Animals ; Biological Specimen Banks ; utilization ; Humans ; Specimen Handling ; methods ; Tissue Fixation ; methods

Objective:To prepare and purify siRNA targeting a proliferation-inducing ligand targeted(APRIL-siRNA),so as to provxde a basis for studying the role of APRIL in human pancreatic cancer.Methods:pET-22b-APRIL was constructed to express APRIL dsRNA of human pancreatic cancer cell line CFPAC-1 in E.coli and the product was purified by chromatography using CF-11 column.APRIL dsRNA was digested by RNaseⅢto prepare APRIL siRNA,then the reaction mixture was loaded onto a DEAE ion exchange chromatography to remove RNaseⅢfrom oligonucleotides,and size exclusion chromatography was used to purify 21 bp siRNA.The purified APRIL siRNA was used to transfect Chinese hamster ovary(CHO)cells and the expression of APRIL in CHO cells was observed under fluorescence microscope Results:APRIL dsRNA was successfully expressed in E.coli after IPTG induction and was purified by CF-11 column.dsRNA was hydrolyzed with RNaseⅢand was purified by DEAE ion exchange chromatography and size exclusion chromatography.15% nondenaturing PAGE and 12% SDS- PAGE confirmed that RNaseⅢwas removed from oligonucleotides and 21 bp siRNA was purified with size exclusion chromatography.It was also found that APRIL siRNA obviously depressed APRIL expression in CHO cells.Conclusion:We have successfully constructed APRIL siRNA targeting APRIL gene of CFPAC-1 cells with in vitro transcription,which provides a basis for knock-down of APRIL gene in CFPAC-1 cells.

Objective To observe the effects of soybean,selenium and spirulina on hemoglobin(Hb)of rats intoxicated with fluorine and aluminiums.Methods According to body weight,84 SD rats were randomly divided into control group,high aluminum group,high fluorine group,high fluorine-aluminum group,high fluorine-aluminium intoxicated rats strengthened with soybean group,high fluorine-aluminium intoxicated rats strengthened with selenium group and high fluorine-aluminium intoxicated rats strengthened with spirulina group,12 in each group.Rats in the control group and the high aluminum group were fed with feed containing 5.2 mg/kg of fluorine and 6.8 mg/kg of aluminum.In other groups,fluorine wag 106 mg/Kg and aluminum 19.7 mg/kg.Fluorine and aluminum concentration in the drinking water of the control group and the high fluorine group were 0.69 mg/L and 0.20 mg/L,respectively.In other groups' drinking water,these values were 0.69 mg/L and 90.2 mg/L,respectively.Ninety days later,Hb concentration of the whole blood was tested.Results Hb concentration of the control group,the high aluminum group,the high fluorine group,and the high fluorine-aluminum group were (160.8±6.3),(142.2±15.9),(156.1±4.9)and(145.2±6.2)g/L,respectively.Fluorine had an effect on the concentration of Hb(F=29.56,P<0.05).The Hb concentration of the high fluorine-aluminum group,the strengthened with soybean group,the strengthened with selenium group and the strengthened with Spirulina group were(145.2±6.2),(150.7±17.7),(156.8±14.5),(154.5±17.8)g/L,respectively.Though the concentration of Hb had increased,there was no significant difference between the four groups(χ2=3.304,P>0.05).Conclusions High-dose fluorine could cause varied decrease in the concentration of Hb.However,aluminum has neitherantagonistic effect nor synergistic effect on the Hb of fluorotic Rat.Soybean,selenium and Spirulina show a trend to increase fluorotic rat's Hb,but they has no evident antagonistic effect.

