin) is water phase, the other's (including free ephedrine) is ether phase. Results: The yield proportion of ephedrine is 0.72%, and porphyrin copper sodium is 1.8%.Conclusion: It is practicable to extract porphyrin copper sodium with ephedrine from Herba Ephedrae.

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.

The aims of the present study were to estimate the affinity between 3,5-()-bis(3-methoxy-4-hydroxybenzal)-4-piperidinone hydrochloride (C0818) and heat shock protein 90 (Hsp90) and to investigate the inhibitory effects of this compound on Hsp90 ATPase activity. Fluorescence spectroscopy was used to examine the affinity between varying concentrations of C0818 and Hsp90, N-Hsp90, M-Hsp90 and C-Hsp90. Fluorescence intensities were recorded in the range of 290-510 nm at 293, 303 and 310 K, respectively. A colorimetric assay for inorganic phosphate (based on the formation of a phosphomolybdate complex and the subsequent reaction with malachite green) were used to examine the inhibitory effects of C0818 on Hsp90 ATPase activity. The equilibrium dissociation constantvalue of C0818 was found to be 23.412±0.943 μmol/L. The interaction between C0818 and Hsp90 was driven mainly by electrostatic interactions. C0818 showed the strongest affinity with C-Hsp90. These results conclusively demonstrate the inhibitory activity of C0818 on the activity of Hsp90 ATPase.