OBJECTIVETo investigate the effect of prostaglandins E2 (PGE2) in enhancing vascular endothelial growth factor (VEGF) expression in a rat macrophage cell line and the effect of the media from PGE2-inuced rat macrophages on angiogenetic ability of human umbilical vein endothelial cells (HUVECs) in vitro.

METHODSWestern blotting and qPCR were employed to investigate the expressions of VEGF protein and mRNAs in rat macrophage cell line NR8383 stimulated by PGE2 in the presence or absence of EP2 receptor inhibitor (AH6809) and EP4 receptor inhibitor (AH23848). Conditioned supernatants were obtained from different NR8383 subsets to stimulate HUVECs, and the tube formation ability and migration of the HUVECs were assessed with Transwell assay.

RESULTSPGE2 stimulation significantly enhanced the expression of VEGF protein and mRNAs in NR8383 cells in a dose-dependent manner. The supernatants from NR8383 cells stimulated by PGE2 significantly enhanced tube formation ability of HUVECs (P<0.05) and promoted the cell migration. Such effects of PGE2 were blocked by the application of AH6809 and AH23848.

CONCLUSIONPGE2 can dose-dependently increase VEGF expression in NR8383 cells, and the supernatants derived from PGE2-stimulated NR8383 cells can induce HUVEC migration and accelerate the growth of tube like structures. PGE2 are essential to corpus luteum formation by stimulating macrophages to induce angiogenesis through EP2/EP4.

Animals ; Cell Line ; Cell Movement ; Cells, Cultured ; Culture Media, Conditioned ; pharmacology ; Dinoprostone ; pharmacology ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; Humans ; Macrophages ; chemistry ; Neovascularization, Pathologic ; RNA, Messenger ; Rats ; Receptors, Prostaglandin E, EP2 Subtype ; metabolism ; Receptors, Prostaglandin E, EP4 Subtype ; metabolism ; Vascular Endothelial Growth Factor A ; Xanthones ; pharmacology

OBJECTIVETo investigate the effects of culture supernatant of human amnion mesenchymal stem cells (hAMSCs-CS) on biological characteristics of human fibroblasts.

METHODS(1) hAMSCs were isolated from deprecated human fresh amnion tissue of placenta and then sub-cultured. The morphology of hAMSCs on culture day 3 and hAMSCs of the third passage were observed with inverted phase contrast microscope. (2) Two batches of hAMSCs of the third passage were obtained, then the expression of vimentin of cells was observed with immunofluorescence method, and the expression of cell surface marker CD90, CD73, CD105, and CD45 was detected by flow cytometer. (3) hAMSCs-CS of the third passage at culture hour 72 were collected, and the content of insulin-like growth factor Ⅰ (IGF-Ⅰ), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF) were detected by enzyme-linked immunosorbent assay. (4) Human fibroblasts were isolated from deprecated human fresh prepuce tissue of circumcision and then sub-cultured. Human fibroblasts of the third passage were used in the following experiments. Cells were divided into blank control group and 10%, 30%, 50%, and 70% hAMSCs-CS groups according to the random number table (the same grouping method below), with 48 wells in each group. Cells in blank control group were cultured with DMEM/F12 medium containing 2% fetal bovine serum (FBS), while cells in the latter 4 groups were cultured with DMEM/F12 medium containing corresponding volume fraction of hAMSCs-CS and 2% FBS. The proliferation activity of cells was detected by cell counting kit 8 and microplate reader at culture hour 12, 24, 48, and 72, respectively, and corresponding volume fraction of hAMSCs-CS which causing the best proliferation activity of human fibroblasts was used in the following experiments. (5) Human fibroblasts were divided into blank control group and 50% hAMSCs-CS group and treated as in (4), with 4 wells in each group, at post scratch hour (PSH) 0 (immediately after scratch), 12, 24, 48, and 72, the migration distance of cells was observed and measured with inverted phase contrast microscope. (6) Human fibroblasts were grouped and treated as in (5), with 3 battles in each group, and apoptosis rate of cells was detected by flow cytometer. Data were processed with analysis of variance of factorial design, analysis of variance for repeated measurement, one-way analysis of variance, LSD test, and t test.

