60,≤60 year age groups and control group was 2.49?1.82,1.82?1.58,0.90?1.38, significantly decreased(P

Objective To determine whether sulfasalazine stimulates hepatic stellate cell (HSC-T6) apoptosis and its possible mechanism. Methods CCK-8 assay, acridine orange/ethidium bromide(AO/EB) and Annexin Ⅴ FITC/PI were used to determine cell growth and cell apoptosis. The expression of NF-?B P65, phospho-IKK and phospho-I?B was detected by Western blotting. The nuclear translocation of HSC-T6 P65 was observed with laser confocal microscopy. Results Sulfasalazine displayed a strong growth inhibition and promoting apoptosis effect on HSC-T6 cells in a dose and time-dependent manner. Sulfasalazine, but not 5-aminosalicylic acid or sulfapyridine, inhibited the activation of NF-?B by down-regulating the expressions of P-IKK, P-I?B and the nuclear translocation of P65. Conclusion Sulfasalazine can inhibit NF-?B activity and promote apoptosis in HSC-T6 cells, where the Rel/NF-?B/I?B/IKK pathway plays an important role in HSC survival.

Objective To establish a new assay for detecting autoantibody Jo-1, cloning and expressing human autoantigen Jo-1 in E.coli.Methods A full length cDNA of human autoantigen Jo-1 was cloned from cell line HL-60 by RT-PCR. The PCR product was TA cloned, sequenced and inserted into the carrier pGEX-5T.The recombinant plasmid was transformed into E.coli BL-21. The positive clones were identified by restricted enzymes and induced by IPTG. The expression product was analyzed by SDS-PAGE and Western blot.Results The PCR product was about 1 500 bp in size which was in accordance with predicted 1 526 bp and sequencing result showed the same with GenBank′s report.The pGEX-5T- Jo-1 positive clone produced a 75 000 fusion protein which had natural immunogenicity of human autoantigen Jo-1 by SDS-PAGE and Western blot.Conclusion Successfully cloning and expression of human autoantigen Jo-1 laid a foundation for further research work.

Objective To clone,express and identify the nuclear antigen Sm B′in E. coli to establish a new assay for detecting autoanti-body to Sm B′. Methods A full length cDNA of Sm B′was cloned from cell line HL-60 by RT-PCR. The PCR product was TA cloned and sequenced and inserted into the vector pGEX-5T. The recombinant plasmid was transformed into E. coli BL21. The positive clones were identified by restricted enzymes and induced by IPTG. The expression product was analyzed by SDS-PAGE and Western blot. Results The PCR product was about 700 bp in size which was in accordance with predicted 657 bp and sequencing result showed consistent with the sequence in GenBank. The pGEX-5T-Sm B′positive clone produced a 51 000 kD of fusion protein which was immunoreac-tive with anti-Sin B′confirmed by SDS-PAGE and Western blot. Conclusion The successful cloning and expression of nuclear antigen Sm B′laid a foundation for further research work.

Objective To clone and express human autoantigen Sm B'in methylotrophie yeast Pichia Pagtoris.Methods The gene Sm B' was cloned bv PCR The PCR product wag inserted into the vector pPIC9k.The recombinant plasmid pPIC9k.Sm B' was transformed into yeast Sm D1168 by electroporation.The positive clones were screened in MD plates.The high copy number transformants were rapidly selected by using G418 and were induced by methan01.Supematants after induction were analyzed by SDS-PAGE and western blot.Sera collected from thirty patients with SLE.thirty patients with mixed connective tissue disease(MCTD)and thirty healthy volunteers were detected by immunodot and immunoblot.Results The PCR product wag about 700 bD in size which Wag in accordance with predicted 657 bp.The pPIC9k-Sm B'showed the same seqencing result with GenBank's report and restriction enzyme analysis confirmed our prediction.The pPIC9k-Sm B' positive clone produced a 32 000 protein which had natural immunogenicitv of human autoantigen Sm B'by SDS-PAGE and western blot.The positive rate of immunodot and IBT were 46.7%(42/90)and 51.1%(46/90),respectively.The agreement between immunodot and IBT was very close(Kappa value=0.911 2,P＜0.01).Conclusion Successfully cloning and expression of human autoantigen Sm B' in methylotmphic yeast Pichia Pagtoris hid a foundation for further research work.

Objective To obtain recombinant human pancreatic alpha-amylase protein (AMY2A). Methods Human pancreatic AMY2A cDNA was synthesized by RT-PCR using total RNA from human pancreatic tissues and a couple of primers designed according to the know sequence of human pancreatic alpha-amy lase gene, then digested with BamH Ⅰ and Kpn Ⅰ and inserted into the prokaryotic expression plasmid pGEX-5T vector. Construct The prokaryotic expression vector pGEX-5T-AMY2A was constructed and transformed into E. coli BL21 cell . Protein expressed under the induction of IPTG. The inclusion bodies were isolated and solubilized with urea and washed denatured and refolded. The fusion protein wes purified by affinity chrom atography with glutathione agarose. Results Sequence and restrction analysis revealed AMY2A gene was cloned in frame into pGEX-5T, SDS-PAGE profile showed a clear protein band with a relative molecular weight of 84000 and western blot indicated that the expressed product specifically reacted to polyclonal anti -human pancreatic AMY2A genes. Conclusion Human pancreatic AMY2A gene was successfully cloned,expressed and purification.

