Objective: To investigate the role of human umbilical vein endothelial cells (HUVEC) in supporting hematopoiesis and the expansion of hematopoietic stem/progenitor cells in vitro. Methods: According to the fact that HUVEC supernatant has colony stimulating activity shown by methylcellulose colony-forming assay and HUVEC can maintain the survival of mononuclear cells for at least four weeks in vitro, CD34+ cells from umbilical cord blood were seeded with (HUVEC group) or without (control group) HUVEC monolayer. Every week cells were collected and counted, the frequency of CFU-GM was measured by using methylcellulose colony-forming assay, and the percentage of CD34+ and CD41a+ cells was measured by flow cytometry. Results: In control group，all the CD34+ cells died in two weeks. However, in HUVEC group,most nucleated cells and CD34+ cells were expanded by 68.1±14.8 fold and 6.6±1.4 fold，respectively at the third week while CFU-GM expansion reached its peak (5.7±2.1 fold) at the week 2. Moreover, the percentage of CD41a+ cells was enhanced significantly, reaching a maximum (15.6%) at the week 3. Conclusions:HUVEC can support hematopoiesis in vitro and expand the hematopoietic progenitor cells and CD41a+ cells in direct contact coculture.

Ten isoflavonoids were isolated from the heartwoods of Caragana changduensis Lion f. by means of various column chromatographic techniques. Based on the detailed spectral data analysis (MS and NMR), as well as comparison with the literatures, their chemical structures were determined as 7,2'-dihydroxy-8,4'-dimethoxyisoflavone (1), 4'-hydroxy-7,3'-dimethoxyisoflavone (2), 5, 7, 4'-trihydroxy-2',5'-dimethoxyisoflavone (3), prunetin (4), afrormosin (5), odoratin (6), genistein (7), texasin (8), pratensein (9), and 6,7,3'-trihydroxy-4'-methoxyisoflavone (10). Among them, compounds 1-3 and 9-10 were isolated from the Caragana genus for the first time. All the compounds were obtained from this species for the first time. In the preliminary assays, compounds 1, 2, 6, and 7 possessed significant inhibitory effects on NO production, with IC50 values of 48.12, 25.32, 62.71, 43.59 μmol x L(-1), respectively.
Animals ; Caragana ; chemistry ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; pharmacology ; Isoflavones ; chemistry ; isolation & purification ; pharmacology ; Macrophages ; drug effects ; metabolism ; Mice ; Molecular Structure ; Nitric Oxide ; antagonists & inhibitors ; metabolism ; RAW 264.7 Cells

Pharmaceutical preparations, particularly as a "secret recipe" of traditional Chinese medicine in medical institutions, are the product of China's medical and health industry, and they are also an important means of competing of different medical institutions. Although pharmaceutical preparations have advantages and characteristics than institutes for drug and pharmaceutical companies, the quality standards of pharmaceutical preparations in medical institutions has not reached the desired level over the years. As we all know, the quality of pharmaceutical preparations is important to ensure the efficacy, especially under the environment of people pay more sttention on drug safety and effectiveness and contry increase emphasis on the stste of pharmaceutical preparations. In view of this, we will improve the grade, stability, and clinical efficacy of pharmaceutical preparations by the advanced equipment, testing instruments and the process dynamic quality control technology. Finally, we hope we can provide new ideas for the quality control of pharmaceutical preparations.
Drug Compounding ; standards ; Medicine, Chinese Traditional ; standards ; Quality Control