RESULTS(1) On culture day 3, most hAMSCs were in large form, and spindle-shaped with much prominences like fibroblasts or in flat polygonal shape. hAMSCs of the third passage were spindle-shaped. The expression of vimentin of hAMSCs of the third passage was strongly positive, and the expressions of surface markers CD90, CD73, and CD105 of the cells were positive, while the expression of CD45 of the cells was negative. (2) The content of IGF-Ⅰ, VEGF, EGF, and bFGF in hAMSCs-CS were respectively (11.7±1.0), (316±68), (6.1±0.4), and (1.49±0.05) pg/mL. (3) At culture hour 12-72, the proliferation activity of human fibroblasts in each hAMSCs-CS group was significantly higher than that in blank control group (with P values below 0.01), and the proliferation activity of human fibroblasts in 50% hAMSCs-CS group was the highest. (4) The width of scratch in two groups was nearly the same at PSH 0. The migration distance of cells in 50% hAMSCs-CS group was significantly longer than that in blank control group at PSH 12-72 (with P values below 0.01). (5) The apoptosis rate of human fibroblasts in blank control group was (16.2±2.4)%, which was significantly higher than that in 50% hAMSCs-CS group [(7.4±3.6)%, t=6.710, P<0.01].

CONCLUSIONShAMSCs-CS can promote proliferation and migration of human fibroblasts and inhibit the apoptosis of human fibroblasts.

Amnion ; cytology ; Apoptosis ; Cell Movement ; Cell Proliferation ; Cells, Cultured ; Culture Media, Conditioned ; chemistry ; Enzyme-Linked Immunosorbent Assay ; Epidermal Growth Factor ; metabolism ; Female ; Fibroblast Growth Factor 2 ; metabolism ; Fibroblasts ; cytology ; drug effects ; Flow Cytometry ; Humans ; Insulin-Like Growth Factor I ; metabolism ; Male ; Mesenchymal Stromal Cells ; chemistry ; Pregnancy ; Vascular Endothelial Growth Factor A ; metabolism

Infection of schistosomiasis japonica may eventually lead to liver fibrosis, and no effective antifibrotic therapies are available but liver transplantation. Hedgehog (HH) signaling pathway has been involved in the process and is a promising target for treating liver fibrosis. This study aimed to explore the effects of pentoxifylline (PTX) on liver fibrosis induced by schistosoma japonicum infection by inhibiting the HH signaling pathway. Phorbol12-myristate13-acetate (PMA) was used to induce human acute mononuclear leukemia cells THP-1 to differentiate into macrophages. The THP-1-derived macrophages were stimulated by soluble egg antigen (SEA), and the culture supernatants were collected for detection of activation of macrophages. Cell Counting Kit-8 (CCK-8) was used to detect the cytotoxicity of the culture supernatant and PTX on the LX-2 cells. The LX-2 cells were administered with activated culture supernatant from macrophages and(or) PTX to detect the transforming growth factor-β gene expression. The mRNA expression of shh and gli-1, key parts in HH signaling pathway, was detected. The mRNA expression of shh and gli-1 was increased in LX-2 cells treated with activated macrophages-derived culture supernatant, suggesting HH signaling pathway may play a key role in the activation process of hepatic stellate cells (HSCs). The expression of these genes decreased in LX-2 cells co-cultured with both activated macrophages-derived culture supernatant and PTX, indicating PTX could suppress the activation process of HSCs. In conclusion, these data provide evidence that PTX prevents liver fibrogenesis in vitro by the suppression of HH signaling pathway.
Animals ; Antigens, Helminth ; isolation & purification ; pharmacology ; Cell Culture Techniques ; Cell Differentiation ; drug effects ; Cell Line ; Culture Media, Conditioned ; chemistry ; pharmacology ; Gene Expression Regulation ; Hedgehog Proteins ; agonists ; antagonists & inhibitors ; genetics ; immunology ; Hepatic Stellate Cells ; cytology ; drug effects ; metabolism ; Humans ; Liver Cirrhosis ; metabolism ; parasitology ; prevention & control ; Macrophage Activation ; drug effects ; Macrophages ; cytology ; drug effects ; immunology ; Models, Biological ; Monocytes ; cytology ; drug effects ; metabolism ; Pentoxifylline ; pharmacology ; Phosphodiesterase Inhibitors ; pharmacology ; RNA, Messenger ; genetics ; immunology ; Schistosoma japonicum ; chemistry ; Signal Transduction ; Tetradecanoylphorbol Acetate ; pharmacology ; Zinc Finger Protein GLI1 ; genetics ; immunology ; Zygote ; chemistry

