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1.
Article in Chinese | WPRIM | ID: wpr-882219

ABSTRACT

Objective:To establish a comet test method for detection of genotoxicity of three reference chemicals in rat liver cells. Methods:6-10 week old Sprague Dawley rats were randomly divided into 4 groups, with normal saline (0.9% NaCl solution) as negative control group. Animals in three test groups were treated, respectively, with ethyl methanesulfonate (EMS) 200 mg/kg, N-methyl-N-nitrosourea (MNU) 50 mg/kg, and D-mannitol 2 000 mg/kg. There were 10 animals in each group, 5 males and 5 females. The animals received two times (21 h interval) of test compounds through intragastric administration, and their clinical symptoms and body weight changes were recorded during the experiment. The rats were sacrificed 3 h after the last exposure. The liver was weighed, then used to prepare single-cell suspensions for the alkaline comet test which determines the average tail DNA content percentage (DNA%) of hepatocytes and other comet indicators. Results:(1) D-mannitol, EMS and MNU did not show significant toxicity in the whole animal. (2) The mean values of tail DNA content percentage (DNA%) of rat hepatocytes in EMS [(60.07±24.69)%] and MNU [(41.66±22.35)%] groups were higher than that in the negative control group [(2.32±1.39)%] and the difference was statistically significant (P<0.05). The difference between D-mannitol group [(3.06±3.30)%] and the negative control group was not significant (P>0.05). Conclusion:This laboratory has established a comet test method using hepatocytes from treated rats. Among three testing chemicals, EMS and MNU have displayed genotoxicity by this assay, but no genotoxicity was observed in D-mannitol treated animals.

2.
Article in English | WPRIM | ID: wpr-773988

ABSTRACT

OBJECTIVE@#To investigate apoptotic effects of berberine, a significant alkaloids component existing in Rhizoma coptidis, and its possible acting mechanism in insulinoma cells.@*METHODS@#Different concentrations of berberine were used to treat mouse insulinoma (MIN6) cells for various period of time. The viability and apoptosis of the cells were analyzed using methylthiazolyldiphenvl-tetrazolium bromide assay, flow cytometry and enzyme-linked immuno sorbent assay. Changes in the relating pro- and anti-apoptosis proteins were detected by western-blotting.@*RESULTS@#The half-maximal inhibitory concentration (IC) of berberine was 5.7 μmol/L on MIN6 cells viability for 16 h. Berberine caused a 20% reduction (P<0.05) in cell number after only 4-h incubation; which reached 50% after 24 h (P<0.01). Berberine treatment for 16 h significantly increased the level of DNA fragmentation. The flow cytometry showed the apoptotic rate increased 2.9- and 4.6-fold after treating with berberine (5 μmol/L) for 8 and 16 h, while 3- and 8.7-fold after 10 μmol/L treatment for 8 and 16 h (P<0.01). Berberine treatment dramatically elevated the expression ratio of Bax to Bcl-2. Meanwhile, berberine notably increased the apoptosis-inducing factors and cytochrome C transforming from the mitochondria to the cytoplasm. Apoptotic protease-activating factor 1 (Apaf-1) was subsequently activated after cytochrome C release. Furthermore, caspase-3 and poly adenosine diphosphate-ribose polymerase were also activated to trigger apoptosis cascade.@*CONCLUSION@#High concentration (5 and 10 μmol/L) of berberine could induce the apoptosis of MIN6 cells through cytochrome C/Apaf-1/caspase-3 and apoptosis inducing factor (AIF) pathway.

3.
Article in English | WPRIM | ID: wpr-281396

ABSTRACT

Objective To construct a model of Seasonal Autoregressive Integrated Moving Average (SARIMA) for forecasting the epidemic of Japanese encephalitis (JE) in Xianyang, Shaanxi, China, and provide valuable reference information for JE control and prevention. Methods Theoretically epidemiologic study was employed in the research process. Monthly incidence data on JE for the period from Jan 2005 to Sep 2014 were obtained from a passive surveillance system at the Center for Diseases Prevention and Control in Xianyang, Shaanxi province. An optimal SARIMA model was developed for JE incidence from 2005 to 2013 with the Box and Jenkins approach. This SARIMA model could predict JE incidence for the year 2014 and 2015. Results SARIMA (1, 1, 1) (2, 1, 1)was considered to be the best model with the lowest Bayesian information criterion, Akaike information criterion, Mean Absolute Error values, the highest R, and a lower Mean Absolute Percent Error. SARIMA (1, 1, 1) (2, 1, 1)was stationary and accurate for predicting JE incidence in Xianyang. The predicted incidence, around 0.3/100 000 from June to August in 2014 with low errors, was higher compared with the actual incidence. Therefore, SARIMA (1, 1, 1) (2, 1, 1)appeared to be reliable and accurate and could be applied to incidence prediction. Conclusions The proposed prediction model could provide clues to early identification of the JE incidence that is increased abnormally (≥0.4/100 000). According to the predicted Results in 2014, the JE incidence in Xianyang will decline slightly and reach its peak from June to August.

