BACKGROUND: Previous studies have demonstrated that LRP16 is an estrogen-responsive gene. Its expression level is strongly associated with the proliferation and invasive growth of human breast cancer cells.OBJECTIVE: To construct a LRP16 targeting vector and screen mouse embryonic stem cell clones with homolougous recombination of an inactive LRP16 gene.DESIGN: Constructing an inserting inactivation target by inserting SA-RIES-β geo expression cassette.SETTING: Bioregulatory Laboratory of the Third Medical Department of Kyushu University in Japan and Department of Molecular Biology, General Hospital of Chinese PLA.MATERIALS: The materials used here were mainly provided by the Bioregulatory Laboratory, the Third Medical Department of Kyushu University in Japan. The mouse genomic library in pBeloBAC11 Vector was purchased from lnvitrogen Corp. The competent TopF10 was purchased from Beijing Tiangen Biotech Corp. pcDNA3.1(+) vector was kept in our laboratory. Mouse ES cells were provided by Kyushu University.METHODS: The experiment was performed in Kyushu University and Department of Molecular Biology of PLA General Hospital from November 2004 to May 2005. Targeting sequence of LRP16 gene was obtained from 129 mouse genomic Bacterial Artificial Chromosomes library based on polymerase chain reaction (PCR) screening. The SA-RIES-β geo fragment was inserted within LRP16 fifth exon to inactivate LRP16. ES cells were screened with G418 and the homologously recombinant clone was identified by Southern blot analysis.MAIN OUTCOME MEASURES: Clones with homologous recombination.RESULTS: The LRP16 fragment including exon 5 to 11 was subcloned into the pBluescript SK Ⅱvector. Restriction map demonstrated that the SA-IRES-β geo fragment was correctly inserted into the LRP16 fifth exon. Southern blot results showed that there was an ES clone with targeting sequence homologously inserted.CONCLUSION: A LRP16 gene targeting vector is constructed and a homologous recombinant is obtained.

Objective To explore the immunogenicity of recombinant chimeric 6Aβ15-T including the Aβ1-15 epitope and a T-helper epitope formulated with different adjuvants and to evaluate its feasibility as a candidate vaccine for Alzheimer disease (AD).Methods The recombinant chimeric antigen 6Aβ15-T formulated with Al adjuvant, Freund′s adjuvant or MF59 adjuvant was administered to two strains of mice .The 6Aβ15-T-immunized group without adjuvants ( Mock) and non-immunized group (Control) were included in this study as control groups .The specific antibody and cellular immune response of the chimeric antigen were evaluated .Results In BALB/c strain mice, three types of adjuvants could substan-tially boost the immunogenicity of chimeric antigen 6Aβ15-T and produce a high level of specific-Aβ(β-amyloid) antibod-ies.In C57BL/6 strain mice, the existence of adjuvants enhanced the immune response of 6Aβ15-T antigen, but the mice in Mock group also produced a strong antibody response .In two strains of mice, prevalence of anti-AβIgG1, which was an indicator of Th2 polarization, was observed in the 6Aβ15-T-immunized mice.Additionally, the Al adjuvant induced a high-er level of IgG1 antibody titers, and the ratio of IgG1/IgG2a was the largest.As expected, the 6Aβ15-T antigen formulated with or without adjuvants induced PADRE-specific, but not Aβ42-specific T cellular immune response .Conclusion The 6Aβ15-T antigens formulated with different types of adjuvants could induce strong Th 2-polarized Aβ42-specific antibody re-sponses without activating self-reactive Aβ42-specific T cells in two strains of mice .The results suggested that the recombi-nant chimeric antigen 6Aβ15-T is a good candidate vaccine for AD .

Lack of proper study models has brought difficulties in the study of the mechanism of viral infection, life cycle and pathogenic mechanism of hepatitis C virus (HCV) and also become the major obstacles in development of efficient vaccine and new drugs for hepatitis C. In recent years, the establishment of robust HCV cell culture infection system and HCV transgenic animal provide powerful tools for the analysis of host virus interactions, which facilitate the discovery of antiviral drugs and vaccines for this important human pathogen.
Animals ; Animals, Genetically Modified ; virology ; Disease Models, Animal ; Genotype ; Hepacivirus ; genetics ; Hepatitis C ; virology ; Mice ; Mice, Transgenic ; virology

BACKGROUNDP-glycoprotein (P-gp) encoded by ATP-binding cassette sub-family B member 1 (ABCB1) gene is a kind of ATP-dependent drug transporter, which plays important roles in multidrug resistance (MDR) of human cancers, such as osteosarcoma. Curcumin is a natural phenolic coloring compound originating from the rhizomes of Curcuma longa, which is proved to possess antitumor biological activities including reversion of MDR. However, the effect and molecular mechanisms of curcumin to osteosarcoma MDR remain unclear.