To explore whether hypoxic condition could promote the olfactory mucosa mesenchymal stem cells (OM-MSCs) to differentiate into neurons with the olfactory ensheathing cells (OECs) supernatant and the potential mechanisms.
 Methods: The OM-MSCs and OECs were isolated and cultured, and they were identified by flow cytometry and immunofluorescence. The OM-MSCs were divided into three groups: a 3%O2+ HIF-1α inhibitors (lificiguat: YC-1) + OECs supernatant group (Group A) , a 3%O2 + OECs supernatant group (Group B) and a 21%O2 + OECs supernatant group (Control group). The neurons, which were differentiated from OM-MSCs, were assessed by immunofluorescence test. The mRNA and protein expression of hypoxia-inducible factor-1α (HIF-1α), βIII-tubulin and glial fibrillary acidic portein (GFAP) were detected by quantitative polymerase chain reaction (Q-PCR) and Western blot. The potassium channels were analyzed by patch clamp.
 Results: The neurons differentiated from OM-MSCs expressed the most amount of βIII-tubulin, and the result of Q-PCR showed that HIF-1α expression in the Group B was significantly higher than that in the other groups (all P<0.05). Western blot result showed that the βIII-tubulin protein expression was significantly higher and GFAP protein expression was obviously decreased in the Group B (both P<0.05). The patch clamp test confirmed that the potassium channels in the neurons were activated.
 Conclusion: Hypoxic condition can significantly increase the neuronal differentiation of OM-MSCs by the OECs supernatant and decrease the production of neuroglia cells, which is associated with the activation of HIF-1 signal pathway.
Blotting, Western ; Cell Differentiation ; physiology ; Cells, Cultured ; Culture Media, Conditioned ; chemistry ; pharmacology ; Flow Cytometry ; Glial Fibrillary Acidic Protein ; metabolism ; Hypoxia ; physiopathology ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Indazoles ; pharmacology ; Mesenchymal Stem Cells ; physiology ; Neurogenesis ; physiology ; Neuroglia ; metabolism ; physiology ; Neurons ; physiology ; Olfactory Mucosa ; Potassium Channels ; Signal Transduction ; Tubulin ; metabolism

OBJECTIVETo test the effect of bifunctional molecule IL2-GMCSF in promoting the activation of dendritic cells (DCs) cultured in tumor conditioned medium.

METHODSWe prepared a tumor conditioned medium using mouse melanoma cell line B16F10 supplemented with IL2-GMCSF, GM-CSF, IL-2, or the combination of the latter two. After culturing mouse DC cell line DC2.4 in the conditioned medium for 24 h, the DCs were examined for phagocytosis, proliferation, maturation phenotype, cytokine secretion, and signal pathway activation.

RESULTSDC2.4 cells displayed characteristics of immature DCs. After cell culture in the conditioned medium, the cells showed enhanced phagocytosis but significantly suppressed cell proliferation activity. Culture in the conditioned medium also promoted DC cell maturation and secretion of macrophage-derived chemokine (MDC), but inhibited IL-12 secretion. Supplementation of the conditioned medium with IL2-GMCSF promoted phagocytosis, proliferation, maturation, and cytokine (including both IL-12 and MDC) secretion of DC2.4 cells. Compared with GM-CSF, IL2-GMCSF induced a higher level of NF-κB signal pathway activation but suppressed STAT3 activation.

CONCLUSIONCompared with GM-CSF, IL2-GMCSF can better promote DC activation in the context of tumor-induced immune suppression, and thus shows potentials in anti-tumor therapy.