4.
Chinese Medical Journal ; (24): 429-435, 2011.
Article in English | WPRIM | ID: wpr-321489

ABSTRACT

<p><b>BACKGROUND</b>Transforming growth factor-β1 (TGF-β1) is known to have a role in keloid formation through the activation of fibroblasts and the acceleration of collagen deposition. The objective of this current study was to isolate TGF-β1 phage model peptides from a phage display 7-mer peptide library to evaluate their therapeutic effect on inhibiting the activity of keloid fibroblasts.</p><p><b>METHODS</b>A phage display 7-mer peptide library was screened using monoclonal anti-human TGF-β1 as the target to obtain specific phages containing ectogenous model peptides similar to TGF-β1. Enzyme-linked immunosorbent assay (ELISA) was performed to select monoclonal phages with good binding activity, which underwent DNA sequencing. MTT assay and apoptosis assessment were used to evaluate the biological effects of the phage model peptides on keloid fibroblasts. Immunofluorescence assay was employed to show the binding affinity of the model peptides on phages causing keloid fibroblasts. Quantitative real-time PCR analysis was carried out to detect the expressions of nuclear factor κB (NF-κB) mRNA, connective tissue growth factor (CTGF) mRNA and TGF-β receptor II (TβRII) mRNA in keloid fibroblasts.</p><p><b>RESULTS</b>Specific phages with good results of ELISA were beneficiated. Four phage model peptides were obtained. The data of MTT showed that TGF-β1 and one phage model peptide (No. 4) could promote keloid fibroblasts proliferation, however, three phage model peptides (No. 1 - 3) could inhibit keloid fibroblasts proliferation. The results of apoptosis assessment showed that the three phage model peptides could slightly induce the apoptosis in keloid fibroblasts. The data of immunofluorescence assay revealed that the model peptides on phages rather than phages could bind to keloid fibroblasts. The findings of quantitative real-time PCR analysis suggested that the expressions of NF-κB mRNA and CTGF mRNA in the three phage model peptide groups decreased, while the expression of TβRII mRNA slightly increased.</p><p><b>CONCLUSIONS</b>Three phage model peptides isolated from a phage display 7-mer peptide library can inhibit keloid fibroblasts proliferation and induce the apoptosis in keloid fibroblasts. They can inhibit the activity of keloid fibroblasts by blocking TGF-β1 binding to its receptor and then regulating the expressions of NF-κB, CTGF and TβRII.</p>


Subject(s)
Apoptosis , Cell Line , Cell Proliferation , Enzyme-Linked Immunosorbent Assay , Fibroblasts , Cell Biology , Fluorescent Antibody Technique , Humans , Peptide Library , Peptides , Allergy and Immunology , Pharmacology , Polymerase Chain Reaction , Transforming Growth Factor beta1 , Allergy and Immunology
5.
Chinese Journal of Hematology ; (12): 536-539, 2010.
Article in Chinese | WPRIM | ID: wpr-353595

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the quantitative and qualitative changes of TCRVα24(+)Vβ11(+) natural killer T (NKT) cells from bone marrow (BM) of aplastic anemia (AA) after in vitro stimulation of α-galactosylceramide (α-Galcer).</p><p><b>METHODS</b>NKT cells in the bone marrow mononuclear cells (BMMNCs) from either AA patients or healthy controls were enumerated with flow cytometry. BMMNCs were cultured in RPMI1640 medium supplemented with either α-Galcer and rhIL-2 or α-Galcer, rhIL-2 and rhG-CSF. The proliferative capacity of NKT cells was determined by NKT cell numbers before and after in vitro culture. Expression of intracellular IFNγ and IL-4 in activated NKT cells was analyzed with flow cytometry.</p><p><b>RESULTS</b>In AA group, the percentage of NKT cells in BMMNCs was (0.19 ± 0.09)%. Addition of rhG-CSF into the α-Galcer/rhIL-2 culture medium resulted in significantly reduced expansion of NKT cells (67.45 ± 29.42-fold vs 79.91 ± 40.56 fold, P < 0.05). Meanwhile, addition of rhG-CSF reduced IFNγ positive NKT cells \[(37.45 ± 7.89)% vs (62.31 ± 14.67)%, P < 0.01\] and increased IL-4 positive NKT cells \[(55.11 ± 12.13)% vs (27.03 ± 9.88)%, P < 0.01\]. In healthy control group, the percentage of NKT cells in BMMNCs was (0.25 ± 0.12)%. Addition of rhG-CSF into the α-Galcer/rhIL-2 culture medium also significantly reduced expansion of NKT cells (97.91 ± 53.22-fold vs 119.58 ± 60.49-fold, P < 0.05), reduced IFNγ positive NKT cells \[(28.65 ± 10.63)% vs (50.87 ± 12.66)%, P < 0.01\], and increased IL-4 positive NKT cells \[(66.53 ± 14.96)% vs (31.11 ± 10.07)%, P < 0.01\].</p><p><b>CONCLUSION</b>Compared to those from healthy controls, BMMNCs from AA patiants have a reduced fraction of NKT cells, which possesses a decreased potential to expand in vitro in response to α-Galcer stimulation, and produce more IFNγ(+) NKT1 cells. rhG-CSF, in combination with α-Galcer, confers polarization of NKT cells towards IL-4(+) NKT2 subpopulation.</p>


