Objective：To set up a system for the culture of Dendrobium chrysotoxum in vitro. Method：Tissue culture，fire fly luminescence and phenol-H2SO4 method. Result：The embryo could germinate with or without light, the MS, 1/2MS, B5, N6 mediums are suitable to the growth and the differentiation of sprout with light, 0.5　mg*L－1NAA and 1　mg*L－16-BA, and ATP have regular changes, the content of polysaccharide was 2.833% in plant and 7.254% in sprout. Conclusion：The light has no effects on the embryo germination, but the phytohormone，nitrogen source and organized elements are important to the growth and differentiation of the sprout which should be transferred to the MS, 1/2MS, B5, N6 mediums in time supplemented with NAA与6-BA, ATP may be served as the dynamic indication of nourishment demand in the plant. The content of polysaccharide in the sprout is higher and can be utilized.

With the combined applications of steam distillation, water extraction and alcohol precipitation, liquid-liquid extraction and column chromatography over macroporous resin, a splitted-fractions method of the chemical constituents of Poria cocos was established. The unoverlapping property of the fractions of P. cocos was qualitatively analysed by using principal component analysis and cluster analysis. With angle cosine, squared euclidean distance and the overlapping analysis of peak area of crude herbs, the unoverlapping property of the fractions of P. cocos was half-quantitatively analysed. The chemical components of P. cocos was divided into the fractions of polysaccharide, petroleum ether, ethyl acetate, alcohol eluate from macroporous resin and water eluate from macroporous resin. Non similarity degree among each chemical fraction was above 80% and main chemical components were identified. The established method for splitting fractions of P. cocos has good stability and repeatability and all chemical components in P. cocos could be completely divided into six fractions. It is the first time that the author half-quantitatively analyse the unoverlapping property of the chemical fractions of P. cocos.
Chromatography, High Pressure Liquid ; Cluster Analysis ; Medicine, Chinese Traditional ; Poria ; chemistry

OBJECTIVETo investigate the chemical constituents of effective part of Epimedium brevicornum.

METHODThe sample was extracted with ethanol and purified by macroporous resin. The structures were identified by HPLC-MS3 experiments.

RESULTNine compounds were identified from the effective part of E. brevicornum.

CONCLUSIONThe method is simple and rapid for the identification of the flavonoids from E. brevicornum.

Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Epimedium ; chemistry ; Flavonoids ; chemistry ; isolation & purification ; Molecular Structure ; Plants, Medicinal ; chemistry ; Reproducibility of Results

AIMTo detect the hepatotoxic pyrrolizidine alkaloids (HPA) in the genus Ligularia Cass..

METHODSThe alkaloid extracts of Ligularia plant materials were detected and analyzed by the method of combination of TLC, and LC/MSn.

RESULTSAmong 22 species of Ligularia Cass., HPA were detected in 18 species with LC/MSn, and no HPA was detected in the remaining 4 species.

CONCLUSIONHPA was first detected with LC/MSn in L. tongelensis and other 15 species of Ligularia Cass.; HPA from these plants should be isolated, separated and identified and it is necessary to study the activities and toxicities of the HPA. The types and kinds of HPA from different species and sources are different, they should be detected separately.

Asteraceae ; chemistry ; classification ; Chromatography, Thin Layer ; Molecular Structure ; Plants, Medicinal ; chemistry ; Pyrrolizidine Alkaloids ; analysis ; chemistry ; Species Specificity ; Spectrometry, Mass, Electrospray Ionization

OBJECTIVETo investigate approach and possibility of transferring basic fibroblast growth factor (bFGF) gene into rabbit bone marrow stromal cells (BMSCs).

METHODSThe eukaryotic expression vectors harboring bFGF cDNA were constructed and transfected into rabbit BMSCs mediated by liposome. The transcription and expression of bFGF gene in the transfected BMSCs were detected by means of morphological observation, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and RT-PCR. The changes in the biological characteristics of the transfected MSCs were also observed.

RESULTSStable overexpression of bFGF protein was detected in the transfected BMSCs, which showed differentiation towards chondrocyte lineage.

CONCLUSIONStable expression of bFGF gene in transfected BMSCs can induce cell differentiation into chondrocyte lineage.

Animals ; Bone Marrow Cells ; cytology ; metabolism ; Female ; Fibroblast Growth Factor 2 ; biosynthesis ; genetics ; Gene Expression ; Genetic Vectors ; genetics ; Male ; Rabbits ; Stromal Cells ; cytology ; metabolism ; Transfection

OBJECTIVETo evaluate the biocompatibility of polylactic-co-glycolic acid (PLGA) for culturing bFGF gene-transfected bone marrow stromal cells (BMSCs) and assess the feasibility of this cell complex for repairing cartilage defect in rabbits using tissue engineering method.

