Objective To evaluate the expression of Toll-like receptor(TLR)on the monocyte- derived dendritic cells(DC)from chronic hepatitis B(CHB)patients and to analyze the expression pro- file and significance of the TLR such as TLR3,TLR4,TLR?,TLR8 and TLRg,which are associat- ed with immune response to viral infection.Methods Peripheral blood mononuclear cell(PBMC) centrifugated by the hydroxyethyl starch(HES)centrifugation were cultured and induced into DC by granulocyte-maerophage colony stimulating factor(GM-CSF)and interleukin-4(IL-4),and their mor- phology and phenotype were detected by the inverted microscope and flow cytometry respectively. Monocyte-derived DC were obtained from 10 chronically hepatitis B virus(HBV)-infected patients and 15 healthy volunteers.TLR3,TLR4,TLR7,TLRS,TLR9 expression on immature and mature DC were analyzed by FACS Calibur.DC was pulsed with HBcAg on day 3 and 5,then DC maturation and ability to process HBcAg and to stimulate autogeneic T cells were evaluated.Results Monocyte- derived DC developed different TLR expression patterns as they went through different maturation stages.TLR7,TLR8 expressions on immature DC and TLR3,TLR7 expressions on mature DC were lower in CHB than in control(for TLR7,TLR8 expression on immature DC:75.9%,1.0%vs 98.4%,15.4%,P

Objective To explore the effet of Yangxue Qingnao granule (YXQNG) on seizures and cognition function of pentylenetetrazole (PTZ)-kindled chronic epileptic rats models, expression of Cav3.2 in the hippocampus and the temporal lobe of these rats, and EEG features of the rats. Methods Forty healthy adult male SD rats were equally divided into 4 groups at random: PTZ group, VPA treatment group, VPA+YXQNG treatment group, normal saline (NS)-control group (n=10). PTZ solution was intraperitoneally injected for 8 weeks to induce the kindling model in the above 3 groups except the NS-control group. VPA by intragastric administration was given to the rats in the VPA treatment group 1 h before PTZ injection; YXQNG and VPA by intragastric administration were given to the rats in the VPA+YXQNG treatment group 1.5 h before PTZ injection. Behavioral changes of the rats were observed 8 weeks after PTZ injection; accuracy rate of response of the rats were examined by electric maze test;EEG was performed; and the expression ofT-type Ca2+ channel protein (Cav3.2) in the temporal lobe and hippocampus was detected by immunohistochemical staining. Results Rats in the PTZ group appeared grade Ⅳ or Ⅴ seizures for 3 consecutive d, and rats in the VPA treatment group, VPA+YXQNG treatment group appeared grade 0-Ⅱ seizures. The accuracy rate of response of the rats in the VPA+YXQNG treatment group was significantly higher than that in the PTZ group (P＜0.05). EEG indicated that paradoxical discharge was noted in rats of PTZ group when seizures appeared, and the total power at the time was obviously higher than that before PTZ injection (P＜0.05). The D-value of total power of EEG in rats of the VPA treatment group and VPA+YXQNG treatment group before and after treatment was significantly higher than that in the PTZ group (P＜0.05). And the level of Cav3.2 in the temporal and hippocampus in rats of the VPA treatment group and VPA+YXQNG treatment group was significantly lower than that in the PTZ group (P＜0.05); as compared with that in the VPA treatment group, the expression of Cav3.2 in the temporal and hippocampus in rats of the VPA+YXQNG treatment group was significantly reduced (P＜0.05). Conclusion The combination use of YXQNG and VPA can decrease the seizure stage, the paradoxical discharge of the brain and the level of Cav3.2 in brain tissue,and improve the cognitive function of the PTZ-kindled rats, indicating that using VAP and YXQNG simultaneously can treat epileptic seizure and protect the neurons.

OBJECTIVETo evaluate colon targeting characteristic of Kuikang colon targeted pellets (KCP) with determination of residual baicalin and baicalein concentration in gastrointestinal tract (GIT).