METHODSWe established a human osteosarcoma drug-resistant cell line MNNG/HOS/MTX by pulse exposure to methotrexate (MTX) and verified that the new cell lines were cross-resistant to other anticancer agents. Then, according to the cytotoxicity assay, we reversed MDR of MNNG/HOS/MTX by 30 µmol/L curcumin, and detected the mechanisms of curcumin reversing MDR through Real-time PCR, Western blotting assay, and Rhodamine123 (Rh123) transport test. Finally, we evaluated the effect of curcumin reversing MDR in vivo by MNNG/HOS/MTX cells xenograft-nude mice model.

RESULTSMNNG/HOS/MTX was proved to be a human osteosarcoma MDR cell line. MTT tumor chemosensitivity test indicates that 30 µmol/L curcumin attenuates the half maximal inhibitory concentration (IC50) and resistance index (RI) to MTX, diamminedichloroplatinum (DDP), adriamycin (ADM), ifosfamide (IFO), and epirubicin (EPI) in MNNG/HOS/MTX cells (P < 0.05). Real-time PCR and Western blotting assays demonstrated that curcumin down-regulated P-gp expression of MNNG/HOS/MTX cells. Rh123 transport test showed that curcumin inhibited the transport function of P-gp in vitro. In vivo studies showed that curcumin displayed the features of sensitizing antitumor drugs and inhibiting the proliferation, invasion, and metastasis of osteosarcoma MDR cells.

CONCLUSIONDown-regulation of P-gp and inhibition of the function of P-gp efflux pump may contribute to MDR reversion induced by curcumin in vitro and in vivo.

ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; metabolism ; Animals ; Antineoplastic Agents ; therapeutic use ; Cell Line, Tumor ; Curcumin ; Drug Resistance, Multiple ; genetics ; Drug Resistance, Neoplasm ; genetics ; Humans ; Male ; Methotrexate ; therapeutic use ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Osteosarcoma ; drug therapy ; genetics

OBJECTIVETo detect the cell-surface-expressed nucleolin and investigate its tumor suppressing effect on the growth of hepatocellular carcinoma cells.

METHODSTo detect cell-surface-expressed nucleolin in the hepatocellular carcinoma cells by immunofluorescence and flow cytometry. To down-regulate the nucleolin expression level in hepatocellular carcinoma cells by RNA interference. The tumor-suppressing effect of cell-surface nucleolin on hepatocellular carcinoma cells was assessed by MTT and transwell chamber assays.

RESULTSNucleolin was expressed in the nuclei, cytoplasm and on the cell surface of hepatocellular carcinoma cells. ShRNA markedly decreased the nucleolin expression level in the cytoplasm and on the cell surface (P < 0.01), but the nuclear nucleolin remained unchanged. After downregulation of cell-surface nucleolin, MTT assays showed that the cell growth rate of hepatocellular carcinoma cells in the shRNA interference group was significantly inhibited as compared with that in the control group (P < 0.01). The transwell chamber assay showed that the mean transmembrane cell number in the shRNA interference group was significantly lower than that in the control group.

CONCLUSIONThe results of this study show that downregulation of cell-surface nucleolin expression inhibits the growth of hepatocellular carcinoma cells in vitro.

Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cell Membrane ; metabolism ; Cell Movement ; Cell Nucleus ; metabolism ; Cell Proliferation ; Cytoplasm ; metabolism ; Down-Regulation ; Humans ; Liver Neoplasms ; genetics ; metabolism ; pathology ; Phosphoproteins ; metabolism ; RNA Interference ; RNA, Small Interfering ; pharmacology ; RNA-Binding Proteins ; metabolism

OBJECTIVETo observe the pathological and clinical characters of nonalcoholic steatohepatitis (NASH).

METHODSLiver biopsy tissues taken from 97 patients negative for common viral detection with serological test and immunohistochemistry as well as in situ hybridization, were observed by routine light microscopic examination. And the clinical data of patients with NASH was analyzed.

RESULTSAmong the chronic hepatitis patients with unknown etiology, the detection rate of NASH was 15.5% (15/97). The characteristic lesions in NASH patients included macrovesicular steatosis in zone 3 of lobules, hepatocytes ballooning degeneration, lobules diffused with acute and chronic inflammation, and perisinusoidal fibrosis. Grading and staging according to Brunt's method, histological lesions could be accounted for G1S1 in 7 patients, G2S2 in 3 patients, G3S1 in 4 patients and G3S2 in 1 patient. There were 14 patients with mild to moderate elevation of transaminase, 10 with hyperlipemia, 8 with diabetes and 9 with fatty liver by ultrasonography.

CONCLUSIONNonalcoholic steatohepatitis is a common form of unknown etiology chronic liver disease with certain clinic-pathological features.

