OBJECTIVETo establish a method for isolating and culturing mouse dental follicle cells and to study the phenotype characteristics of dental follicle cells.

METHODSMandibular first molars from 9 day old Balb/c mice were digested with 1% trypsin, subsequently, and the dental follicle was enucleated from the tooth germ and cultured. The shape and ultrastructural appearance of dental follicle cells were observed under phase-contrast microscope and scanning electron microscope (SEM). Immunocytochemistry was used to detect the expression of alkaline phosphatase (ALP), bone sialoprotein (BSP) and osteopontin (OPN).

RESULTSThree types of cells were isolated: some were cuboidal/polygonal; some were elongated, spindle-shaped, fibroblast-like cells; and a minor, third cell type was very thin and elongated. The cytoplasm of the first two cell types was filled with abundant granules. The cells were pleomorphism under SEM and had many filipodia and microvilli. According to whether there were filaments overlying the surface, the cells could be divided into two subtypes. Some but not all follicle cells expressed ALP, BSP and OPN.

CONCLUSIONThe cultured dental follicle cells consisted of several cell phenotypes and had the potential of differentiating into cementoblasts, periodontal ligament fibroblasts and osteoblasts.

Alkaline Phosphatase ; Animals ; Cell Culture Techniques ; Cell Line ; Cells, Cultured ; Dental Cementum ; Dental Sac ; Fibroblasts ; Integrin-Binding Sialoprotein ; Mice ; Mice, Inbred BALB C ; Molar ; Osteoblasts ; Osteopontin ; Periodontal Ligament ; Phenotype ; Tooth Germ

Objective To establish qualitative and quantitative analysis methods for identification and determination of multi-constituent of Chanfukang granules (CFKG) using high performance liquid chromatography-time-of-flight mass spectrometry (HPLC-TOF-MS) and high performance liquid chromatography-triple quadrupole mass spectrometry (HPLC-MS/MS),and a preliminary pharmacokinetic study was carried out.Methods An Agilent ZORBAX Eclipse Plus C1s column (100 mm × 3.0 mm,1.8 μm) and a Waters XBridge(R) BEH C18 column (150 mm × 4.6 mm,2.5 μm) were used with 0.1％ formic acid aqueous solution-acetonitrile as mobile phases by gradient elution.The compounds were detected by electrospray ion source in both positive and negative mode with multiple reaction monitoring (MRM) mode.SD rats were ig with CFKG (0.5,5 g/kg).Blood samples of 0.2 mL were taken from jugular vein at different time points and plasma components and contents were determined by HPLC-MS/MS.Results Twenty-four constituents were identified by HPLC-TOF-MS qualitative analysis and thirteen constituents were quantitatively detected by HPLC-MS/MS.The established HPLC-MS/MS method had a good separation,and all the legal verification met the requirements.The content of stachydrine,Leonurine,and astragaloside Ⅳ were high,which may be the main active ingredient The pharmacokinetic results showed that,only when the dose was 5 mg/kg,stachydrine and Leonurine can detected.Elimination of stachydrine was slow and elimination of Leonurine was fast.Conclusion A rapid and efficient method for studying the chemical constituents was established,which could provide reference for the quality control of Chanfukang granules.

OBJECTIVETo compare the hybrid between species of Dendrobium huoshanense and its parents on growing, physiologic indexes and content of medicinal components, and provide theoretical basis for species quality improvement.

METHODThe chlorophyll content, the photosynthesis rate, the polysaccharides content and the alkaloids content were measured by anhydrous ethanol method, Cl-310 photosynthesis determination system, colorimetry of concentrated sulphuric acid-phenol and acid dyes colorimetry respectively.

RESULTThe growth of hybrid was close to D. moniliforme, and apparently higher than D. huoshanense. The chlorophyll content and the photosynthesis rate of one-year-hybrid were markedly higher than its parents. The content of polysaccharides and alkaloids in two-year-stem and three-year-stem of hybrid were close to that of D. huoshanense.

CONCLUSIONThe hybrid integrates superiority of parents on growth and accumulation of medicinal components opens vast vistas for development and utilization.