METHODThe baicalin and baicalein were assayed by HPLC. The recovery differences of the drug between KCP and conventional pellets from GIT were investigated, three and six hours after administration.

RESULTThe baicalin recovery of KCP (70%) from rat GIT was higher than that of CP (about 20%). Most of KCP were intact at 3 h after oral administration, and distributed in lower ileum. It indicated that release site of KCP was in lower ileum and colon. Six hours later, a small amount of baicalin was recovered in intestime, which showed that the release of baicalin from KCP was complete.

CONCLUSIONThe determination of residual baicalin in rat GIT was feasibility for evaluating KCP. The result confirmed KCP of colon targeting property.

Animals ; Colon ; metabolism ; Drug Delivery Systems ; Drug Implants ; Drugs, Chinese Herbal ; administration & dosage ; metabolism ; Flavanones ; metabolism ; Flavonoids ; metabolism ; Ileum ; metabolism ; Logistic Models ; Rats ; Rats, Wistar

Bone Neoplasms ; metabolism ; pathology ; surgery ; Diagnosis, Differential ; Female ; Humans ; Immunohistochemistry ; Langerhans Cell Sarcoma ; metabolism ; pathology ; surgery ; Middle Aged ; S100 Proteins ; metabolism ; Talus ; Vimentin ; metabolism

To study the molecular mechanisms of nitric oxide donor sodium nitroprusside (SNP) -induced apoptosis in K562 human leukemia cell line, the different concentrations of SNP and different time of culture were used to treat K562 cell. At the same time, potassium ferricyamide (PFC) was used as control, blank was designed in experiment. Cell apoptosis was analysed by cell morphology, DNA agarose gel electrophoresis, DNA content, and annexin-V/PI labeling method. The TdT-mediated dUTP nick end labeling (TUNEL) assay was used to quantify in situ cell apoptosis. Reactive oxygen species (ROS) in cells and mitochondrial transmembrane potential (DeltaPsim) were labeled by dihydrorhodamin 123, 2', 7'-dichlorodihydrofluorescein diacetate and rhodamin 123/PI. bcl-2, bax, bad, p53 gene proteins and mitochondrial membrane protein were analysed by flow cytometry. The results showed that the K562 cell apoptosis was confirmed by typical cell morphology, DNA fragment, sub-G(1) phase, TUNEL and annexin-V/PI labeling. A majority of K562 cells were arrested in G(0)/G(1) phase. During the process of SNP-induced apoptosis in K562 cell, the mean fluorescence intensity of ROS in cells was significantly higher than those in blank and PFC control, while the DeltaPsim reduced. The expression of p53, bax, bad, Fas protein and mitochondrial membrane protein increased and bcl-2 protein decreased after SNP treatment. It is concluded that SNP induces K562 cell apoptosis through increasing ROS in cells, expressing the p53, bax, bad, Fas protein and mitochondrial membrane protein and decreasing bcl-2 protein, opening the mitochondrial permeability transition pore and reducing DeltaPsim. Furthermore, the Fas was activated during the apoptosis process.
Apoptosis ; drug effects ; Dose-Response Relationship, Drug ; Humans ; In Situ Nick-End Labeling ; K562 Cells ; Membrane Potential, Mitochondrial ; drug effects ; Nitric Oxide Donors ; pharmacology ; Nitroprusside ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Reactive Oxygen Species ; metabolism ; Tumor Suppressor Protein p53 ; metabolism ; bcl-2-Associated X Protein ; metabolism ; fas Receptor ; biosynthesis

OBJECTIVETo determine the effects of nerve growth factor (NGF) on proliferation of hepatic stellate cells (HSCs) and investigate the related molecular mechanism.

METHODSAfter incubating cultured HSCs for 24 h with different concentrations of NGF (100, 200 or 400 ng/mL), the cell proliferation was observed by XTT colorimetric assay and cell cycle was detected by flow cytometry. Morphological changes in response to a 24 h exposure to 100 ng/mL NGF were observed by transmission electron microscopy.