The effects of RNAi-mediated gene silencing of LRlG1 on proliferation and invasion of the human glioma cell line U251-MG and the possible mechanisms were explored in this study. The plasmids pGenesil2-LRIG1-shRNA1 and pGenesil2-LRIG1-shRNA2 were transfected into U251-MG glioma cells respectively by using Lipofectamine 2000 and the transfected cells in which the LRIG1 expression was stably suppressed were selected by G418. The cells transfected with negative shRNA served as control. The expression levels of LRIG1 mRNA and protein were measured by qRT-PCR and Western blotting, respectively. The cell cycle was analyzed by flow cytometry. The results showed that LRIG1 mRNA expression was reduced by 70% and 58% and LRIG1 protein expression by 58% and 26% in U251-MG cells transfected with pGenesil2-LRIG1-shRNAl and pGenesil2-LRIG1-shRNA2 relative to the negative shRNA-transfected U251-MG cells. The proliferative capacity of the LRIG1 specific siRNA-transfected cells was stronger than that of control cells. Cell cycle analysis showed that silencing LRIG1 significantly increased the percentage of S phase cells and the proliferation index (P<0.01). Moreover, silencing LRIG1 could promote the invasion of U251-MG cells (P<0.05). These findings suggested that LRIG1-targeting siRNA can exert a dramatically inhibitory effect on RNA transcription and protein expression of LRIG1, and LRIG1 down-regulation could promote the proliferation of U251-MG cells, arrest U251-MG cells in S phase, and enhance the invasion of U251-MG cells.

Objective To evaluate the acute effects of mirtazapine on sleep polysomnographic variables in patients with major depressive disorder (MDD) using polysomnography (PSG). Methods Twenty-five MDD patients took mirtazapine 15 mg an hour before bedtime during the first three days and then 30 mg during the following four days. Polysomnographic and clinical data were collected at baseline and on the 7th day. Results The scores on the Athens Insomnia Scale (AIS,7.92±3.86,t=10.255,P=0.000), the Hamilton Anxiety Rating Scale (HAMA,6.84±5.57,t=6.137, P=0.000) and the Hamilton Depression Rating Scale (HAMD-17,9.80±4.41,t=12.132,P =0.000) decreased rapidly after a 7-day medication. PSG showed mirtazapine administration significantly increased the total sleep time (402.46±80.75,t=-2.990,P=0.006), the sleep efficiency (76.17%±10.65%,t=-2.750,P=0.011), and the slow wave sleep percentages(19.66%±11.43%,t=3.236, P=0.004) and decreased the wake time after sleep onset (80.38±48.02,t=2.972,P =0.007). However, there was no significant difference in the sleep latency, the number of awakening, the rapid eye movemert (REM) sleep latency, the ratio of REM sleep and the frequency of REM sleep episode. Conclusion Mirtazapine as monotherapy in the treatment of MDD has relieved depressive symptoms rapidly and significantly, increased the total sleep time, the sleep efficiency and the slow wave sleep percentages thus to achieve better sleep quality.

OBJECTIVETo clone Helicobacter pylori adhesin (hpaA) gene,to construct the expression vector of the gene and to identify immunogenicity of the fusion protein.

METHODSThe hpaA gene from a clinical isolate Y06 of H.pylori was amplified by high fidelity PCR. The nucleotide sequence of the target DNA amplification fragment was sequenced after T-A cloning. The expression vector pET32a with inserted hpaA gene was constructed. hpaA fusion protein was expressed in E.coli strain BL21DE3 induced by IPTG at different dosages. Western blot using antibody against whole cell of H.pylori as well as immunodiffusion assay using antiserum of rabbit against the fusion protein was applied to determine immunogenicity of the fusion protein.

RESULTSIn comparison with the reported corresponding sequences, the homology of nucleotide sequence of the cloned hpaA gene was from 94.25% approximate, equals 97.32%, while the homology of its putative amino acid sequence was as high as 95.38% approximate, equals 98.46%. The expression output of HpaA fusion protein in pET32a-hpaA-BL21DE3 system was approximately 40% of the total bacterial proteins. HpaA fusion protein was able to combine with antibody against whole cell of H.pylori and induce rabbit to preduce high titer antibody after the animal was immunized with the protein.

CONCLUSIONAn expression system with high efficiency of H.pylori hpaA gene has been established successfully. The expressed HpaA fusion protein with satisfactory immunogenicity and immunoreactivity can be used as antigen in H.pylori vaccine.

