AIM:To explore a novel method to isolate human nucleus pulposus mesenchymal stem cells (hNP-MSCs) in vitro and to identify their biological characteristics.METHODS:The explant culture method was employed to isolate hNP-MSCs from nucleus pulposus tissue obtained by percutaneous endoscopic lumbar discectomy (PELD).The isolated cells were passaged for purification and cultured in vitro followed by morphological observation.The cell proliferation ability was detected by CCK-8 assay.Growth curves of the cells were drawn and surface antigens were detected by flow cytometry.The cells at the 3rd~6th passages were induced for adipogenic, osteogenic and chondrogenic differentiation, and examined by oil red O staining, alizarin red staining and Alcian blue staining.RESULTS:The cells with self-renewal were obtained from nucleus pulposus tissue obtained by PELD.The results of flow cytometry analysis revealed that the cells were positive for CD29, CD44, CD90, CD73 and CD105, but negative for CD34 and CD45.The proliferative capacity was consistent with the growth characteristics of MSCs and multilineage differentiation potential was identified.CONCLUSION:A novel method to efficiently isolate and culture hNP-MSCs,PELD combined with explant culture method,was established, which would promote the study of regenerative medicine based on hNP-MSCs.

Antineoplastic Agents, Phytogenic ; therapeutic use ; Colorectal Neoplasms ; drug therapy ; pathology ; Ginsenosides ; therapeutic use ; Humans ; Lymph Nodes ; pathology ; Lymphatic Metastasis ; prevention & control

GJ-4 is crocin enrichments extracted from Gardenia jasminoides J. Ellis, and our previous studies have shown that GJ-4 significantly improved learning and memory impairment induced by Aβ in mice. Herein, a memory deficit model was developed by injecting okadaic acid (OA) into the lateral ventricle of mice, and the neuroprotection and underlying mechanism of GJ-4 on neuronal injury caused by Tau hyperphosphorylation were investigated. The Animal Care & Welfare Committee, Institute of Materia Medica, CAMS & PUMC has approved all procedures (No.00000318). GJ-4 at different doses was intragastric administration to mice for 16 days. Step-down test and Morris water maze test showed that GJ-4 could significantly improve OA-induced memory impairment in mice, and reduced the loss of Nissl bodies in the hippocampus of mice. GJ-4 could also decrease the phosphorylation level of Tau protein at Ser396, Thr231 and Ser404 via increasing protein phosphatase 2A (PP2A) activity and inhibiting glycogen synthase kinase-3β (GSK-3β) activity. Besides, further researches indicated that GJ-4 could inhibit the level of oxidative stress in the brain of OA mice, reduce neuronal apoptosis and inhibit the neuroinflammation mediated by activation of astrocytes in the hippocampus of mice, and eventually achieve its effects in improving learning and memory impairment in mice. According to these findings, we anticipated that GJ-4 might be a potential therapeutic drug for Alzheimer's disease.

BACKGROUND: The immature dendritic cell (imDC) can induce immunological tolerance and has widely application in the field of organ transplant. At present, the methods of inducing imDC are insufficient, so the new induction method is demanding.OBJECTIVE: To investigate the effect of sodium butyrate (SB) on the maturation and immunological function of human peripheral blood-derived imDC.DESIGN: Controlled observation and in vitro cytological trial.SETTING: Department of Hepatobiliary Surgery in the Second Affiliated Hospital of Sun Yat-sen University.MATERIALS: Five samples of human peripheral blood were obtained from the healthy volunteers (aged 20-23 years) of Sun Yat-sen University, totally 500 mL. Then peripheral blood mononuclear cells (PBMCs) and lymphocytes were isolated within 2 hours.METHODS: The experiment was carried out in the Medical Research Center of the Second Hospital Affiliated to Sun (1 mmol/L) was added for induction, while those supplemented with maturation promoting factor lipopolysaccharide (LPS)the beginning of induction, while LPS was added on the sixth day for second stimulation.MAIN OUTCOME MEASURES: Cell morphological change, flow cytometry was used to detect DC phenotype,FITC-labeled Dextran was used to detect the endocytosis of DC, the production of IL-12 was determined by means of enzyme-linked immunosorbent assay, and the proliferation of lymphocyte induced by DC was assayed with mixed lymphocyte reaction.expressions of CD80, CD83 and HLA-DR were significantly lower in the imDC of routine induction group following SB maturity promoting, compared with LPS group (P＜0.01). On the sixth day, LPS was added into the SB-induced imDC,Endocytosis of DC: The imDC of routine induction group possessed a significantly lower endocytic activity after induced by LPS, and there were extremely significant differences compared with blank control group and SB maturation Production of IL-12: The production of IL-12 in the mDC induced by LPS was significantly higher than that in control group, SB maturation promoting group and SB induction group, the mDC induced by LPS in routine induction group stimulated significantly stronger proliferation of lymphocyte (P＜0.01).