RESULTSNGF significantly inhibited HSC proliferation (P less than 0.05) in a dose-independent manner. The optical densities of the XTT colorimetric assay were 0.66+/-0.03 for 100 ng/mL NGF, 0.69+/-0.03 for 200 ng/mL NGF, and 0.66+/-0.03 for 400 ng/mL NGF, all of which were significantly lower than that of the control group (0.73+/-0.01; P less than 0.05). All concentrations of NGF led to significantly higher numbers of HSCs in the G2 phase (100 ng/mL: 14.83+/-5.41%, 200 ng/mL: 14.73+/-2.50%, and 400 ng/mL: 14.87+/-2.06%), compared to that detected in the control group (7.47+/-4.39%; P less than 0.05). Twenty-four hours of exposure to 100 ng/mL NGF caused morphological changes indicative of apoptosis.

CONCLUSIONNGF inhibits the proliferation of HSCs, possibly by arresting the cells in the G2 phase of the cell cycle. NGF-inhibited cells may also undergo apoptosis.

Animals ; Apoptosis ; Cell Cycle ; Cell Proliferation ; drug effects ; Cells, Cultured ; Flow Cytometry ; Hepatic Stellate Cells ; cytology ; drug effects ; Nerve Growth Factor ; pharmacology ; Rats

OBJECTIVETo study the role of extracellular matrix (ECM) in neural differentiation of mouse embryonic stem cells (ESCs).

METHODSMouse ESCs were incubated in the ESC conditioned medium, and the formation of embryonic bodies (EBs) were induced in bacteriological dishes using high-concentration all-trans retinoic acid (RA). The EBs were seeded on different matrixes (gelatin, fibronectin, and laminin/poly-L-ornithine) to test their impact on neural differentiation of the ESCs using immunofluorescence assay. The effect of laminin/poly-L-ornithine on the growth of neurites was evaluated with fluorescence microscopy.

RESULTSHigh-concentration RA activated and accelerated the differentiation of ESCs toward nestin-positive neural progenitor cells. Fibronectin supplement in the matrix dose-dependently promoted ESC differentiation into neural progenitor cells, while laminin/poly-L-ornithine increased the growth of the neurites and induced the maturation of the differentiated neural cells.

CONCLUSIONECM plays an important role in neural differentiation of mouse ESCs, and application of FN produces the most conspicuous effect during the differentiation of the ESCs into the neural progenitor cells;laminin/poly-L-ornithine is the most effective during their differentiation into neural cells.

Animals ; Cell Differentiation ; drug effects ; Cells, Cultured ; Culture Media ; Embryonic Stem Cells ; cytology ; drug effects ; Extracellular Matrix ; physiology ; Fibronectins ; pharmacology ; Laminin ; pharmacology ; Mice ; Neurons ; cytology ; drug effects ; Peptides ; pharmacology ; Tretinoin ; pharmacology

Objective To investigate the gene mutations of calcium-and integrin-binding protein 2 (CIB2) 196C＞T, 272T ＞ C and 297C ＞ G carried by students with non-syndromic hearing impairment from special educational schools in Yunnan Province. Methods The experimental group included 337 students with non-syndromic hearing impairment who failed to carry deafness gene with GJB2 (35 del G, 176_191 del 16,235delC, 299_300 del AT), GJB3 (C538T,G547A), mtDNA 12S rRNA (A1555G, C1494T), and SLC26A4 (IVS7_2A＞G, A2168G) . The control group consisted with 150 healthy people. Genomic DNA was isolated from peripheral blood with EDTA anti-coagulate. The subject's DNA fragments including CIB2 196C＞T, 272T ＞ C and 297C＞ G were amplified by polymerase chain reaction (PCR), and subsequently analyzed by direct sequencing to identify deafness-associated mutations. Results Both in the experimental group and control group, we failed to find the mutation of CIB2 196C＞T, 272T＞C and 297C＞G in all individuals. Conclusion Mutations in CIB2 gene 196C＞T, 272T＞C and 297C＞G are not a frequent cause of non-syndromic hearing loss among deaf people in Yunnan province. It provided important information for deafness with formulating landscape of gene screening in this region.