Adhesins, Bacterial ; Animals ; Antibodies, Bacterial ; blood ; Bacterial Vaccines ; immunology ; Base Sequence ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Helicobacter pylori ; immunology ; Hemagglutinins ; genetics ; immunology ; Humans ; Polymerase Chain Reaction ; Rabbits ; Recombinant Fusion Proteins ; immunology ; Vaccines, Synthetic ; immunology

Objective To investigate the effects of hemodialysis (HD) and peritoneal dialysis (PD) on the complications and outcomes after renal transplantation. Methods Clinical data of 402 renal transplant recipients maintained on dialysis for more than 3 months were retrospectively studied and divided into 2 groups: HD group(n=303)and PD group(n=99). Among them, 345 recipients were followed up for an average of (30.2±15.2) months. The impact of HD and PD on the acute rejection, delayed graft function (DGF), infection, chronic rejection and the graft and patient survival rates were analyzed. Results The mean dialysis duration was significantly longer in PD group and the hepatitis B infection rate was significantly higher in HD group. There were no signiticant differences between the HD and PD groups in regarding to primary disease for end-stage renal disease, age, gender, blood pressure, hemoglobin, HLA match, hot and cold ischemia time, and hepatitis C vires infection. The incidence of DGF, acute and chronic rejection, and cytomegalovirus and other infections between HD and PD groups were not significantly different. However, the graft loss happened more frequently in hepatatis B patients than that in non hepatitis B patients (19.23% vs 8.86%, P=0.021), and the post-transplant infection ocurred less in non hepatits B patients with PD. The acute rejection episodes were higher in HD patients who received pretransplant dialysis for more than 12 months (P<0.05). The overall recipients survival rates of HD and PD groups were similar (1-year: HD 94.34%, PD 91.25%;5-year: HD 92.83%, PD 90%), and the same as the graft survival rates in HD and PD groups (1-year: HD 93.21%, PD 96.25%;5-year: HD 87.17%, PD 91.25%). Conclusions The influences of PD and HD on the complications after renal transplantaton, 1-year and S-year recipients and graft survival rates are similar, so both HD and PD can be chosen as the pretransplant dialysis modality. As the incidence of acute rejection increases with time in HD, it is better to shorten the time of pretransplant dialysis to decrease the complication.

Reactive oxygen species (ROS) is involved in autoimmune destruction of islet beta cells, which has been proven to be an important underlying pathogenesis for insulin dependent diabetes mellitus (IDDM). Calcitonin gene-related peptide (CGRP) is a widely distributed neuropeptide, which has been found to play an important role in protecting myocytes from ROS. We hypothesized that exogenous CGRP gene administration before the pathogenic stage of insulitis might suppress the production of ROS and provide a hopeful therapeutic intervention for autoimmune diabetes. We performed CGRP gene transfer by injecting naked plasmid directly into skeletal muscles of mice with electroporation enhancement to achieve a continuous expression of CGRP in skeletal muscles, and thereby its secretion into the circulation. The effect of CGRP gene transfer on the pathogenesis of diabetes was studied in autoimmune diabetic mice induced by multiple low dose streptozotocin (MLDS). The CGRP gene therapy decreased morbidity of autoimmune diabetes, and significantly ameliorated hyperglycemia in these mice. CGRP gene transfer inhibited the production of ROS and malondialdehyde (MDA). In addition, it enhanced the activity of catalase (CAT) and superoxide dismutase (SOD) significantly. The data suggest that intramuscular CGRP gene transfer ameliorates autoimmune destruction of islet beta cells, resulting in significant reduction in diabetes incidence of MLDS diabetes mice. CGRP benefits might be mediated at least in part by inhibiting the oxidative stress in islet beta cells of these mice.
Animals ; Calcitonin Gene-Related Peptide ; administration & dosage ; genetics ; therapeutic use ; Diabetes Mellitus, Experimental ; prevention & control ; Diabetes Mellitus, Type 1 ; prevention & control ; Gene Transfer Techniques ; Genetic Therapy ; Injections, Intramuscular ; Islets of Langerhans ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Pancreas ; metabolism ; Reactive Oxygen Species ; metabolism ; Superoxide Dismutase ; metabolism ; Transgenes ; genetics

OBJECTIVETo investigate the value of Diffusion-Weighted Imaging (DWI) in the diagnosis of early stage liver diffuse lesions.

