Objective:To construct GM-CSF and EB virus LMP2A fusion gene recombinant adenovirus and identificate it , using recombinant adenovirus to do immunologic study.Methods:With RT-PCR and Western blot to detect GC 2A gene expression of the recombinant adenovirus , with ELISA to analysis antibodies of vAd-GC2A stimulation in murine body , lactate dehydrogenase assay to detect the mouse cellular immunity effect , thereby the recombinant adenovirus immune effect was initially analysis.T-test method was used to do preliminary analysis and evaluation of the immune effect of recombinant adenovirus .Results:GC2A fragment amplified by PCR, the size was 1 961 bp and it was the expected.The results showed, the recombinant adenovirus vAd-GC2A proteins can be recognized by sera antibody of GM-CSF ( LMP2A); the results of ELISA showed that vAd-GC2A can stimulate the generation of antibodies.EBV-positive tumor cell killing rate (%) by CTL induced recombinant adenovirus was (66.7 ±6.9)%, at the same time the adenovirus type 5 wild strains and PBS were (24.3 ±2.5)%and(32.4 ±3.1)%only(P≤0.05).Conclusion:Fusion gene has been successfully constructed , vAd-GC2A in mice have humoral and cellular immune function.The data suggest that the vAd-GC2A should play a potent role in preventing the positive EB virus tumor.

OBJECTIVE To study the molecular mechanism of cisplatin(DDP)by which HeLa cell growth and proliferation are inhibited. METHODS Cultured HeLa cells were treated with DDP 0.02-75 μmol · L-1 for 24 or 48 h. CCK-8 assay was used to determine the cell proliferation. The wound scratch assay was used to detect the cell migration and invasion. Flow cytometry was used to detect the cell cycle arresting. q-PCR was used to test the expression of metastasis suppressor gene 1 (MTSS1)mRNA. Western blot was used to determine protein levels of MTSS1,phosphorylated-extra?cellular signal-regulated kinase(p-ERK) and phosphorylated-serine-threonine kinase(p-AKT). RESULTS Following the treatment with DDP for 24 or 48 h,the proliferation of HeLa cells was inhibited significantly (P<0.05),the value of the half inhibitory concentration (IC50) of cells was 4.14 and 11.82 μmol · L-1. Migration and invasion activity of HeLa cells were reduced according to the wound scratch assay(P<0.05). Flow cytometry results showed that the cell cycle was arrested at S phase. q-PCR results showed that MTSS1 mRNA expression changed with DDP in a concentration-dependent manner (r24 h=-0.965,P<0.01;r48 h=-0.953,P<0.01). Western blot showed that the protein levels of MTSS1,p-ERK and p-AKT expression declined significantly with the increase in DDP concentrations(p-ERK:r24 h=-0.875,P<0.01;r48 h=-0.966,P<0.01. p-AKT:r24 h=-0.831,P<0.01;r48 h=-0.863,P<0.01. MTSS1:r24 h=-0.969,P<0.01;r48 h=-0.988,P<0.01). CONCLUSION DDP treatment inhibits HeLa growth and proliferation by interfering with the MTSS1 expression and disturbing the activation of ERK and AKT signaling pathways.