Subject(s)
Anemia, Aplastic , Metabolism , Bone Marrow , Metabolism , Humans , Interleukin-4 , Metabolism , Killer Cells, Natural , Cell Biology , Natural Killer T-Cells
6.
Chinese Medical Journal ; (24): 1195-1200, 2010.
Article in English | WPRIM | ID: wpr-352593

ABSTRACT

<p><b>BACKGROUND</b>Keratinocyte growth factor (KGF) significantly influences epithelial wound healing. The aim of this study was to isolate KGF phage model peptides from a phage display 7-mer peptide library to evaluate their effect on promoting epidermal cell proliferation.</p><p><b>METHODS</b>A phage display 7-mer peptide library was screened using monoclonal anti-human KGF antibody as the target. Enzyme linked immunosorbent assay (ELISA) was performed to select monoclonal phages with good binding activity. DNA sequencing was done to find the similarities of model peptides. Three-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, immunofluorescence assay and quantitative real-time PCR analysis were employed to evaluate the effect of the phage model peptides on epidermal cells.</p><p><b>RESULTS</b>Thirty-three out of fifty-eight (56.9%) of the isolated monoclonal phages exhibited high binding activity by ELISA. Ten of fifteen obtained phage model peptides were similar to KGF or epidermal growth factor (EGF). MTT assay data showed that four (No. 1 - 4) of the ten phage model peptides could promote epidermal cell proliferation. The expression of keratinocyte growth factor receptor (KGFR) mRNA in the KGF control group and the two phage model peptide groups (No. 1 and No. 2) increased. Expression of c-Fos mRNA and c-Jun mRNA in the KGF control group increased, but did not increase in the four phage model peptide groups (No.1 - 4).</p><p><b>CONCLUSION</b>Four phage model peptides isolated from the phage display 7-mer peptide library can safely promote epidermal cell proliferation without tumorigenic effect.</p>


Subject(s)
Cell Proliferation , Cells, Cultured , Enzyme-Linked Immunosorbent Assay , Epidermis , Cell Biology , Fibroblast Growth Factor 7 , Chemistry , Pharmacology , Humans , Peptide Library , Peptides , Chemistry , Pharmacology , Polymerase Chain Reaction , Receptor, Fibroblast Growth Factor, Type 2 , Genetics
7.
Article in Chinese | WPRIM | ID: wpr-317894

ABSTRACT

<p><b>OBJECTIVE</b>By means of the detection of the numbers of CD34(+) cells and eosinophils (EOS), and the level of IL-5 in peripheral blood from normal controls and patients with allergic rhinitis pre- or post-treatment, the role of EOS-stem cells paths for treatment effect in allergic rhinitis (AR) was studied so as to find the convenient and quick indicators which could be used to evaluate the clinical therapeutic effect and adjust the methods of the hormone therapy.</p><p><b>METHODS</b>There were 2 groups. (1) experimental group: 44 patients, including 24 males and 20 females with a age range of 7 to 68 years old. The patients received the treatment of fluticasone spurt for four weeks. (2) CONTROL GROUP: 30 cases, from normal health examination. The EOS numbers, CD4(+) cell numbers and the IL-5 level were examined in control group as well as before and after therapy in experimental group.</p><p><b>RESULTS</b>The IL-5 level and CD numbers before therapy in experimental group were (88.25 +/- 33.47) ng/L, (9.24 +/- 2.15)/10(5), significantly higher than that after therapy and experimental group which were (44.34 +/- 16.32) ng/L, (6.31 +/- 1.83)/10(5) and (31.24 +/- 8.43) ng/L, (3.47 +/- 1.32)/10(5) respectively. The IL-5 level was positively correlated with the CD34(+) number pre-and post treatment in experimental group (r = 0.64, P <0.01; (r = 0.61, P <0.01). The EOS number was positively correlated with the level of IL-5 (r = 0.64, P <0.01).</p><p><b>CONCLUSIONS</b>IL-5 and CD34(+) cells can be regarded as indicators to evaluate the therapeutic effect. The EOS, CD34(+) cells and the level of IL-5 in the peripheral blood are correlated with the pathogenesis of AR, suggesting that there is a related path between the local nasal tissue of AR patients and the marrow.</p>


