<Emphasis Type="Italic">In vitro</Emphasis> anti-hepatitis B virus effect of <Emphasis Type="Italic">Hypericum perforatum L</Emphasis>.

The anti-hepatitis B virus(HBV)effects and its mechanisms of the ethanol extracts of Hypericum perforatum L.(EHP)in vitro were explored.HepG2 2.2.15 cells,a stable HBV-producing cell line,were cultured as the model system to observe the anti-HBV effect.The viral antigens of cellular secretion,HBsAg and HBeAg,were determined by enzyme linked immunosorbent assay(ELISA).The quantity of HBV-DNA released in the supernatant was assayed by real-time PCR.In order to understand the mechanisms of the suppression of H...     

Fluoroquinolone resistance and mutation patterns in <Emphasis Type="Italic">gyrA</Emphasis> and <Emphasis Type="Italic">parC</Emphasis> genes in <Emphasis Type="Italic">Neisseria gonorrhoeae</Emphasis> isolates from Shanghai,China

In order to study the resistance of Neisseria （N.） gonorrhoeae to the fluoroquinolone and detect mutation patterns of quinolone resistance-determining regions （QRDRs） of clinical isolates in Shanghai, China, a total of 80 clinical isolates of N. gonorrhoeae were consecutively collected from Shanghai. The MIC of fluoroquinolone for the isolates was examined by using the agar dilution method and the mutation profiles of the QRDRs of gyrA and parC were analyzed by sequencing and restriction fragment length polymorphism （RFLP）. Chi-square test was used for comparison of the t：nutation patterns. The results showed that： （1） High percentages of the 8 isolates were resistant to ciprofloxacin （95.0%）, ofloxacin （95.0%） and lomefloxacin （97.5%）, only one strain was susceptible to the ciprofloxacin. （2） Sensitive strains had a substitute of Asp95→Ala in the gyrA, and all isolates that were resistant or intermediated to the ciprofloxacin, had a double mutation in the gyrA （Ser91, Ala 92 and Asp95）. Some strains also had a mutation in the parC. （3） The MICs of these isolates were significantly associated with the mutation patterns in the gyrA and parC. A double mutation of gyrA combined with parC87 mutation was a predominant pattern in Shanghai and could mediate high level resistance to ciprofloxacin. It suggests that mutations in the QRDRs of gyrA and parC may be responsible for the fluoroquinolone resistance. And fluoroquinolone could not be used as the first line antibiotics for gonorrhea treatment any more in Shanghai, China.     

<Emphasis Type="Italic">In vitro</Emphasis> study on human trophoblast cells infected with HCMV

Human trophoblast cells were isolated and cultured in vitro in order to investigate possible pathogenesis of intrauterine infection caused by HCMV.Trophoblast cells were obtained by compound enzymes digestion and discontinuous percoll gradient.Cells and purity were identified by using immunocytochemistry assay with anti-CK7,Vim and β-hCG antibodies.HCMV AD169 strain replication in isolated trophoblast cells and cell apoptosis were detected at different time points post infection(p.i.).The results showed tha...     

Reversal of multi-drug resistance by vector-based-ShRNA-Mdr1 <Emphasis Type="Italic">In Vitro</Emphasis> and <Emphasis Type="Italic">In Vivo</Emphasis>

In order to investigate the effects of vector-based hairpin small interference RNA （shRNA） on the reversal of multi-drug resistance （mdr） of A2780/Taxol cells, a novel vector pEGFP-HI/mdrl containing mdrl-shRNA targeting at position 2943-2963 of mdrl was designed and synthesized. Subsequently, A2780/Taxol cells were transfected with pEGFP-H1/rndrl, and the expression ofmdrl mRNA and P-gp was detected by using RT-PCR and Western blot respectively. MTT was used to measure the 50% inhibition concentration （IC50） of Taxol to A2780/Taxol cells. The results showed that at the 24th and 48th h after transfection, the expression of mdrl mRNA was decreased to （52.1±1.0）% and （0.01±1.7）%, and that of P-gp decreased to （88.3±2.1）% and 0%, respectively. At the 48th h after transfection, the relative reversal rate of A2780/Taxol cells to Taxol was 69.54%. In vivo, the nude mice xenografts were injected with pEGFP-H1/mdrl, and then administrated Taxol. The tumor volume in pEGFP-H1/mdrl-transfected group was significantly reduced as compared with that in blank control group or pEGFP-Hl-transfected group （807.20±103.16 vs 1563.78±210.54 or 1480.78±241.24 mm^3, both P〈0.01）. These results suggested that transfection of pEGFP-HI/mdrl could efficiently down-regulate the expression of mdrl mRNA and P-gp in A2780/Taxol cells, and effectively restore the sensitivity of A2780/Taxol ceils to Taxol both in vitro and in vivo.     