Alkaloids ; analysis ; Chlorophyll ; analysis ; Dendrobium ; chemistry ; classification ; genetics ; growth & development ; Hybridization, Genetic ; Photosynthesis ; Plants, Medicinal ; chemistry ; classification ; genetics ; growth & development ; Polysaccharides ; analysis

The purpose of this study was to investigate the repair of the osteoarthritis(OA)-induced cartilage injury by transfecting the new TGF-β3 fusion protein (LAP-MMP-mTGF-β3) with targeted therapy function into the bone marrow-derived mesenchymal stem cells (MSCs) in rats. The recombinant of pIRES-EGFP-MMP was constructed by combination of DNA encoding MMP enzyme cutting site and eukaryotic expression vector pIRES-EGFP. LAP and mTGF-β3 fragments were obtained from rat embryos by RT-PCR and inserted into the upstream and downstream of MMP from pIRES-EGFP-MMP respectively, so as to construct the recombinant plasmid of pIRES-EGFP-LAP-MMP-mTGF-β3. pIRES-EGFP-LAP-MMP-mTGF-β3 was transfected into rat MSCs. The genetically modified MSCs were cultured in medium with MMP-1 or not. The transfected MSCs were transplanted in the rat OA models. The OA animal models were surgically induced by anterior cruciate ligament transaction (ACLT). The pathological changes were observed under a microscope by HE staining, Alcian blue, Safranin-fast Green and graded by Mankin's scale. pIRES-EGFP-LAP-MMP-mTGF-β3 was successfully constructed by means of enzyme cutting and sequencing, and the mTGF-β3 fusion protein (39 kD) was certified by Western blotting. Those genetically modified MSCs could differentiate into chondrocytes induced by MMP and secrete the relevant-matrix. The transfected MSCs could promote chondrogenesis and matrix production in rat OA models in vivo. It was concluded that a new fusion protein LAP-MMP-mTGF-β3 was constructed successfully by gene engineering, and could be used to repair the OA-induced cartilage injury.

Adolescent ; Adult ; Aged ; Apoptosis ; Cell Cycle ; Cell Division ; Child ; Female ; Glioma ; chemistry ; pathology ; Humans ; Immunohistochemistry ; Inhibitor of Apoptosis Proteins ; Male ; Microtubule-Associated Proteins ; analysis ; Middle Aged ; Neoplasm Proteins ; Prognosis ; Proto-Oncogene Proteins c-bcl-2 ; analysis

OBJECTIVETo study the difference in the pharmacokinetics of emodin in Zhiganning capsules and Rhizoma Polygontum Cuspidatum by nonaqueous RP-HPLC.

METHODThe rats were orally administered with the extraction of Rhizoma Polygontum Cuspidatum and Zhiganning capsules. After hydrolysis and extraction, the content of emodin in the plasma is determined by Nonaqueous RP-HPLC.

RESULTThe concentration-time profiles of emodin fit two-compartment model. The pharmacokinetics parameters including, t1/2alpha, AUC(0-infinity), CL(s) and C(max) of emodin in the group of Rhizoma Polygontum Cuspidatum were significantly different from these in the group of its compounds.

CONCLUSIONThere is a significant difference in pharmacokinetics of emodin between zhiganning capsules and the extraction of Rhizoma Polygontum Cuspidatum.

Animals ; Area Under Curve ; Capsules ; Drug Combinations ; Drugs, Chinese Herbal ; administration & dosage ; chemistry ; isolation & purification ; Emodin ; isolation & purification ; pharmacokinetics ; Female ; Male ; Plants, Medicinal ; chemistry ; Polygonum ; chemistry ; Rats ; Rats, Sprague-Dawley ; Rhizome ; chemistry

Child ; Electrocardiography ; Humans ; Male ; Ventricular Fibrillation ; physiopathology

