Adult ; Cell Separation ; methods ; Endothelial Cells ; cytology ; Humans ; Stem Cells ; cytology

BACKGROUND:There are numerous studies on bone marrow mesenchymal stem cells(BMSCs)from small animals such as rats and rabbits,but no few reports addressing BMSCs from big animals.OBJECTIVE:To observe in vitro cultured goat BMSCs,and to understand its biological properties.METHODS:A healthy Chinese goat aged ten months was obtained to extract 5 mL fresh bone marrow from the posterior superior iliac spine by puncture following anesthesia.Using the whole bone marrow method,the samples were incubated in a sterile plastic culture flask and added with DMEM/F12 containing 10% fetal bovine serum.Following 80% 90% confluence,cells were digested by trypsin.Cells at passage 3 in logarithmic phase were collected and frozen,and then recovered.Changes in cell morphology were observed using an inverted microscope.Cell growth curve was measured using MTT assay.The potential of osteogenic differentiation was examined utilizing Von Kossa's staining.RESULTS AND CONCLUSION:The primary cultured BMSCs were cultured with adherent growth.The cells were spindle form.Cell morphology following passage 3 was similar,showing long spindle shape.Following freezing and recovery,cell adherence was slower compared with subculture cells,and no significant difference was detected in cell morphology and viability compared with subculture cells.Growth cycle was similar in passage 3-passage 5 cells.BMSCs entered lag phase at days 2 and 3,logarithmic phase at day 3,and platform phase at days 6 and 7,and then growth speed was slow.Goat BMSCs were positive for Von Kossa's stain at 3 weeks following osteogenic induction.Results verified that cultured goat BMSCs showed strong genetic stability and proliferation ability,and differentiated into osteoblasts.

At present, majority of the small molecular drugs used in clinics target proteins, they exert the efficacy through the binding to specific sites on the target protein. However, the "druggable" protein targets account for a small portion of the total number of proteins, and "non-druggable" proteins account for 80%, because of not having suitable drug binding sites. In the central rule, RNA is located in the upstream of proteins and controls the transcription of proteins. The research of small molecule drugs targeting RNA can solve the problem of protein "undruggable proteins" in some extent. This review summarizes the representative research achievements of small molecular drugs targeting RNA in recent years, and the screening methods applied to this field, with the focuses on the latest progress of small molecular drugs targeting novel coronavirus RNA.

Objective To predict the CTL epitopes of Tat exon 1 region in HIV-1 CRF07_BC strains, which were prevailing in China. Methods Total of 236 plasma samples were from the 3rd National HIV Molecular Epidemic Survey (NMES3). All the subjects were infected with HIV-1 CRF07_BC viruses. The tat exon 1 region was amplified by reverse transcription reaction and nested polymerase chain reaction (nested-PCR), then the PCR products were sequenced. The distribution of CTL epitopes of this region were predicted by on-line software BIMAS HLA Peptide Binding Predictions and statistics software. Results To-tal of 236 CRF07_BC strains were from 16 provinces, mainly in intravenous drug asers(58.9%)and then sex(25.0%). It was showed that there were 12 CTL epitopes of 236 Tat exon 1 region of CRF07_BC strains mainly located in proline-rich region, cysteine-rich region and core-region. Those epitopes were banded by 5 HLA presenting molecules in genotype(A * 2501 ,A * 2902, B * 15,B * 5301 and Cw * 1203) and 6 HLA presenting molecules in serotype (B53, B58 ,B57 ,A3 ,A68 and Cw12). The frequency of single amino acid substitution was more than 50% in 7 CTL epitopes. Conclusion The CTL epitopes in Tat exon 1 of CRF07 _BC strains were located in different functional regions, and there were some amino acid variations in them.