<Emphasis Type="Italic">cagA</Emphasis> and <Emphasis Type="Italic">vacA</Emphasis> status and influence of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> infection on serum oxidative DNA damage in Iranian patients with peptic ulcer disease

Background  

Infection with Helicobacter pylori strains may result in different pathological manifestations and increased oxidative stress leading to a strong inflammatory response in gastric mucosa.     

A novel sequence-based <Emphasis Type="Italic">coa</Emphasis> genotyping method to discriminate nosocomial methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolates

Aim  

To evaluate a novel sequence-based coa typing method in classification for nosocomial methicillin-resistant Staphylococcus aureus.     

The role of brain-derived neurotrophic factor in mouse oocyte maturation <Emphasis Type="Italic">in vitro</Emphasis>

Brain-derived neurotrophic factor (BDNF) can promote developmental competence in mammalian oocytes during in vitro maturation (IVM),but the role of BDNF in oocyte maturation at cellular level is not still clear.In this study,mouse cumulus-enclosed oocytes subjected to IVM were fertilized and cultured to blastocyst stage.Meiotic spindle configuration and cortical granules distribution during oocyte maturation in vitro were assessed by using immunofluorescence and laser confocal microscopy.The results showed that BDNF contributed to the complete preimplantation development of mouse oocytes compared to the control oocytes (13.78% vs.5.92%;P<0.05).Further,BDNF did not accelerate nuclear maturation of IVM oocytes.For the BDNF-treated oocytes at meiosis Ⅰ,Meiotic spindle areas were significantly smaller and the number of cytoplasmic microtubule organizing centers was greater than that in the control,and the percentages of oocytes showed spindles positioned near the oolemma and a well-formed cortical granule-free domain were significantly higher than that of the control.These morphological characteristics of the BDNF-treated oocytes were much closer to the oocytes matured in vivo than those of the control oocytes.In conclusion,BDNF can promote the developmental competence of mouse IVM oocytes,by improving the meiotic spindle configuration and location and cortical granules distribution at meiosis Ⅰ.     

Patients with cervical <Emphasis Type="Italic">Ureaplasma Urealyticum</Emphasis> and <Emphasis Type="Italic">Chlamydia Trachomatis</Emphasis> infection undergoing IVF/ICSI-ET: The need for new paradigm

Genital tract infections with ureaplasma urealyticum (UU) and chlamydia trachomatis (CT) are the most frequent sexually-transmitted disease worldwide. UU and CT infections are considered to be the leading cause for infertility and adverse pregnancy outcomes. However, little is known about the specific effect of cervical UU and CT infections on the etiology of female infertility, as well as the pregnancy outcomes of the patients undergoing in vitro fertilization/intracytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET). In order to find the association between cervical UU and/or CT infection and pregnancy outcomes, we conducted a retrospective case-control study on the patients undergoing IVF/ICSI-ET with cervical UU and/or CT infection. A total of 2208 patients who received IVF/ICSI-ET were enrolled in this study. Data on the general conditions, pregnancy history and clinical pregnant outcomes were analyzed in terms of the cervical UU and CT detection. Our results revealed that cervical UU and CT infections were the risk factors for ectopic pregnancy and tubal factor-induced infertility. Moreover, the pregnancy rate, abortion rate, ectopic pregnancy rate and premature birth rate in patients with UU and/or CT infections showed no significant difference when compared with the control group. We recommend that cervical UU and CT detection should be an optional item for infertility patients and clinical UU detection should differentiate the subtypes of cervical UU. Positive cervical UU and CT infections should not be taken as strict contraindications for IVF/ICSI-ET.     

Influence of silencing TRAF6 with shRNA on LPS/TLR4 signaling <Emphasis Type="Italic">in vitro</Emphasis>

This study investigated the influence of silencing TRAF6 with shRNA on lipopolysaccharide(LPS)/toll-like receptor(TLR)-4 signaling pathway in vitro.Four plasmids(pGCsi-TRAF6-shRNA1,2,3,4) containing different shRNA sequences were designed and synthesized.The proliferation of RAW264.7 cells after transfected with these plasmids was measured by MTT assay.Inflammatory cellular models were established by LPS stimulation.Levels of TNF-α,IL-1β and TGF-β1 in the supernatants,mRNA expressions of TRAF6,IL-6 and COX-...     