The stem cell leukemia (SCL) gene is a new oncogene related with leukemogenesis. To explore the effects of antisense oligonucleotides of SCL on leukemic cells, SCL antisense phosphorothioate oligodeoxynucleotides (AS-PS-ODN) were used to treat K562 and CEM leukemic cell lines to observe the effects on proliferation, differentiation, apoptosis and SCL mRNA expression in the cells. The results showed that incubation of K562 or CEM cells with AS-PS-ODN at different concentrations led to inhibition of cell proliferation, and the inhibitory effects varied with the incubation time. The positive rate of benzidine staining in K562 cells increased significantly after the inhibition with AS-PS-ODN, compared with S-PS-ODN treatment. The characteristics of apoptosis were observed in K562 cells treated with AS-PS-ODN, but not in CEM cells. Expression of SCL mRNA in K562 and CEM cells and SIL-SCL mRNA in CEM cells decreased after incubation of AS-PS-ODN. It is concluded that SCL AS-PS-ODN inhibits specifically the proliferation of K562 and CEM cells, also decreases the level of SCL and SIL-SCL mRNA expression. AS-PS-ODN enhances erythroid differentiation and induces premature apoptosis in K562 cells.
Apoptosis ; drug effects ; Basic Helix-Loop-Helix Transcription Factors ; Cell Division ; drug effects ; DNA-Binding Proteins ; antagonists & inhibitors ; physiology ; Humans ; K562 Cells ; Oligonucleotides, Antisense ; pharmacology ; Proto-Oncogene Proteins ; antagonists & inhibitors ; physiology ; RNA, Messenger ; analysis ; T-Cell Acute Lymphocytic Leukemia Protein 1 ; Transcription Factors ; antagonists & inhibitors ; physiology

OBJECTIVETo investigate the relationship between alterations of p16INK4a and p14ARF genes and gastric carcinogenesis.

METHODSTumors and gastric tissues neighboring carcinoma from 48 patients with gastric cancer were studied. Homozygous deletion, mutation, methylation of the CpG islands, mRNA expression of p16INK4a and p14ARF genes were assessed by polymerase chain reaction (PCR), PCR-single strand conformation polymorphism (SSCP), PCR based methylation assay, and reverse transcription (RT)-PCR.

RESULTS(1) The overall homozygous deletion rate of p16INK4a and p14ARF was 35.4% (17/48), and no homozygous deletion was examined in all the gastric tissues neighboring tumor. (2) There was no point mutation of p16INK4a and p14ARF in 31 gastric cancers without homozygous deletion and in the matched gastric tissues adjacent to tumor. (3) Methylation of the CpG islands of p16INK4a and p14ARF was detected in 47.9% (23/48) of gastric cancers, while methylation was observed only in 2 of 48 gastric tissues neighboring cancers with a significant difference (P <0.01). (4) The rate of p16INK4a mRNA loss was 47.9% (23/48) in gastric cancer, and the cases of combined methylation of exons 1alpha and 2 had a higher loss rate (100%, 6/6) of p16INK4a mRNA than those of methylation form the other exons (11.8%, 2/17) (P <0.01). The loss rate of p14ARF mRNA was 45.8% (22/48) in gastric cancer, and patients with combined methylation of exons 1beta and 2 had a higher loss rate (100%, 3/3) of p14ARF mRNA than those of the methylation from the other exons (15%, 3/20) (P < 0.05). (5) The combined loss of p16INK4a and p14ARF mRNAs was examined in 1 (5.6%) of 18 cases of well and moderately-differentiated carcinomas, and 11 (36.7%) of 30 cases of poorly and not-differentiated carcinomas with significant difference (P <0.05).

CONCLUSIONp16INK4a and p14ARF genes were frequently inactivated by homozygous deletion and methylation of the 5' CpG islands in gastric cancer, which might have played an important role in the development of gastric cancer.

Adenocarcinoma ; genetics ; Adolescent ; Adult ; Aged ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; DNA Methylation ; Female ; Gene Deletion ; Genes, p16 ; Humans ; Male ; Middle Aged ; RNA, Messenger ; analysis ; Stomach Neoplasms ; genetics ; Tumor Suppressor Protein p14ARF ; genetics