Transcriptions are regulated by transcription factors. Natural transcription factors usually consist of at least two functional domains: a DNA-binding domain and an effector domain. According to this, novel artificial transcription factors are designed to up or down regulate transcription and expression of a target gene. The Cys2-His2 zinc finger domain is a DNA-binding module that has been widely used as the DNA-binding domain in artificial transcription factors. Each zinc finger domain, which comprises about 30 amino acids that adopt a compact structure by chelating a zinc ion, typically functions by binding 3 base pairs of DNA sequence. Several zinc fingers linked together would bind proportionally longer DNA sequences. According to the "bipartite complementary" library strategy, a pair of zinc finger phage display libraries were constructed. After construction of the libraries, a 9bp sequence (5'-GCAGAGGCC-3') on the promoter of SV40 was chosen as a target for next step. After parallel selection, PCR amplification, desired fragments recovery, re-ligation, and additional rounds of selection, phage enzyme-linked ELISA experiments were performed to identify specific binding clones displaying the zinc fingers with predetermined sequence-specificity to our target sequence. Then two clones with strong ELISA signals were chosen to be tested for binding both to its full target site (5'-GCAGAGGCC-3') and to sites containing single transition mutations. The binding specificity of one of the two clones (clone 3) was shown to be fairly good. The three-finger DNA-binding domain targeted to SV40 promoter, that is, zinc finger sequences on clone 3, was fused to KOX1 suppression domain KRAB and cloned into pcDNA3.1 (+) (which expression product was artificial transcription factor). The zinc fingers (which expression product was the DNA-binding domain of artificial transcription factor) and KRAB domain only (which expression product was effector domain of artificial transcription factor) were also cloned separately into the same expression vector. All constructs contained an N-terminal nuclear localization signal. Every of the vectors (including pcDNA3.1 (+) without inserting sequences) were cotransfected with pGL3-Control and pRL-TK and the activity of luciferase was used to indicate the function of product from transfected expression vectors. Our artificial transcription factor was proved to repress the expression of reporter gene efficiently,while with only DNA-binding domain or effector domain the repression was not remarkable. By adding different effector domains and changing the DNA-binding domain, artificial transcription factor would have a wide range of potential applications.
Enzyme-Linked Immunosorbent Assay ; Genes, Synthetic ; genetics ; physiology ; Models, Theoretical ; Peptide Library ; Polymerase Chain Reaction ; Promoter Regions, Genetic ; genetics ; Transcription Factors ; chemical synthesis ; chemistry ; metabolism ; Zinc Fingers ; genetics ; physiology

Objective To study the epidemic situation and characteristic of subtypes of HIV-1 strains prevalent in Hebei Province. Methods Viral RNA was extracted from plasma samples, HIV-1 genes (env and gag ) were amplified by RT and nested-PCR using specific primer pairs and sequenced directly.The acquired sequences were compared with international subtypes references and their phylogenetic-trees were analyzed to determine the subtype. Results Among 154 HIV-1 antibody positive cases , 148 HIV-1 gene fragments were amplified and analyzed. There were 6 kinds of HIV-1 subtypes and recombinants, moreover unidentified 2 cases in 148 samples, of which 61 (41.2%) cases of B', 59(39.9% ) cases of CRF01_AE, 16( 10.8% ) cases of CRF07_BC, 6(4.1% ) cases of CRF08_BC, and 2( 1.4% ) cases of C and B01 each. HIV-1 B01 was detected firstly in Hebei Province. Conclusion Six HIV-1 subtypes were identified in Hebei Province. B' and CRF01_AE are the primary subtypes and recombinants of HIV-1 existed in Hebei Province. The surveillance of HIV-1 gene variation should be paid more attention to.

OBJECTIVETo explore the mechanism by which HBV X gene(HBx) inhibits apoptosis of human hepatoma cell line HepG2 in terms of miRNA.

METHODSThree cell lines were prepared: HepG2 cells stably transfected with HBx (HepG2/HBx), HepG2 cells stably transfected with pcDNA3.1 (HepG2/pcDNA3.1) and HepG2 cells. Flow cytometry was adopted to measure the apoptosis of these three cells and Taqman fluorescence quantitative PCR was used to examine miR-192 expression. After HepG2 cells was transfected with miR-192, the apoptosis was analyzed by flow cytometry and the expressions of p53 and PUMA at mRNA and protein levels were evaluated by SYBR Green quantitative PCR and Western blot, respectively.

RESULTSCompared with HepG2/pcDNA3.1 cells (11.46% ± 0.69%) and HepG2 cells (12.5% ± 0.66%), the apoptosis rate of HepG2/HBx cells (2.37% ± 0.35%) was significantly reduced (F = 171.722, P < 0.01). The level of miR-192 was 49.1% ± 5.9% in HepG2 cells, which was dramatically down-regulated (F = 14.319, P = 0.019) as compared to the other two groups (HepG2/pcDNA3.1: 98.0% ± 8.9%; HepG2: 100%). Compared with HepG2 cells transfected with miR-NC (10.74% ± 1.15%), transfection of miR-192 into HepG2 cells led to increased apoptosis (15.74% ± 1.17%) (F = 18.415, P = 0.013) and higher p53 and PUMA expressions at mRNA (p53: 1.68 ± 0.12 vs 0.90 ± 0.09, F = 43.115, P = 0.003, PUMA: 1.66 ± 0.10 vs 0.98 ± 0.06, F = 22.541, P = 0.009) and protein (p53: 3.07 vs 1, PUMA: 2.13 vs 1) levels.

CONCLUSIONHBx could inhibit apoptosis of HepG2 cells through down-regulation of miR-192 which induces apoptosis of HepG2 cells.