Subject(s)
Adolescent , Adult , Aged , Androstadienes , Therapeutic Uses , Anti-Allergic Agents , Therapeutic Uses , Antigens, CD34 , Metabolism , Bone Marrow Cells , Case-Control Studies , Child , Eosinophils , Allergy and Immunology , Female , Fluticasone , Humans , Interleukin-5 , Blood , Male , Middle Aged , Rhinitis, Allergic, Perennial , Blood , Drug Therapy , Stem Cells , Young Adult
8.
Chinese Journal of Epidemiology ; (12): 1030-1035, 2007.
Article in Chinese | WPRIM | ID: wpr-322846

ABSTRACT

<p><b>OBJECTIVE</b>The purpose of this study was to observe the association between chronic glomerulonephritis (CGN) and human leukocyte antigen (HLA) on DNA level in order to identify susceptible and protective genes and to further explain the possible pathogenesis of CGN.</p><p><b>METHODS</b>1073 renal transplantation patients with Han ethnicity were included in this study. All patients were recruited from three provincial Hospitals during the past ten years. The control group contained 7418 healthy Han volunteer donors from Shandong Hematopoietic Stem Cell Data Bank of China. We collected data about the polymorphism of HLA-I , II and DRB1. Gene frequency (GF), relative risk (RR) and correlation test were analyzed using statistical software. Some patients carrying the susceptible genes were followed up for 1,3 and 5 years, and compared their survival rate respectively.</p><p><b>RESULTS</b>The frequency of HLA-A23, A25,B15, B40, B53 and DRB1 * 18 alleles increased significantly in CGN patients than in controls, showing that they might be the suspicious susceptibility genes of CGN. After the follow-up periods, the prognosis of patients with the susceptible genes was worse than the controls. The frequency of haplotypes of A23-B44-DRB1 * 18, A25-B15-DRB1 * 07, A3-B70-DRB1 * 11, A68-B13-DRB1 * 04, A11-B10-DRB1 * 12 increased significantly in CGN patients than in controls. There were 8 lower frequencies alleles (including A20, A22, A35, A36, A38, B21, B73 and B78) in CGN patients that were not found in the control group. The frequencies of the HLA-A32, A33, B50, B58, B60, B71, DRB1 * 16 alleles decreased significantly in CGN patients than in controls, showing that they might be the protective genes of CGN.</p><p><b>CONCLUSION</b>Our data showed that there might be corresponding susceptibility and protective genes of CGN in Han population, in Shandong. There was significant association between the five common haplotype and CGN in Shandong population. However, the prognosis of the patients with the susceptibility genes was worse than the controls.</p>


Subject(s)
Adolescent , Adult , Aged , Alleles , Case-Control Studies , China , Female , Gene Frequency , Glomerulonephritis , Genetics , HLA-DR Antigens , Genetics , HLA-DRB1 Chains , Haplotypes , Histocompatibility Antigens Class I , Genetics , Histocompatibility Antigens Class II , Genetics , Humans , Male , Middle Aged , Polymorphism, Genetic , Young Adult
9.
Chinese Medical Journal ; (24): 1187-1191, 2007.
Article in English | WPRIM | ID: wpr-240243