ObjectiveTo study the effect of fluorine on proliferation of osteoblast through extra cellular signal-regulated protein kinase(ERK) signaling pathway.MethodsMouse osteoblasts(MC3T3-E1) were cultured in vitro with different concentrations of fluoride for 24 and 48 h (the concentrations of Fˉ were 0,200,400,600,1000,2000,4000,8000,10 000 μmol/L,respectively).The optimum concentration for promotion of cell proliferation was determined by methylthiophene tetrazolium(MTT) assay.According to the optimum concentration,the cells were randomly divided into three groups:control group (0 μmol/L Fˉ); fluorine group (400 μmol/L Fˉ); fluorine and MAPK inhibitor PD98059 group(400 μ mol/L Fˉ + 10 μ mmol/L PD98059).Cell cycle was detected by flow cytometry after 48 h culture.The expression of P-ERK protein was determined by Western blotting and immunofluorescence.ResultsThe optimum concentration of fluorine for proliferation of osteoblasts was 400 μ mol/L.Compared with the control group[(76.12 ± 10.08)％,(2.06 ± 0.31)％],the number of cells in G0/G1 phase[(63.04 ± 8.12)％] reduced and the number of cells in S phase[(9.13 ± 2.08)％] increased in fluorine group (all P ＜ 0.05) ; but the number of cells in G0/G1 phase [(92.11 ± 9.01 ) ％] in fluorine and mitogen-activated protein kinases (MAPK) inhibitor PD98059 group was significantly increased(P ＜ 0.05 ).Western blotting results showed that:compared with the control group[(100.00 ± 0.00)％],the expression of P-ERK protein in fluorine group[(131.24 ± 13.88)％] was significantly higher(P ＜ 0.05 ),but the expression of P-ERK protein in fluorine and MAPK inhibitor PD98059 group [(91.33 ± 9.68 )％] was not significantly changed(P ＞ 0.05).The results of immunofluorescence were similar to that of Western blotting.ConclusionsFluorine at the concentration of 400 μmol/L can promote the proliferation of osteoblasts.ERK signaling pathway has played a key role in the proliferation of osteoblasts.

To investigate the possible involvement of Ras signaling in the hematopoietic differentiation of embryonic stem cells (ES cells), ES cells were transfected with RasN17, the dominant-negative mutant of Ras. Western blot was used to test the effect of RasN17 expression on Erk1/2 and Akt phosphorylation, semi-quantitative RT-PCR was used to detect expression of gene related to hematopoiesis in differentiation of ES cells. The results showed that the expression of RasN17 in the ES cells remarkably downregulated the phosphorylation of Erk1/2 and Akt simultaneously. Moreover, the expression of several markers related with hematopoiesis including Runx1, SCL and beta-major globin, were significantly suppressed in the EB expressing RasN17, whereas the transcription of Flk1, a gene required earlier than SCL in development of hematopoietic and endothelial lineages, was not influenced. It is concluded that the activation of Ras is pivotal for in vitro hematopoietic differentiation of ES cells.
Animals ; Cell Differentiation ; physiology ; Cells, Cultured ; Embryonic Stem Cells ; cytology ; Genes, ras ; Hematopoiesis ; physiology ; Hematopoietic Stem Cells ; cytology ; Mice ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Phosphorylation ; Signal Transduction ; ras Proteins ; physiology

OBJECTIVE:To provide reference for promoting the rational use of essential medicines in primary medical institutions.METHODS:Twenty six rural primary medical institutions (13 township health centers,13 village clinics) were randomly selected from 2 poverty-stricken county (city) in Huanggang city of Hubei province.The utilization of essential medicine was investigated and analyzed statistically through exporting hospital information system data and on-site interviews.RESULTS:The utilization rate of essential medicine in sample township health centers was 76.9％,and the amount of essential medicine accounted for 93.0％;the utilization rate of essential medicine in sample village clinics was 89.7％.The utilization rate of variety from essential medicine list was 53.6％ in sample township health centers and 25.0％ in sample village clinics;the utilization rate of variety from National Essential Medicine List was higher than that of Provincial Essential Medicine List Supplement.The amount of 5 major categories as antimicrobial agents,cardiovascular agent in sample primary medical institutions accounted for 64.7％ of chemical agents.The top ten medicines in the list of amount were all essential medicine,9 of which were national essential medicines.There were 2.8 kinds of medicines in each outpatient prescription averagely in sample township health centers.The proportion of antibiotic prescription was 44.2％.CONCLUSIONS:The utilization rate of essential medicines in township health centers of this area is lower than WHO recommended value,and the ratio of amount meets the policy requirements.The utilization rate of variety from Provincial Essential Medicine List Supplement is in low level in primary medical institutions;the amount of anti-microbial drugs is in high level;the rationality of medicine use in prescriptions needs to be improved.It is suggested to adjust the type of provincial supplement list dynamically according to actual situation and control the price and amount of main categories strictly,the rationality of prescription.