To investigate the pharmacokinetic characteristics and absolute bioavailability of ginkgolide A (GA), ginkgolide B (GB) and bilobalide (BB) in rats. In this experiment, a high-performance liquid chromatography-tandem mass spectrometry (LC-MS/ MS) method was established to determine the plasma concentrations of GA, GB and BB in rats after rats were administrated with the three drugs through ig and iv respectively. The main pharmacokinetic parameters and absolute bioavailability of three ginkgolide compounds were obtained by using pharmacokinetic software DAS 2. 0. After the inject of GA, GB and BB, the results showed Cmax at (513.9 ± 116.9), (701.3 ± 76.0), (5,255.6 ± 476.8) µg · L(-1) and AUC0.24h of (960.9 ± 268.5), (779.5 ± 140.6), (7,409.3 ± 1,181.1) µg · h · L(-1), respectively; after the oral administration, the results showed Cmax at (522.9 ± 39.9), (146.8 ± 31.6), (2,711.9 ± 588.9) µg · L(-1) and AUC0-24 h of (1,760.4 ± 300.7), (636.6 ± 180.3), (16,651.4 ± 1,306.5) µg · h · L(-1), respectively. The absolute bioavailability of GA, GB and BB in rats was (61.1 ± 10.4)%, (27.2 ± 7.7)%, (56.2 ± 4.4)%, respectively. The method established in this experiment has a good specificity and sensitivity and so can be used to study the pharmacokinetics and absolute bioavailability of GA, GB and BB in rats.
Animals ; Biological Availability ; Chromatography, High Pressure Liquid ; Cyclopentanes ; pharmacokinetics ; Furans ; pharmacokinetics ; Ginkgolides ; pharmacokinetics ; Lactones ; pharmacokinetics ; Male ; Rats ; Rats, Sprague-Dawley ; Tandem Mass Spectrometry

OBJECTIVETo investigate the influence of the rate of morphologically normal sperm (MNS) on the clinical outcomes of conventional in vitro fertilization (IVF) in patients with one retrieved oocyte.

METHODSFrom January 2013 to January 2015, a total of 256 couples with one retrieved oocyte underwent conventional IVF in our center. According to the rate of MNS, the patients were divided into two groups: MNS < 4% (134 cycles) and MNS ≥ 4% (122 cycles). We compared the rates of no transferrable embryo cycles, fertilization, cleavage, normal fertilization, abnormal fertilization, high-quality embryo and transferrable embryo between the two groups. A total of 75 fresh embryo transfer cycles were performed, 43 in the MNS < 4% group and the other 32 in the MNS ≥ 4% group. We also compared the rates of implantation, clinical pregnancy and abortion between the two groups.

RESULTSThere were no statistically significant differences between the two groups in the rates of no transferrable embryo cycles, fertilization, cleavage, normal fertilization, abnormal fertilization, high-quality embryo and transferrable embryo (P > 0.05). The rates of implantation, clinical pregnancy and abortion exhibited no remarkable differences either in the fresh embryo transfer cycles between the two groups (P > 0.05).

CONCLUSIONThe rate of MNS does not affect the clinical outcomes of conventional IVF in patients with one retrieved oocyte.

Abortion, Spontaneous ; Cleavage Stage, Ovum ; Embryo Implantation ; Female ; Fertilization ; Fertilization in Vitro ; methods ; statistics & numerical data ; Humans ; Male ; Oocyte Retrieval ; Pregnancy ; Pregnancy Rate ; Single Embryo Transfer ; statistics & numerical data ; Sperm Count ; Spermatozoa ; physiology

OBJECTIVETo develop a therapeutic vaccine against human tumors associated with human papillomavirus type 16E6E7 (HPV16E6E7) which is modified from a Chinese patient of the cervical cancer which possessing the antigenicity and no transforming activity, and explore more active vaccine for inducing cellular immunity with mouse co-stimulatory molecular B7-1 gene.

METHODSThe modified E6E7 gene expression plasmid pVR1012-fmE6E7 was constructed and transfected Cos-7 cells, and the E7 protein specific expression was testified by immunofluorescence assay. C57BL/6 mice were immunized intramuscularly with pVR1012-fmE6E7 alone or in combination with B7-1 gene expression plasmid (pcDNA3.1-B7-1). The activity of cytotoxic T lymphocytes (CTLs) was analyzed with 51Cr specific release assay and the specific antibody in sera was analyzed by indirect ELISA. HPV16 positive C57BL/6 tumor cells C3 were inoculated subcutaneously in the vaccinated mice to assay the growth of transplanted tumors.

RESULTSThe specific CTLs and antibody from immunized mice were induced efficaciously by the E6E7 gene immunization, and co-administration of B7-1 gene could significantly enhanced the CTLs immune responses of fmE6E7, and protected 33% immunized mice against C3 tumor cells challenge. In contrast, all the mice immunized only with fmE6E7 gene developed transplanted tumors after C3 cells challenge. There was no difference in E7 specific antibody responses between mice immunized with the E6E7 gene only and co-administration with B7-1 gene.