Adult ; Biopsy ; Fatty Liver ; pathology ; Female ; Humans ; Liver ; pathology ; Male ; Middle Aged

OBJECTIVETo separate and identify cyclopeptides of tubers of Rubia schumanniana.

METHODThe 70% methanol extracts from tubers of Rubia schumanniana were separated and purified by silica gel, RP-18, Sephedax LH-20 and HPLC. Their structures were identified by spectral analysis.

RESULTNine cyclopeptides were separated and identified as RA- II (1), RA-V (2), RA-VIII (3), rubiyunnanin C (4), RA-X (5), RY-II (6), RA- I (7), RA-XIII (8) and RA-XIII-OMe (9), respectively.

CONCLUSIONAll of nine cyclopeptides were separated from R. schumanniana for the first time.

Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Molecular Structure ; Peptides, Cyclic ; analysis ; isolation & purification ; Rubia ; chemistry

The paper is to report the development of a high-performance liquid chromatographic/tandem mass spectrometry (HPLC-MS/MS) method for the determination of icaritin (ICT) in rat plasma. After precipitated with acetonitrile from the plasma, ICT was isolated chromatographically on a Dikma C18 column. The mobile phase consisted of acetonitrile-water-acetic acid (72 : 28 : 1.5, v/v/v). Electrospray ionization (ESI) source was applied and operated in the positive ion mode. Multiple reaction monitoring (MRM) mode with the transitions of m/z 387 --> m/z 313 and m/z 331 --> m/z 315 were used to quantify ICT and the internal standard, respectively. The linear calibration curve was obtained in the concentration range of 2.5-1,000 ng x mL(-1). The lower limit of quantification was 2.5 ng x mL(-1). The inter- and intra-day precision (RSD) were less than 9.63%, and the accuracy (relative error) was within +/-7.42%. The method was proved to be suitable for the pharmacokinetics of ICT, which offers advantages of high sensitivity and selectivity.
Administration, Oral ; Animals ; Chromatography, High Pressure Liquid ; methods ; Epimedium ; chemistry ; Female ; Flavonoids ; administration & dosage ; blood ; isolation & purification ; pharmacokinetics ; Male ; Plants, Medicinal ; chemistry ; Rats ; Rats, Wistar ; Reproducibility of Results ; Sensitivity and Specificity ; Spectrometry, Mass, Electrospray Ionization ; methods ; Tandem Mass Spectrometry ; methods

OBJECTIVETo investigate the effects of hepatitis C virus (HCV) on transcription regulation genes of host cells by gene chip assays in cultured cells with intact HCV genome.

METHODSHuh-7 hepatoma cells were cultured and infected with in vitro constructed HCV. The total RNAs, proteins and cell culture supernatants of HCV infected cells and control cells were isolated. Proteins and cell culture supernatants were used to detect the HCV replication and protein expression in cell culture system. The HCV protein expression was detected with Western blotting. Released HCV from infected cells was analyzed by real-time fluorescence quantitative PCR. Total RNA was qualified using 10 g/L agarose gel electrophoresis. cRNA was synthesized, fluorescence labeled and purified, then hybridized with Agilent oligo microarray (20173 probes). Differential expression of genes related to transcription in cell culture system was analyzed.

RESULTHCV was positive in cell culture supernatants and HCV protein expression was also positive according to Western blotting results. Eleven up-regulated and 11 down-regulated genes related to transcription were found after Agilent gene chip screening.

CONCLUSIONIntact hepatitis C virus cell culture system provides an useful tool for study on the affects of HCV infection on transcription regulation genes in host cells.

Cell Line, Tumor ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Genome, Viral ; Hepacivirus ; genetics ; growth & development ; Hepatocytes ; virology ; Humans ; Liver Neoplasms ; genetics ; pathology ; virology ; Transcription, Genetic

To identify the active constituents in vitro and blood-absorbed ingredients in vivo from Yin Chen Hao decoction provides scientific evidence for probing its prevention and treatment mechanism on acute liver injury. An ultrahigh performance liquid chromatography quadrupole-time of flight-mass spectrometry (UPLC-QTOF/MS) method was applied for analysis of Yin Chen Hao decoction and the serum samples of mice with con-A induced acute liver injury after preventive oral administration for 14 days (the use of all laboratory animals in this study was approved by the Ethics Committee of the Naval Medical University, 19YF1459400). A total of 90 chemical constituents were identified from Yin Chen Hao decoction, mainly were flavonoids, terpenoids, tannins, quinones. 5 prototype compounds were identified in the serum, including chrysophanol, deoxyrhapontin-8-O-gallate, mussaenosidic acid, herniarin, emodin. The established UPLC-QTOF/MS method could efficiently and sensitively identify the constituents in vitro and blood-absorbed ingredients of Yin Chen Hao decoction, primarily clarify the material basis of its hepatoprotective effect, and provided a scientific basis for the quality marker selection and the pharmacodynamic material basis research on the decoction.