METHODSBMSCs transfected by bFGF gene were cultured on PLGA matrix to assess the biocompatibility of PLGA. The cell complex was then implanted into the cartilage defect in rabbits, and its effect in cartilage defect repair was evaluated by histological observation and immunohistochemical staining.

RESULTSBMSCs transfected by bFGF gene grew normally on PLGA matrix. After implantation, the complex showed good effect for cartilage defect repair in rabbits.

CONCLUSIONPLGA has good biocompatibility with the transfected BMSCs, and the cell complex can be used for repairing rabbit cartilage defect and may potentially serve as a substitute of cartilage autograft.

Animals ; Biocompatible Materials ; chemistry ; Bone Marrow Cells ; cytology ; Cartilage, Articular ; injuries ; surgery ; Cells, Cultured ; Female ; Fibroblast Growth Factor 2 ; genetics ; Genetic Engineering ; methods ; Implants, Experimental ; Lactic Acid ; chemistry ; Male ; Polyglycolic Acid ; chemistry ; Rabbits ; Random Allocation ; Stromal Cells ; cytology ; Transfection

OBJECTIVETo investigate the changes of gene expression in rat neurons induced by 1.8 GHz radiofrequency electromagnetic fields (RF EMF) and to screen for the RF EMF-responsive genes.

METHODSNewly-born SD rats in 24 hours were sacrificed to obtain cortex and hippocampus neurons. The cells were divided randomly into two groups: the experiment group (the irradiation group) and the control group (the false irradiation group). In the irradiation group, after twelve days' culture, neurons were exposed to 1.8 GHz RF EMF modulated by 217 Hz at a specific absorption rate (SAR) of 2 W/kg for 24 hours (5 minutes on/10 minutes off) while in the false control group, the neurons were put in the same waveguide as in the irradiation group, but were not exposed to any irradiation. The total RNA was isolated and purified immediately after exposure. The affymetrix rat neurobiology U34 assay was used for detecting the changes in gene expression profile according to the manufacturer's instruction. RF EMF-responsive candidate gene was confirmed by using ribonuclease protection assay (RPA).

RESULTSAmong 1200 candidate genes, the expression levels of 34 genes were up or down regulated. Microtubule associated protein 2 (Map2) gene was selected as the candidate and subjected to further analysis. RPA data clearly revealed that Map2 was statistically significantly up-regulated after neurons were exposed to the RF EMF (P < 0.05).

CONCLUSIONThe modulation of gene expression and function of Map2 as a neuron specific cytoskeleton protein is crucial to maintain the normal framework and function of neurons. The finding that 1.8 GHz RF EMF exposure increases the expression of Map2 might indicate some unknown effects of RF EMF on neurons.

Animals ; Animals, Newborn ; Cell Phone ; Cells, Cultured ; Dose-Response Relationship, Radiation ; Down-Regulation ; Electromagnetic Fields ; Female ; Gene Expression ; radiation effects ; Male ; Microtubule-Associated Proteins ; biosynthesis ; genetics ; Neurons ; metabolism ; radiation effects ; Radio Waves ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Up-Regulation

OBJECTIVETo provide scientific basis for quality control of Lindera aggregata.

METHODHPLC analytical method was established using a Lichrospher C18 column and acetonitrile-water (56:44) as the mobile phase, detected at 235 nm.

RESULTThe linear range of linderane is between 0.0642 - 0.5774 microg, the average recovery was 98.4%, RSD1.7% (n = 9).

CONCLUSIONContents of linderane in commercially available and collected samples were from 0.028% to 0.123% and from 0.056% to 0.222% respectively.

Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; analysis ; Lindera ; chemistry ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry ; Quality Control ; Sesquiterpenes ; analysis

OBJECTIVETo prepare cytochrome (CYP)2E1-silenced hepatocytes by lentivirus-mediated RNA interference technology and to investigate the hepatotoxicity of trichloroethylene (TCE) in CYP2E1-silenced hepatocytes.

METHODSShort hairpin RNA fragments were designed and synthesized and were then ligated into the lentiviral vector; single colonies were screened; the plasmid was extracted after PCR and sequence identification and then transferred into L02 hepatocytes; the CYP2E1-silenced hepatocytes were selected; real-time quantitative PCR and Western blot were used to evaluate the interference effects. The obtained CYP2E1-silenced hepatocytes, as well as normal L02 hepatocytes, were treated with TCE (0, 0.25, 0.50, 1.00, 2.00, and 4.00 mmol/L). The cell viability and half maximal inhibitory concentration (IC50) of TCE were measured; the apoptotic rate of cells was measured by flow cytometry; the mRNA expression levels of apoptosis genes and oncogenes were measured by real-time quantitative PCR.