Animals ; Cell Differentiation ; Cell Line, Tumor ; drug effects ; Cell Proliferation ; Chemokine CCL22 ; metabolism ; Culture Media, Conditioned ; chemistry ; Dendritic Cells ; cytology ; drug effects ; Gene Expression Regulation, Neoplastic ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Immune Tolerance ; Interleukin-12 ; metabolism ; Interleukin-2 ; pharmacology ; Melanoma, Experimental ; pathology ; Mice ; NF-kappa B ; metabolism ; Phagocytosis ; STAT3 Transcription Factor ; metabolism ; Signal Transduction

In this study, we aimed to investigate the influences of conditioned medium from human umbilical vein endothelial cells (HUVEC) on cancer stem cell phenotype of human hepatoma cells. HUVEC and human hepatoma cells (MHCC97H) were cultured, respectively, and then the MHCC97H cells were co-cultured with conditioned medium from HUVEC (EC-CM) with Transwell system. Anti-cancer drug sensitivity, colony-formation, migration/invasion ability, expression of cancer stem cell marker and sphere formation were performed to determine the cancer stem cell phenotype in MHCC97H cells. We found that MHCC97H cells co-cultured with EC-CM exhibited significantly higher colony-formation ability and lower sensitivity of anti-cancer drugs 5-FU and Cis. Transwell assay showed that treatment with EC-CM obviously increased migration and invasion of MHCC97H cells. Moreover, increased sphere forming capability and expression of CD133 in MHCC97H cells were observed after co-cultured with EC-CM. These results suggested that EC-CM could promote cancer stem cell phenotype of hepatoma cells.
Antineoplastic Agents ; pharmacology ; Carcinoma, Hepatocellular ; Cell Line, Tumor ; Coculture Techniques ; Culture Media, Conditioned ; Fluorouracil ; pharmacology ; Human Umbilical Vein Endothelial Cells ; chemistry ; Humans ; Liver Neoplasms ; Neoplastic Stem Cells ; cytology ; Phenotype

Flavonoids are a class of important active ingredients in traditional Chinese medicine, pharmacological activity and in vivo process is the focus of research in recent years. Calycosin is the main active ingredients of flavonoids in Astragali Radix, recent studies indicate that it has many kinds of pharmacological activity, but the absorption and transport characteristics in vivo is unclear. The experiment using Caco-2 cell model, with apigenin as internal standard substance, using the method for the determination of drug concentration by HPLC, were studied at different concentrations and absorption transport characteristics of respectively adding different types of protein inhibitors. Data were analyzed by Q test, the results show that low, middle, high concentration of P(app)(BL-AP)/ P(app)(AP-BL) = 1.38 < 1.5, respectively adding different types of protein inhibitors, compared with the control group of P(app)(BL-AP)/ P(app)(AP-BL), there were no significant differences. Calycosin absorption may mainly passive transport, also involved in active transport mechanism, the transport may not be affected by the P-protein, MRP2 protein, SGLT protein.
Absorption ; Biological Transport ; Caco-2 Cells ; Chromatography, High Pressure Liquid ; Culture Media, Conditioned ; chemistry ; Drug Stability ; Drugs, Chinese Herbal ; analysis ; pharmacokinetics ; Humans ; Hydrogen-Ion Concentration ; Isoflavones ; analysis ; pharmacokinetics ; Models, Biological

The paper aimed to study the residue decline dynamic and standards for safety utilization of carbendazim in roots, stems, leaves of Anoectochilus roxburghii and in growth media. Samples extracted with methanol were purified by liquid-liquid extraction and analysed by HPLC. The results showed that average rate of recovery was 82.9% - 95.7% and RSD were 2.0% - 6.3% with add of carbendazim in respectively diverse concentration, which meets inspection requirement of pesticide residue. Two kinds of dosages of carbendazim were treated, varying from recommended dosage (1.0 kg x hm(-2)) to 1.5 times recommended dosage (1.5 kg x hm(-2)). Results of two years test showed that the half-life period of carbendazim were 7.01 - 8.51 d in the growth media of A. roxburghii, 3.58 - 4.27 d in stems and 3.50 - 3.91 d in leaves, 4.93 - 5.71 d in roots. Providing max recommended residue of carbendazim in the cultivation of A. roxburghii is 0.5 mg x kg(-1), sprayed 4 times a year with the dosage of 1.0 kg x hm(-2), 28 days is proposed for the safety interval of the last pesticide application's and harvest's date.
Benzimidazoles ; metabolism ; pharmacology ; Carbamates ; metabolism ; pharmacology ; Chromatography, High Pressure Liquid ; Culture Media, Conditioned ; chemistry ; Dose-Response Relationship, Drug ; Fungicides, Industrial ; metabolism ; pharmacology ; Liquid-Liquid Extraction ; Orchidaceae ; drug effects ; metabolism ; Pesticide Residues ; analysis ; metabolism ; Plant Leaves ; drug effects ; metabolism ; Plant Roots ; drug effects ; metabolism ; Plant Stems ; drug effects ; metabolism

OBJECTIVETo study, at the cytological level, the basic concept of Chinese medicine that "the Kidney (Shen) controls the bone".