Incidence and significance of <Emphasis Type="Italic">FLT3</Emphasis>-ITD and <Emphasis Type="Italic">NPM1</Emphasis> mutations in patients with normal karyotype acute myeloid leukaemia

Background  

Acute myeloid leukaemia (AML) is a heterogeneous clonal disorder of haematopoietic progenitor cells. Approximately half of all adult AML patients have a normal karyotype (NK-AML) and an intermediate risk prognosis.     

Effect of VEGF on neural differentiation of human embryonic stem cells <Emphasis Type="Italic">in vitro</Emphasis>

The effects of vascular endothelial growth factor （VEGF） on neural differentiation of human embryonic stem cells （hESCs） in vitro and the possible mechanism were observed. The hESCs lines, TJMU1 and TJMU2, were established and stored by our laboratory, hESCs differentiated into neuronal cells through embryonic body formation. In this induction process, hESCs were divided into three groups： group A, routine induction; group B, routine induction＋10 ng/mL VEGF; group C, routine in- duction＋10 ng/mL VEGF＋10 ng/mL VEGFR2/Fc. OCT4, Nestin and GFAP in each group were de- tected by RT-PCR, and the cells expressing Nestin and GFAP were counted by immunofluorescence. The percentage of Nestin positive cells in group B was significantly higher than in groups A and C, while the percentage of GFAP positive cells in group B was significantly lower than in groups A and C （P〈0.01）. There was no significant difference between groups A and C （P〉0.05）. It was concluded that VEGF, via VEGFR2, stimulated the neural differentiation of hESCs in vitro.     

Antitumor effect of betulinic acid on human acute leukemia K562 cells <Emphasis Type="Italic">in vitro</Emphasis>

The effects of betulinic acid (BA), a pentacyclic lupane-type triterpene, on the cell viability, cell cycle and apoptosis in human leukemia K562 cells were investigated. The effects of BA on the growth of K562 cells were studied by MTT assay. Apoptosis was assayed through Annexin V/propidium iodide (PI) double-labeled cytometry. The effects of BA on the cell cycle of K562 cells were studied by a PI method. The expression of Bax and capase-3 was detected by using Western blot. The results showed that BA was ...     

Triptolide-induced apoptosis by inactivating nuclear factor-kappa B apoptotic pathway in multiple myeloma <Emphasis Type="Italic">in vitro</Emphasis>

The effect of triptolide on proliferation and apoptosis of human multiple myeloma RPMI-8226 cells in vitro,as well as the roles of nuclear factor-kappa B(NF-κB) and IκBα was investigated.The effect of tritptolide on the growth of RPMI-8226 cells was studied by MTT assay.Apoptosis was detected by Hoechest 33258 staining and Annexin V/PI double staining assay.The expression of NF-κB and IκBα was observed by Western blot and confocal microscopy.The results showed that triptolide inactivated NF-κB apoptotic pathway in human multiple myeloma RPMI-8226 cells.Triptolide at nM range induced proliferation inhibition in a dose-and time-dependent manner and apoptosis in a dose-dependent fashion in RPMI-8226 cells.Besides,we observed the inhibition of NF-κB /p65 in the nuclear fraction was correlated with the increase in the protein expression of IκBα in the cytosol.These results suggested that triptolide might exhibit its strong anti-tumor effects via inactivation of NF-κB/p65 and IκBα.     

Application of decellularized scaffold combined with loaded nanoparticles for heart valve tissue engineering <Emphasis Type="Italic">in vitro</Emphasis>

The purpose of this study was to fabricate decelluarized valve scaffold modified with polyethylene glycol nanoparticles loaded with transforming growth factor-β1(TGF-β1),by which to improve the extracellular matrix microenvironment for heart valve tissue engineering in vitro.Polyethylene glycol nanoparticles were obtained by an emulsion-crosslinking method,and their morphology was observed under a scanning electron microscope.Decelluarized valve scaffolds,prepared by using trypsinase and TritonX-100,were modified with nanoparticles by carbodiimide,and then TGF-β1 was loaded into them by adsorption.The TGF-β1 delivery of the fabricated scaffold was measured by asing enzyme-linked immunosorbent assay.Whether unseeded or reseeded with myofibroblast from rats,the morphologic,biochemical and biomechanical characteristics of hybrid scaffolds were tested and compared with decelluarized scaffolds under the same conditions.The enzyme-linked immunosorbent assay revealed a typical delivery of nanoparticles.The morphologic observations and biological data analysis indicated that fabricated scaffolds possessed advantageous biocompatibility and biomechanical property beyond decelluarized scaffolds.Altogether this study proved that it was feasible to fabricate the hybrid scaffold and effective to improve extracellular matrix microenvironment,which is beneficial for an application in heart valve tissue engineering.