RESULTSThe IC50s of TCE for L02 hepatocytes and CYP2E1-silenced hepatocytes were 15.1 mmol/L and 23.6 mmol/L, respectively. The apoptotic rate increased as the dose of TCE rose in the two types of cells; the CYP2E1-silenced hepatocytes hada significantly lower apoptotic rate than L02 hepatocytes when they were exposed to 2.0 and 4.0 mmol/L TCE (P < 0.05 or P < 0.01). The mRNA expression level of bcl-2 (anti-apoptosis gene) in CYP2E1-silenced hepatocytes was 15% ∼ 60% higher than that in L02 hepatocytes (P < 0.01), while the mRNA expression levels of caspase-3 and caspase-9 (apoptosis genes) in CYP2E1-silenced hepatocytes were 30% ∼ 60% lower than those in L02 hepatocytes (P < 0.01). The mRNA expression level of p53 (cancer suppressor gene) in CYP2E1-silenced hepatocytes was 81 - 278% higher than that in L02 hepatocytes (P < 0.01), while the mRNA expression levels of c-fos and k-ras (oncogenes) in CYP2E1-silenced hepatocytes were 20-68% lower than those in L02 hepatocytes (P < 0.01).

CONCLUSIONCYP2E1-silenced cells can be successfully prepared by lentivirus-mediated RNA interference technology. Silencing CYP2E1 gene can reduce the hepatotoxicity of TCE and inhibit the expression of some apoptosis genes and oncogenes, suggesting that CYP2E1 gene plays an important role in TCE metabolism and is related to the hepatotoxicity of TCE.

Apoptosis ; drug effects ; genetics ; Cell Line ; Cell Survival ; drug effects ; genetics ; Cytochrome P-450 CYP2E1 ; genetics ; metabolism ; Genetic Vectors ; Hepatocytes ; drug effects ; metabolism ; Humans ; Lentivirus ; genetics ; RNA Interference ; Trichloroethylene ; toxicity

OBJECTIVETo investigate the quantitative and qualitative changes of TCRVα24(+)Vβ11(+) natural killer T (NKT) cells from bone marrow (BM) of aplastic anemia (AA) after in vitro stimulation of α-galactosylceramide (α-Galcer).

METHODSNKT cells in the bone marrow mononuclear cells (BMMNCs) from either AA patients or healthy controls were enumerated with flow cytometry. BMMNCs were cultured in RPMI1640 medium supplemented with either α-Galcer and rhIL-2 or α-Galcer, rhIL-2 and rhG-CSF. The proliferative capacity of NKT cells was determined by NKT cell numbers before and after in vitro culture. Expression of intracellular IFNγ and IL-4 in activated NKT cells was analyzed with flow cytometry.

RESULTSIn AA group, the percentage of NKT cells in BMMNCs was (0.19 ± 0.09)%. Addition of rhG-CSF into the α-Galcer/rhIL-2 culture medium resulted in significantly reduced expansion of NKT cells (67.45 ± 29.42-fold vs 79.91 ± 40.56 fold, P < 0.05). Meanwhile, addition of rhG-CSF reduced IFNγ positive NKT cells \[(37.45 ± 7.89)% vs (62.31 ± 14.67)%, P < 0.01\] and increased IL-4 positive NKT cells \[(55.11 ± 12.13)% vs (27.03 ± 9.88)%, P < 0.01\]. In healthy control group, the percentage of NKT cells in BMMNCs was (0.25 ± 0.12)%. Addition of rhG-CSF into the α-Galcer/rhIL-2 culture medium also significantly reduced expansion of NKT cells (97.91 ± 53.22-fold vs 119.58 ± 60.49-fold, P < 0.05), reduced IFNγ positive NKT cells \[(28.65 ± 10.63)% vs (50.87 ± 12.66)%, P < 0.01\], and increased IL-4 positive NKT cells \[(66.53 ± 14.96)% vs (31.11 ± 10.07)%, P < 0.01\].

CONCLUSIONCompared to those from healthy controls, BMMNCs from AA patiants have a reduced fraction of NKT cells, which possesses a decreased potential to expand in vitro in response to α-Galcer stimulation, and produce more IFNγ(+) NKT1 cells. rhG-CSF, in combination with α-Galcer, confers polarization of NKT cells towards IL-4(+) NKT2 subpopulation.

Anemia, Aplastic ; metabolism ; Bone Marrow ; metabolism ; Humans ; Interleukin-4 ; metabolism ; Killer Cells, Natural ; cytology ; Natural Killer T-Cells