CONCLUSIONSThe modified E6E7 gene can be used as target gene for developing DNA vaccine, and B7-1 gene may represent an attractive adjuvant for enhancement of the specific cellular immune responses.

Animals ; Antibodies, Neoplasm ; immunology ; B7-1 Antigen ; genetics ; immunology ; Female ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Oncogene Proteins, Viral ; genetics ; immunology ; Papillomavirus E7 Proteins ; Repressor Proteins ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection ; Uterine Cervical Neoplasms ; immunology ; pathology ; virology ; Vaccines, DNA ; immunology

OBJECTIVETo perform a Meta-analysis on peginterferon with interferon in treatment of HIV patients coinfected with refractory genotype HCV.

METHODSA literature search of Medline was conducted to identify eligible randomized controlled trials. Meta analysis was conducted to evaluate peginterferon and interferon in treatment of coinfected HCV genotype 1 or 4 in HIV patients.

RESULTSix trials of 88 matched the selection criteria. Total 1,131 patients with coinfection of HCV genotype 1 or 4 and HIV were included. Sustain viral response was higher in patients treated with peginterferon plus ribavirin compared with that of interferon plus ribavirin (26 % compared with 8 %) or peginterferon alone (26 % compared with 13 %). Severe adverse effects and withdrawal rates were similar for patients treated with peginterferon and patients treated with interferon.

CONCLUSIONPeginterferon plus ribavirin in treatment of patients with coinfection of genotype 1 or 4 HCV and HIV can achieve higher sustain viral response and the likelihoods of serious adverse effects and withdrawal rates are similar to other therapies.

Adult ; Antiviral Agents ; administration & dosage ; Drug Therapy, Combination ; Female ; Genotype ; HIV Infections ; complications ; drug therapy ; immunology ; Hepacivirus ; classification ; genetics ; Hepatitis C, Chronic ; complications ; drug therapy ; virology ; Humans ; Interferon-alpha ; administration & dosage ; Male ; Polyethylene Glycols ; administration & dosage ; Randomized Controlled Trials as Topic ; Recombinant Proteins ; Ribavirin ; administration & dosage

OBJECTIVETo investigate the effect of early intervention by pravastatin with two different dosage on inflammatory factors and endothelial vasodilator function in patients with unstable angina (UA).

METHODS108 patients with UA were investigated consecutively and divided randomly into three groups (group 20 mg, n = 37; group 10 mg, n = 37; group control, n = 34). Blood samples were examined at admission and 4, 8 weeks after the therapy of pravastatin. Fourty patients of UA were chosen from those three groups (15, 15 and 10 cases respectively). The endothelium-dependent vasodilation and the function of vascular endothelium of them were measured. In the dosage of 20 mg pravastatin group non-endothelium-dependent vasodilation in brachial artery was also tested by ultrasound before and 8 weeks after the therapy. Cardiac events were followed up for 2 months.

RESULTS(1) The use of pravastatin in early admission period of UA could significantly reduce inflammatory factors and improve vascular endothelium function, which was more obviously in the group of 20 mg/d than in group of 10 mg/d. These benefits occurred in 4th week and more obviously in 8th week after the therapy. (2) The lipid lowering therapy in the early stage of admission (24 - 48 h) resulted in the reduction of cardiac events in the hospital.

CONCLUSIONThe use of pravastatin 20 mg/d seems better than that of 10 mg/d in all the fields as above in early admission period of UA patients.

Adult ; Aged ; Angina, Unstable ; drug therapy ; Anticholesteremic Agents ; administration & dosage ; therapeutic use ; Female ; Follow-Up Studies ; Humans ; Inpatients ; Male ; Middle Aged ; Pravastatin ; administration & dosage ; therapeutic use ; Prospective Studies

OBJECTIVETo compare the tubulogenesis capability of malignant glioma-derived microvessel endothelial cells (GDMEC) from human brain with that of ECV304 cells in a three dimentional model and to explore the significance of GDMEC in the study on angiogenesis.