Apoptosis ; Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Down-Regulation ; Genes, Viral ; Hep G2 Cells ; Hepatitis B virus ; genetics ; Humans ; Liver Neoplasms ; genetics ; metabolism ; pathology ; MicroRNAs ; metabolism ; Trans-Activators ; genetics ; metabolism

Objective To investigate the chemical constituents of the root of Wrightia pubescens.Methods Compounds were isolated by the combined use of colum chromatography,preparative HPLC and recrystallization,and their structures were identified by their physicochemical and spectroscopic data.Cytotoxic activities of the compounds were evaluated using MTT method in vitro.Re?sults Twelve compounds 1-12 were isolated from the ethyl acetate soluble part from 75% ethanol extract of the root of W.pubescens, and identified as scopoletin(1),coumarin(2),cleomiscosin B(3),mollugin(4),4-hydroxybenzoic acid(5),vanillic acid(6), vanillin(7),4-hydroxymethyl-5-hydroxy-2H-pyran-2-one(8),β-sitosterol(9),β-daucosterol(10),ursonic acid(11),and me?dioresinol(12). Compound 4 showed cytotoxic activity against HepG2 cells in vitro with IC50of 8.0 mg/L. The IC50of compound 11 against MCF-7 and HepG2 cells was 14.7 and 18.2 mg/L,respectively.Conclusion Compounds 1-5,8 and 12 were isolated from the genus Wrightia for the first time. Compounds 6 and 10 were isolated from the title plant for the first time. Compounds 4 and 11 showed cytotoxic activities against tumor cells in vitro.

To further understand triptolide, this paper has introduced the pharmacology, pharmacokinetics, toxicity, the clinic application and semi-synthesis of triptolide on basis of importance and significant contents of reference which have been consulted in the past twenty years. Presently triptolide and Tripterygium wilfordii have been a hot spot of modernization of Chinese traditional medicine. It is very important to develop a new dosage form of high effect and low toxicity by making use of advanced technology according to its characteristics.
Animals ; Anti-Inflammatory Agents, Non-Steroidal ; pharmacology ; Antineoplastic Agents, Alkylating ; pharmacology ; Antispermatogenic Agents ; pharmacology ; Diterpenes ; chemical synthesis ; isolation & purification ; pharmacology ; toxicity ; Epoxy Compounds ; Humans ; Immunosuppressive Agents ; pharmacology ; Phenanthrenes ; isolation & purification ; pharmacology ; toxicity ; Tripterygium ; chemistry

OBJECTIVETo investigate the effect of synthetic non-methylated CpG-ODN combined with recombined HBsAg on the phenotype, function and the expression level of nucleic transcription factor NF-kappa B (NF-kB) and AP-1 of monocyte-derived dendritic cells (DC) in patients with chronic hepatitis B (CHB).

METHODSPurified monocytes were isolated from the peripheral blood of CHB patients and healthy volunteers and cultured with human granulocyte-monocyte colony stimulating factor added together with interleukin-4. On the fifth day of culture, CpG-ODN, HBsAg and other reagents, such as TNF alpha and PBS, were added to the medium, and 18 hours later cells were collected for the detection of surface molecules (HLA-DR/CD86/CD1a). IL-12p70 levels in the supernatant and stimulating capacity to allogenic T lymphocytes were detected. The nucleic proteins of NF-kB and AP-1 in DC were extracted and purified for the gel shift assay.

RESULTSCompared with those of the PBS group, the expression rates of HLA-DR of DC treated with CpG-ODN and/or HBsAg were obviously increased. Both the IL-12p70 level and the stimulating capacity of DC to allogenic T lymphocytes in mixed lymphocyte reaction increased significantly in the CpG-ODN group and in the CpG-ODN/HBsAg combination group (P less than 0.01 and 0.05, respectively). The expression rates of CD1a were raised only in the CpG-ODN/HBsAg combination group. Three kinds of immunological adjuvants, TNF alpha, CpG-ODN and CpG-ODN/HBsAg enhanced the expression of nucleic NF-kB and inhibited the expression of AP-1 in DC.

CONCLUSIONCpG-ODN, like TNF alpha, has remarkable stimulatory effect on the impaired phenotype and function of monocyte-derived DC in patients with CHB; CpG-ODN and HBsAg have a synergetic effect in increasing the antigen presenting function. The regulating ability of CpG-ODN and TNF alpha on the expression levels of NF-kB and AP-1 might be one of the mechanisms of their immunostimulatory effects on DC of the CHB patients.

Adjuvants, Immunologic ; pharmacology ; Adult ; Cells, Cultured ; Dendritic Cells ; metabolism ; HLA-DR Antigens ; metabolism ; Hepatitis B Surface Antigens ; pharmacology ; Hepatitis B, Chronic ; metabolism ; Humans ; Male ; NF-kappa B ; metabolism ; Oligodeoxyribonucleotides ; pharmacology ; Recombinant Fusion Proteins ; Transcription Factor AP-1 ; metabolism