ABSTRACT

<p><b>BACKGROUND</b>Gene therapy has been a hot spot in repair of bone defects in recent years. This study aimed to construct a recombinant plasmid pcDNA3.1-VEGF(165), and to observe the effect of vascular endothelial growth factor 165 (VEGF(165)) gene therapy on bone defects in rabbits.</p><p><b>METHODS</b>Total RNA was extracted from rabbit bone tissues. VEGF(165) cDNA fragment was prepared by reverse transcription and the gene was cloned by polymerase chain reaction (PCR). Plasmid pMD18-T/VEGF(165) combined with pcDNA3.1 was cloned to reconstruct pcDNA3.1-VEGF(165) plasmid. Thirty New Zealand white rabbits weighing (2.50 +/- 0.13) kg were used to establish models of bone defects (1 cm in length) of the bilateral radii. The bone defects were repaired with absorbable gelatin sponge. After the operation, physiological sodium chloride solution was injected into the injured site in one of the forelegs of the rabbits as the control group, and pcDNA3.1-VEGF(165) plasmid (0.2 ml, 200 ng) was injected into the opposite foreleg as the experiment groups. At weeks 1, 2, 4, 6, 8, and 12 after the treatments, the bones were examined by X-ray, and the specimens of the bone defects were collected, stained with HE, and observed under a light microscope. The expression of VEGF(165) mRNA was examined by real-time quantitative polymerase chain reaction (RQ-PCR).</p><p><b>RESULTS</b>The pcDNA3.1-VEGF(165) plasmid with a correct sequence was constructed successfully. Postoperative X-ray found no difference between the two groups at week 1. In the experiment group, callus and synostosis were observed after 2 weeks, and osteosis structure was normal at week 12; these phenomena occurred much later in the control group. In the experiment group, HE staining showed a large amount of newly formed blood vessels after 2 weeks, a number of bone trabeculae with osteoblasts proliferation at 4 weeks, and fresh bone cortex and reformed medullary cavity at 12 weeks; whereas in the control group these structures formed in later phases. The VEGF(165) mRNA in the experiment group was expressed at a low level at week 1, reached the peak at weeks 3, and then decreased to a normal level after 6 weeks.</p><p><b>CONCLUSIONS</b>Local use of pcDNA3.1-VEGF(165) plasmid at bone defects can upregulate the expression of VEGF(165) and accelerate the formation of capillaries and the repair of bone defects. Angiogenesis and osteogenesis can be promoted by a combination of pcDNA3.1-VEGF(165) and gelatin sponge.</p>


Subject(s)
Animals , Bone Diseases , Diagnostic Imaging , Therapeutics , Genetic Therapy , RNA, Messenger , Rabbits , Radiography , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Vascular Endothelial Growth Factor A , Genetics
10.
Article in Chinese | WPRIM | ID: wpr-297115

ABSTRACT

<p><b>OBJECTIVE</b>To examine the effect of pcDNA3.1-VEGF165 vector to the angiogoiesis, expression of collagen type I and type III mRNA in soft tissue injury model.</p><p><b>METHODS</b>Thirty two Sprague-Daulay rats,weighted (180 +/- 20) g, were made tissue injury in the bilateral of vertebral region. Round wound (diameter 12 mm) was made by perforex on the back, removed the skin and 2 mm muscle, one side was experimental group by random and the other as control. The wound was done with sodium chloride (0.2 ml) in the control group, with the recombinant VEGF165 vector (0.2 ml, 200 mg) in the experimental group. The wound healing and other general state of health was observed after the operation. The specimens were obtained at 3,5, 7,14 and 30 days after injury. Draw the materials from the rats at the same time, all samples were divided into two parts. one ( > 0.1 g) was conserved in refrigerator at - 80 degrees C, which was extracted total RNA by TRIZOL, design the primer of rat's collagen type I and type III, RT-PCR analysis indicated that collagen type I, III. The other was fixed by 10% formalin. Examine wound healing of local tissue and count it' s MVD by HE staining.</p><p><b>RESULTS</b>All the rabbits were well alive, no death or infection. Wound healing time was shorter than the control one (14.2, 17.4 d). Inflammatory cell infiltrate, cellula intersitialis, fibroblast, collagen and the density of angiogenesis were more in the experimental group than in the control one. The MVD was significant difference between the two groups at 1, 2 weeks are 63.38 +/- 9.20, 52.72 +/- 7.06 and 76.64 +/- 12.27, 66.84 +/- 9.82 (P < 0.05). The expression of collagen type I , III mRNA was found in the third day, the peak was in the second week and then degression. The collagen type I , III mRNA and beta-actin specificitic belt were found and its initial template volume different, the results was trend of RT-PCR obtained.</p><p><b>CONCLUSIONS</b>The local application of pcDNA3.1-VEGF165 can enhance the expression of collagen type I, III mRNA, enhance angiogenesis and extra cellular matrix, both of which can shorten healing time of tissue injury.</p>


Subject(s)
Animals , Collagen Type I , Metabolism , Collagen Type III , Metabolism , Genetic Vectors , RNA, Messenger , Metabolism , Rats , Rats, Sprague-Dawley , Soft Tissue Injuries , Genetics , Metabolism , Transfection , Vascular Endothelial Growth Factor A , Genetics , Wound Healing
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