METHODSThe GDMEC were isolated from malignant gliomas of human brain and purified by selective binding to the monoclonal antibody against CD105 bound to the magnetic MACS MicroBeads. GDMEC and endothelial-like cell line ECV304 were compared with their capabilities of formatting tubule-like structure (TLS) in the three dimentional collagen matrix, with or without inducement by various concentration of vascular endothelial growth factor (VEGF).

RESULTSThe obtained GDMEC had a high purification (98%) and could be successfully cultured in vitro. GDMECs formed more TLS than ECV304 cells of the same number and at the same time points. VEGF could induce rapid formation of TLS in a dose-dependent manner, however, ECV304 cells were less response to VEGF stimulation.

CONCLUSIONSGDMEC could maintain their endothelial characteristics and potential capability of angiogenesis. They were more response to VEGF than ECV304, therefore, more suitable for in vitro studies on tumor angiogenesis.

Brain Neoplasms ; blood supply ; Cells, Cultured ; Dose-Response Relationship, Drug ; Endothelial Cells ; drug effects ; Endothelium, Vascular ; cytology ; Glioma ; blood supply ; Humans ; Immunomagnetic Separation ; Microcirculation ; pathology ; Neovascularization, Pathologic ; Vascular Endothelial Growth Factors ; administration & dosage ; pharmacology

Objective To study the effect of OK-432 in combination with IL-2 on Lewis lung cancer (LLC) growth and bcl-2 expression in C57BL/6 mice. Methods Forty C57BL/6 mice bearing LLC were divided into 4 groups and injected with 0.9% NaCl solution, OK-432, IL-2 and OK-432/IL-2. The inhibition of tumor growth after the drug administration and the expression of bcl-2 in paraffin-embedded primary tumor tissues were observed with SABC. Results The growth of LLC in the group with combined treatment was significantly inhibited. ( with inhibition rate of 60%, P < 0.05). The expression of bcl-2 in this group was significantly lowered compared with control group (P < 0.05). Conclusions OK-432 and IL-2 has distinct synergetic anti-tumor effect, possibly by attacking the tumor cells directly or indirectly, and the improved interaction of cells due to inhibition of bcl-2 gene expression and induction of tumor cell apoptosis may also contribute to this effect.

OBJECTIVE We have recently reported that cysteinyl leukotriene (CysLT) signaling plays an important role in microglial interleukin (IL)-1β secretion and subsequent neurotoxicity. The present study aimed to examine microglial morphological changes and the upstream molecular underlying IL-1βproduction in CysLT receptor agonist leukotriene D4 (LTD4)-treated BV2 microglia in vitro. METHODS Twenty-four hours after murine microglial BV2 cells were stimulated with LTD4 (1-100 nmol·L- 1), the cell proliferation and morphology were observed. The expression level of cysteinyl aspartate-specific protease 1 (CASP1) protein was measured by Western blotin BV2 cells. In addition, BV2 cells were pretreated with or without CysLT1 receptor antagonist montelukast for 1 h and the effects of monte-lukaston LTD4-stimulated microglial activation and CASP1 expression were evaluated. RESULTS The number of BV2 cells had an increasing tendency after 24 h treatment with LTD4, but no significant differences were observed between the control and LTD4-treated cells (P>0.05). Under basal and resting conditions, BV2 microglial cells displayed a ramified morphology. However, LTD4 at 100 nmool · L- 1 drove microglial morphological changes from a ramified towards an amoeboid shape. The expression of CASP1 protein was significantly upregulated in 100 nmool·L-1 LTD4-treated BV2 microglia (P<0.01). Furthermore, pretreatment with CysLT1 receptor antagonist montelukast prevented cell morphological changes and suppressed the increased CASP1 expression in LTD4-treated BV2 cells (P<0.05). CONCLUSION CysLT receptor agonist LTD4 induces morphological changes and CASP1 expressionin BV2 microglia, which can be inhibited by CysLT1 antagonist. These results suggest the involvement of CysLT signaling in microglial morphological changes and CASP1 expression.