AIM: To investigate the immunological function of sodium butyrate-induced immature dendritic cells in vitro.METHODS: The human monocyte-derived dendritic cells were induced in the presence of human granulocyte macrophage-colony stimulating factor(GM-CSF) and interleukin-4 (IL-4), combined with sodium butyrate. The immunological function of sodium butyrate-induced dendritic cells was detected by the FCM, endocytic activity, T cells stimulatory proliferation capacity, and interleukin-12 (IL-12) production.RESULTS: Sodium butyrate could down-regulate the major histocompatibility complex(MHC) class II and costimulatory molecules of dendritic cells, increase the endocytic activity, induce a stage of T-cell anergey, and inhibit the T helper cell type 1-skewing factor IL-12 production. CONCLUSION: Sodium butyrate inhibits the maturation of dendritic cells and induces production of immature dendritic cells, which may help to explore the machenism of its epigenitic modification.

Adult ; Aged ; Cell Cycle Proteins ; analysis ; Coal Mining ; Cyclin-Dependent Kinase Inhibitor p27 ; Female ; Humans ; Immunohistochemistry ; Lung ; chemistry ; pathology ; Male ; Middle Aged ; Occupational Diseases ; metabolism ; Pneumoconiosis ; metabolism ; Tumor Suppressor Proteins ; analysis

BACKGROUNDIn vitro fertilization (IVF) researches have suggested that cystathionine beta synthase (CBS) is involved in oocyte development. However, little is known about the regional and cellular expression patterns of CBS in the ovary. The purpose of this study was to analyze the localization of CBS in mice ovaries and to investigate the expression profile during follicular development.

METHODSWe used in situ hybridization and immunohistochemical analysis to determine CBS expression in the ovaries of female Balb/c mice. Then the follicles were collected from F1 (C57BL x Balb/c) mice and cultured in vitro. With the method of semi-quantitative RT-PCR, we also investigated the expression profile of CBS during follicular development.

RESULTSCBS was absent in the oocytes, although it was ubiquitously expressed in the ovary with the strongest expression in follicular cells at all stages. In late antral follicles, CBS expression was markedly higher in granulosa cells located close to the antrum and in cumulus cells around the oocyte. The semi-quantitative RT-PCR showed that CBS mRNA was detected in follicles at all stages in vitro. In cumulus-oocyte complexes superovulated, CBS expression also increased rapidly.

CONCLUSIONSCBS was located mainly in the follicular cells in the ovaries. The level of CBS expression is high in follicles during folliculogenesis in mice. Differences in the CBS expression profile between oocyte and follicular cells suggest a role for CBS as a mediator in interactions between oocyte and granulosa cells.

Animals ; Cystathionine beta-Synthase ; analysis ; genetics ; Female ; Gene Expression Profiling ; Immunohistochemistry ; In Situ Hybridization ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Ovarian Follicle ; physiology ; Ovary ; enzymology ; RNA, Messenger ; analysis

AIMTo observe protective effects and involved mechanism of ginsenoside Rb3 on hypoxic/ischemic brain injury, using cultured hippocampal neurons, rat hippocampal slices and intact animals.

METHODS(1) Mice were tightly closed in glasses of 150 ml, and then survival time of them was observed. (2) During simulated ischemia and after reoxygenation, changes of orthodromic population spikes (OPS) in the area CA1 of hippocampal slice were investigated. (3) By using histochemistry, the expressions of NOS in CA1 area of rat hippocampus after hypoxic exposure were observed. (4) Using LDH detection, tests of total NOS, iNOS and cNOS activity, the protective effects of ginsenoside Rb3 were investigated on cultured hippocampal neurons treated with hypoxia.

RESULTS(1) Given ginsenoside Rb3 (10 mmol/L), mice survived significantly longer than that of control group. (2) The occurrence of HIP (hypoxic injury potentials) decreased after administration of ginsenosides Rb3 (60 micromol/L) in many slices, while the recovery rate and amplitude of OPS after reoxygenation were significantly higher than those of the control group. (3) In CA1 area of rat hippocampus, NOS-positive neurons increased at the end of 24 h hypoxia and further 24 h reoxygenation, while the number of NOS-positive neurons decreased after treatment with ginsenoside Rb3. (4) The LDH leakage rate of cultured rat hippocampal neurons increased at the end of hypoxia, while it decreased after treatment with Rb3. Moreover the total NOS, especially iNOS activity of these neurons also decreased.