Objective To investigate molecular diagnostic method for hereditarymethemoblobinemia. Methods The cDNA coding sequence of NADH-cytochrome b5 reductase (b5R) from 3 patients with hereditary methemoglobinemia was analyzed by direct sequencing of RT-PCR products and the genomic DNA of b5R gene by PCR-restriction endonuclease digestion or PCR-sequencing. Results The b5R cDNA of patient A was T/C heterozygous at nucleotide 527 and G/A heterozygous at nucleotide 608. The b5R cDNA of patient B was G/A heterozygous at both nucleotide 170 and nucleotide 179. The b5R cDNA of patient C was G/A heterozygous at nucleotide 608 and C/T heterozygous at nucleotide 791. Result of genomic DNA analysis was in agreement with that of cDNA approach. Conclusion The method for molecular diagnosis of hereditary methemoglobinemia was established and 3 novel b5R gene mutations were identified in compound heterozygosity in 3 Chinese patients.

An ultra-high performance liquid chromatography-mass spectrometry ( UPLC-MS/MS) method was developed for the determination of 8 kinds of cephalosporins, cefoperazone, cefquinome, cefalonium, cefazolin, cefapirin, Ceftiofur, cefpirome and cefalexin, in edible parts of aquatic products. The samples were extracted with acetonitrile-water and cleaned up by multi-walled carbon nanotubes ( MwCNTs) SPE cartridge. All the target compounds were separated on an Acquity Xselect CSH C18 column with gradient elution by using acetonitrile and 0. 1% formic acid aqueous as eluent, and detected by UPLC-MS/MS under ESI+ ionization and MRM mode. Under optimized conditions, this method had a good linearity (R2≥0. 995) and the limits of quantification were in the range of 2-10 μg/kg ( S/N=10 ) . The recoveries of the method for the target compounds spiked at three different levels were 67. 3%-94. 2% with the relative standard deviations (RSDs) of 3. 3%-14%. The method had the characteristics of low cost, high accuracy and good precision, and could meet the requirements of cephalosporins determination.

A method was developed for the determination of tetrodotoxin in marine organisms by high perfor-mance liquid chromatography-mass spectrometry with immunoaffinity column. The samples were extracted with 1% acetic acid methanol solution and diluted with phosphate buffer at pH 7-8. After cleaned up by immuno-affinity column, the samples were analyzed by LC-MS/MS and quantitatively determined by external standard method. The chromatographic separation was performed on an ACQUITY UPLC BEH Amide column with gradient elution by using acetonitrile and 5 mol/L ammonium acetate solution containing 0. 1% formic acid as mobile phase. Detection was carried out by electrospray positive ionization mass spectrometry in the multiple reaction monitoring mode. Linear ranges of TTX was in the range of 0. 3 -20. 0 μg/L with correlation coeffi-cient more than 0. 997. The quantification limit of the method was 0. 3 μg/kg. The recoveries of standard addition for tetrodotoxin were 88. 7%-102. 3%, and the relative standard deviation was 2. 0%-6. 4%. The method could be used to identify and quantify tetrodotoxin in marine organisms with satisfactory reproducibility and sensitivity.

Objective To explore the effect of fluoxetine on the brain function of bulimia nervosa (BN) patients.Methods Seven female BN patients,who met criteria of the 3rd version Chinese Criteria of Mental Diseases (CCMD-3),accepted functional magnetic resonance imaging (fMRI) examinations before and after the antidepressant treatment (fluoxetine (20 mg/day)) for three months.Seven normal controls accepted the same fMRI examination only at baseline.fMRI imaging was block-design.Blocks of food or non-food stimulus containing pictures selected from International Affective Picture System (IAPS) which were shown by computer automatically.All subjects were evaluated by Hamilton anxiety scale (HAMA),Hamilton depression scale (HAMD17) and Likert Scale-likelihood evaluation to the same pictures in the fMRI imaging blocks.Results The average intensity and volume activated in BN before treatment were both significantly lower than that in the control (P＜0.05).But under stimulus of food pictures,bilateral prefrontal cortex and left amygdala of BN patients were significantly activated.After fluoxetine treatment,the intensity and volume activated both increased significantly (P＜0.01) and the main areas being activated were right temporal,cerebellum and bilateral prefrontal cortex.Conclusion Fluoxetine improves the bulimic symptoms of BN patients and decreases abnormal activation of prefrontal and limbic in these areas.The underline mechanism may be related to functions of serotonin system in prefrontal-limbic path.