METHODSKaempferol was isolated form Rhizoma Drynariae (Gu Sui Bu, GSB) and at several concentrations was incubated with opossum kidney (OK) cells, osteoblasts (MC3T3 E1) and human fibroblasts (HF) at cell concentrations of 2×10(4)/mL. Opossum kidney cell-conditioned culture media with kaempferol at 70 nmol/L (70kaeOKM) and without kaempferol (0OKM) were used to stimulate MC3T3 E1 and HF proliferation. The bone morphological protein receptors I and II (BMPR I and II) in OK cells were identified by immune-fluorescence staining and Western blot analysis.

RESULTSKaempferol was found to increase OK cell growth (P<0.05), but alone did not promote MC3T3 E1 or HF cell proliferation. However, although OKM by itself increased MC3T3 E1 growth by 198% (P<0.01), the 70kaeOKM further increased the growth of these cells by an additional 127% (P<0.01). It indicates that the kidney cell generates a previously unknown osteoblast growth factor (OGF) and kaempferol increases kidney cell secretion of OGF. Neither of these media had any significant effect on HF growth. Kaempferol also was found to increase the level of the BMPR II in OK cells.

CONCLUSIONSThis lends strong support to the original idea that the Kidney has a significant influence over bone-formation, as suggested by some long-standing Chinese medical beliefs, kaempferol may also serve to stimulate kidney repair and indirectly stimulate bone formation.

3T3 Cells ; Animals ; Cell Line ; Culture Media, Conditioned ; Intercellular Signaling Peptides and Proteins ; secretion ; Kaempferols ; pharmacology ; Kidney Tubules ; physiology ; secretion ; Medicine, Chinese Traditional ; Mice ; Opossums ; Osteoblasts ; chemistry

Alzheimer's disease (AD) is the most common cause of age-related dementia. The neuropathological hallmarks of AD include extracellular deposition of amyloid-beta peptides and neurofibrillary tangles that lead to intracellular hyperphosphorylated tau in the brain. Soluble amyloid-beta oligomers are the primary pathogenic factor leading to cognitive impairment in AD. Neural stem cells (NSCs) are able to self-renew and give rise to multiple neural cell lineages in both developing and adult central nervous systems. To explore the relationship between AD-related pathology and the behaviors of NSCs that enable neuroregeneration, a number of studies have used animal and in vitro models to investigate the role of amyloid-beta on NSCs derived from various brain regions at different developmental stages. However, the Abeta effects on NSCs remain poorly understood because of conflicting results. To investigate the effects of amyloid-beta oligomers on human NSCs, we established amyloid precursor protein Swedish mutant-expressing cells and identified cell-derived amyloid-beta oligomers in the culture media. Human NSCs were isolated from an aborted fetal telencephalon at 13 weeks of gestation and expanded in culture as neurospheres. Human NSCs exposure to cell-derived amyloid-beta oligomers decreased dividing potential resulting from senescence through telomere attrition, impaired neurogenesis and promoted gliogenesis, and attenuated mobility. These amyloid-beta oligomers modulated the proliferation, differentiation and migration patterns of human NSCs via a glycogen synthase kinase-3beta-mediated signaling pathway. These findings contribute to the development of human NSC-based therapy for AD by elucidating the effects of Abeta oligomers on human NSCs.
Amyloid beta-Peptides/*pharmacology ; Animals ; Apoptosis ; Cell Aging ; Cell Movement ; Cell Proliferation ; Culture Media, Conditioned/chemistry/pharmacology ; Fetus/cytology ; Glycogen Synthase Kinase 3/*metabolism ; HEK293 Cells ; Humans ; Mice ; Mice, Inbred C57BL ; Neural Stem Cells/*drug effects/metabolism/physiology ; Signal Transduction ; Telomere Shortening