CONCLUSIONGinsenoside Rb3 has a significant protective effect on hypoxic-ischemic injury of neurons, and this involves the stabilization of the cell membrane, the inhibition of the expression and activity of NOS, especially iNOS activity.

Animals ; Cell Survival ; drug effects ; Female ; Ginsenosides ; pharmacology ; Hypoxia-Ischemia, Brain ; metabolism ; Lactate Dehydrogenases ; metabolism ; Male ; Membrane Potentials ; Mice ; Mice, Inbred ICR ; Nitric Oxide Synthase Type II ; metabolism

OBJECTIVETo study the effects of two specific cyclooxygenase inhibitors (SCI), rofecoxib and celecoxib, combined with chemotherapeutic drugs 5-Fu, DDP and VP-16 on gastric cancer cell line BGC-823, and to evaluate whether specific cyclooxygenase inhibitors can be used as a synergetic agent in chemotherapy.

METHODSThe gastric cancer cell line BGC-823 cells were incubated for 48 hours with rofecoxib and celecoxib, 5-Fu, DDP and VP-16 (concentration gradient of 5-Fu, DDP and VP-16:1 microg/ml, 10 microg/ml and 100 microg/ml), or in combination, respectively. MTT working solution was added to each culture and calculated the survival rates of gastric cancer cells. Median-effect principle and Professor Jin's evaluation methods were applied to detect the interaction between the specific cyclooxygenase inhibitors and chemotherapeutic agents.

RESULTSThe inhibition rates of gastric cancer cells were 42.63% +/- 1.26% and 50.67% +/- 2.35% by treatment with 0.1 micromol/L rofecoxib and 50 micromol/L celecoxib, respectively. The inhibition rates of gastric cancer cells by treatment with 5-Fu, DDP and VP-16 at different concentrations (1 microg/ml, 10 microg/ml and 100 microg/ml) were 39.75% +/- 3.14%, 49.96% +/- 2.08%, 87.93% +/- 3.66%; 48.28% +/- 2.08%, 59.46% +/- 1.69%, 88.23% +/- 4.81%; and 29.23% +/- 3.27%, 49.34% +/- 3.75%, 79.24% +/- 2.44%, respectively. However, the inhibition rates showed a synergetic role while combined the two SCI (0.1 micromol/L rofecoxib and 50 micromol/L celecoxib) with chemotherapeutic agent at different concentrations (P <0.05).

CONCLUSIONBoth rofecoxib and celecoxib have an ability to suppress gastric cancer cells in vitro, and the synergetic role becomes evident when rofecoxib and celecoxib are combined with chemotherapeutic agents at different concentrations, which indicate that the two specific cyclooxygenase inhibitors may be used as a chemotherapeutic sensitizer.

Adenocarcinoma ; pathology ; Antimetabolites, Antineoplastic ; pharmacology ; Antineoplastic Agents ; pharmacology ; Celecoxib ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Cisplatin ; pharmacology ; Cyclooxygenase 2 Inhibitors ; pharmacology ; Cyclooxygenase Inhibitors ; pharmacology ; Dose-Response Relationship, Drug ; Drug Synergism ; Etoposide ; pharmacology ; Fluorouracil ; pharmacology ; Humans ; Lactones ; pharmacology ; Pyrazoles ; pharmacology ; Stomach Neoplasms ; pathology ; Sulfonamides ; pharmacology ; Sulfones ; pharmacology

OBJECTIVETo analyze and identify the principal composition of ethyl acetate part of Huangqi Danggui decoction by high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS).

METHODThe analysis conditions are as follows: Zorbax SB C18 (4.6 mm x 250 mm, 5 microm) column; mobile phase (A) water mobile phase (B) acetonitrile, gradient elution; UV detection wavelength 254 nm; ESI source and data acquisition in positive and negative mode.

RESULTThe accurate molecular weights of 20 compounds were measured and identified in ethyl acetate part of Huangqi Danggui decoction. Furthermore, the types of them are as below: flavonoids and flavonoid glycosides, saponins, oligosaccharides, amino acids and phthalides.

CONCLUSIONIt is a rapid and accurate method that the compositions of compound prescription of traditional Chinese medicine can be identified in terms of the separation of high performance liquid chromatography, the accurate molecular weights measured by MS and other information, which can clarify the potential effective compounds of Huangqi Danggui decoction.

Acetates ; chemistry ; Amino Acids ; chemistry ; Benzofurans ; chemistry ; Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; chemistry ; Flavonoids ; chemistry ; Glycosides ; chemistry ; Molecular Weight ; Oligosaccharides ; chemistry ; Saponins ; chemistry ; Spectrometry, Mass, Electrospray Ionization ; methods