Objective:To explore the expression pattern of pyroptosis-related genes(PRGs)in hepatocellular carcinoma(HCC),and analyze the relationship between its expression and tumor prog-nosis and immune microenvironment.Methods:TCGA database was used to analyze the genetic changes and expression patterns of PRGs in primary HCC cells,and cluster analysis was used to i-dentify the pyrogenic subtypes of HCC.To compare the difference of prognosis and immune mi-croenvironment among HCC pyrodeath subtypes.Scorch death score quantified the comprehensive expression of PRGs in each sample,and analyzed the correlation between scorch death score and each immune score.Results:Two pyroptosis-associated subtypes of primary HCC were identi-fied,and the expression pattern of PRGs is closely related to the prognosis of cancer patients and the tumor microenvironment.The subtype with high expression of PRGs had a poor prognosis,and functional enrichment analysis found that some tumor-promoting pathways and PD-1 checkpoint pathways were significantly enriched in this subtype.And various cells and immune checkpoints re-lated to immunosuppression were also enriched in this subtype.By constructing PYROPTO-SIS_score to quantify the comprehensive expression of pyroptosis-related genes in each sample,it was found that PYROPTOSIS_score was significantly positively correlated with tumor-infiltrating macrophages,myeloid-derived suppressor cells,and Treg cells.Conclusion:These results sug-gest that pyroptosis may play a tumor-promoting as well as immunosuppressive role in HCC,pro-viding new insights into the assessment of tumor patient prognosis and the immune microenviron-ment.

Objective To investigate the effect of sevoflurane on mRNA expression of N -methyl -D -aspartate ( NMDA ) receptor subunit in neonatal rat hippocampi.Methods Twenty-four SD rats were devided into 2 groups( n=12 each) via a random number table:sevoflurane group and control group.The 3.0% sevoflurane and oxy-gen inhalated for 6 h respectively.Rats were sacrificed at 24 h after inhalation of sevoflurane or oxygen.Hippocampi were removed to idendtify the mRNA expression of NMDA subunit and the ratio of NR2 B/NR2 A through in situ hybridization.Results Compared with control group, NR2BmRNA and the ratio of NR2B/NR2A increased, and NR2 AmRNA decreased significantly in sevoflurane group ( P<0.05 ).Conclusion Neurotoxicity of sevoflurane in immature hippocampi might be attributed to regulation of mRNA expression of NMDA subunit in neonatal rat.

In the present study, an attempt was made to prove the question whether endothelial cell precursors exist in blood circulation during postnatal period. CD34(+) cells were harvested from G-CSF mobilized adult blood and umbilical cord blood and incubated onto fibronectin/gelatin-coated Petric dishes in the presence of recombinant human vascular endothelial cell growth factor(rhVEGF) and basic fibroblast growth factor(rhbFGF). Endothelial cell lineage was identified by von Willebrand factor(vWF) expression and Ulex europous agglutinin I(UEA-I) binding capacity. The results showed that a firmly adherent cell monolayer formed when CD34(+) cells, but not CD34(-) cells, were cultured for 5 - 6 weeks as described before. Immunocytochemistry and flow cytometry analysis showed that almost all of the adherent cells were vWF-positive and around 90% were able to bind UEA-I specifically. These findings demonstrate that angioblasts exist in the circulation during postnatal life and therefore, vasculogenesis might occur in adults.

This study was aimed to investigate whether mesenchymal stem cells (MSCs) existed in human umbilical cord vein and to establish the methods of isolation and expansion in vitro. The MSCs derived for perfusion of umbilical cord vein (UVMSCs) were collected after parturition of Healthy pregnant women. The morphology of MSCs and their differentiation potential into osteoblast, adipocyte and chondrocyte were observed the phenotype and cell cycle of MSCs were determined by using flow cytometry. The result showed that the mesenchymal stem cells separated from umbilical cord vein gave rise to a population of adherent cells with a typical fibroblast-like morphology. Similarly to bone marrow-derived MSCs, they highly expressed CD29, HLA-ABC, CD166, CD105, CD73 and CD44, and were negative for any hematopoietic and endothelial markers (CD45, CD34, CD14 and CD144). Functionally, they could differentiate into the osteoblast, adipocyte and chondrocyte. It is concluded that MSCs exist in the human umbilical cord vein perfusion. Their read amplification in vitro contribute to clinical applications for cell therapy and tissue engineering.
Cell Differentiation ; Cell Separation ; methods ; Cells, Cultured ; Female ; Fetal Blood ; cytology ; Humans ; Mesenchymal Stromal Cells ; cytology ; Umbilical Veins ; cytology