METHODSDiethylnitrosamine (DEN) was used to induce liver lesions in rats. Sequential DWI studies were performed on the livers from 1 to 14 weeks after DEN was administered through drinking water. Comparing studies with a blank control group was set and pathohistological examinations of the livers were performed.

RESULTSNo obvious routine MRI morphological change was found in either group during this period, but DWI demonstrated heterogeneous changes in the test group at the cirrhosis stage. There was no significant alteration of the apparent diffusion coefficient (ADC) value in the control group during this period (P > 0.05). The ADC values of the test group began to decline from the fifth week. Until the tenth week, the ADC value of the test group decreased drastically and when b = 300 s/mm2 statistic, the results showed an obvious difference between the two groups. There were also differences between the ADC values at the 10th, the 9th and the 1st weeks of the test group (P < 0.05). When b = 600 s/mm2 and 1000 s/mm2, significant differences were found after the sixth week between the two groups (P < 0.01). The main pathohistological liver change in the test group during the 1 to 4 week period after DEN was administered was swelling of hepatocytes; during the 5 to 8 week period it was fibrous tissues hyperplasia, and in the 9 to 14 week period it was cirrhotic nodule formation.

CONCLUSIONMR functional DWI could detect liver diffuse lesions earlier than conventional MR imaging. Measurement of ADC value may be of use in early diagnosis of liver diffuse diseases and for monitoring the changes of the lesions.

Animals ; Chemical and Drug Induced Liver Injury ; Diethylnitrosamine ; Diffusion Magnetic Resonance Imaging ; methods ; Liver Diseases ; diagnosis ; pathology ; Male ; Rats ; Rats, Wistar

BACKGROUND AND OBJECTIVEThe level-Ib lymph node metastasis is rare in nasopharyngeal carcinoma (NPC). When and how this level should be irradiated with precise radiotherapy remains controversial. This study evaluated the prevalence and prognostic significance of level-Ib lymphadenopathy on the prognosis of NPC patients.

METHODSFrom January 1990 and December 1999, 933 newly diagnosed patients with NPC treated at Sun Yat-sen University Cancer Center were randomly selected, examined with computed tomography (CT) imagining for evidence of level-Ib lymphadenopathy before treatment. All patients received radical radiotherapy with or without chemotherapy. The relationship between level-Ib lymphadenopathy and post-treatment outcomes including overall survival (OS), locoregional recurrence-free survival (LRFS), and distant metastasis-free survival (DMFS) were analyzed using Kaplan-Meier methods. The Cox proportional hazards regression model was used to adjust for other prognostic factors.

RESULTSOf the 933 patients, 55 (5.9%) were found to have level-Ib lymphadenopathy, which was associated with carotid sheath involvement, oropharynx involvement and levels, and lateral cervical lymph node involvement. In the subgroup with carotid sheath involvement, with multivariate analysis accounting for all previously known prognostic factors, level-Ib lymphadenopathy was still associated with a risk of decreased OS (RR, 2.124; P<0.001), DMFS (RR, 2.168; P<0.001), and LRFS (RR, 1.989; P=0.001).

CONCLUSIONLevel-Ib lymphadenopathy in the patients with carotid sheath involvement is an independent prognostic factor.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Carcinoma, Squamous Cell ; diagnostic imaging ; drug therapy ; pathology ; radiotherapy ; Chemotherapy, Adjuvant ; Child ; Cobalt Radioisotopes ; therapeutic use ; Female ; Humans ; Lymph Nodes ; pathology ; Lymphatic Metastasis ; Male ; Middle Aged ; Nasopharyngeal Neoplasms ; diagnostic imaging ; drug therapy ; pathology ; radiotherapy ; Neck ; pathology ; Neoplasm Metastasis ; Neoplasm Recurrence, Local ; Particle Accelerators ; Pharynx ; pathology ; Prognosis ; Proportional Hazards Models ; Radiography ; Radioisotope Teletherapy ; Retrospective Studies ; Survival Rate ; Young Adult