Animals ; Cell Proliferation ; drug effects ; Female ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; drug effects ; Nitric Oxide ; metabolism ; Rats ; Rats, Sprague-Dawley ; Tamoxifen ; analogs & derivatives ; pharmacology

AIMTo investigate the effects of tamoxifen on proliferation of human breast cancer Bcap-37 cells and cervical carcinoma HeLa cells and to explore it's possible mechanism.

METHODSThe techniques of cell culture, growth curves, flow cytometry and laser scanning confocal microscope were used.

RESULTSTamoxifen (10(-6) mol/L) shifted the growth curve of Bcap-37 cells downward, and shifted the growth curve of HeLa cells upward. Tamoxifen (10(-8) - 10(-6) mol/L) inhibited the proliferation of Bcap-37 cells in a dose-dependent manner, but stimulated the proliferation of HeLa cells in a dose-dependent manner. Bcap-37 cells appeared apoptosis when treated with tamoxifen (10(-6) mol/L), and the same dose stimulated the proliferation of HeLa cells at GI/S phases. The apoptotic rate of Bcap-37 cells was 97.5%. It blocked G1 phase of HeLa cells from 55.5% to 32.8%, and increased the S phase from 29.0% to 49.4%. Tamoxifen (10(-6) mol/L) also increased the releasing of calcium in Bcap-37 and HeLa cells.

CONCLUSIONTamoxifen can significantly influence the proliferation of breast cancer and cervical carcinoma cells possibly by affecting cell cycle and stimulating the releasing of Ca2+ in the cells.

Breast Neoplasms ; drug therapy ; pathology ; Cell Proliferation ; drug effects ; Female ; HeLa Cells ; Humans ; Tamoxifen ; pharmacology ; therapeutic use ; Tumor Cells, Cultured ; Uterine Cervical Neoplasms ; drug therapy ; pathology

Primer Express 2.0 software was used to design the primers and the MGB probe. With the plasmid pHaMDR1/A containing mdr1 cDNA as the template, we established a real-time fluorescent quantitative polymerase chain reaction system, which, at the template concentration of 3.061 x 10(3) to 3.061 x 10(9) cps/ml, had a correlation coefficient of 0.988243 between template concentration and threshold cycle value. This PCR method allows sensitive, specific and quantitative detection of human mdr1 gene.
ATP-Binding Cassette, Sub-Family B, Member 1 ; analysis ; genetics ; DNA Primers ; Female ; Fluorescent Dyes ; Fluorometry ; methods ; Genes, MDR ; genetics ; Humans ; Male ; Polymerase Chain Reaction ; methods ; Taq Polymerase

OBJECTIVETo study the effect of siRNA targeting ADAM17 (ADAM17-siRNA) on the proliferation of prostate cancer PC-3 cells.

METHODSAfter transfecting PC-3 cells with ADAM17-siRNA 1 and ADAM17-siRNA 2, we detected the expressions of ADAM17 mRNA and protein by RT- PCR and Western blotting, respectively. We measured the changes in the proliferation and DNA synthesis of PC-3 cells by MTT and bromodeoxyuridine (BrdU) incorporation assay, examined the cell cycle profile by flow cytometry, and determined the expressions of the genes associated with PC-3 cell proliferation by Western blotting.

RESULTSBoth ADAM17-siRNA 1 and 2 effectively reduced the expressions of ADAM17 mRNA and protein in the PC-3 cells. Knockdown of ADAM17 with the two siRNAs significantly inhibited cell proliferation as compared with the control group (0.43 +/- 0.57 and 0.44 +/- 0.64 vs 0.80 +/- 0.51, P < 0.05) and down-regulated DNA synthesis (0.48 +/- 0.43 and 0.54 +/- 0.59 vs 0.79 +/- 0.72, P < 0.05). The cell cycle profile showed that the cell population of the G1 phase was markedly higher in both the ADAM17-siRNA groups than in the control ([61.83 +/- 2.41]% and [59.78 +/- 1.92]% vs [41.38 +/- 1.53]%, P < 0.05), but that of the S phase remarkably lower in the former two than in the latter ([23.64 +/- 2.56]% and [25.24 +/- 1.86]% vs [33.51 +/- 1.47]%, P < 0.05), with a concomitant decrease in the expression of the cell cycle protein cyclin D1 and increase in the cyclin-dependent kinase inhibitor p21.

CONCLUSIONADAM17-siRNA can effectively inhibit the proliferation of PC-3 cells by up-regulating cyclin D1 and down-regulating p21 protein, and ADAM17 has a potential value in the gene therapy of prostate cancer.

ADAM Proteins ; genetics ; metabolism ; ADAM17 Protein ; Cell Line, Tumor ; Cell Proliferation ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Down-Regulation ; Humans ; Male ; Prostatic Neoplasms ; genetics ; metabolism ; pathology ; RNA Interference ; RNA, Messenger ; genetics ; RNA, Small Interfering ; genetics ; Signal Transduction ; Transfection

OBJECTIVETo investigate the incidence and the gene mutation frequencies and patterns of β-thalassemia (β-Thal) in ethnic Han children in Chongqing city.

METHODA total of 1726 children were screened by using automatic hemocytic analyzer, cellulose acetate electrophoresis and fetal hemoglobin alkali denaturation test. Samples with mean corpuscular volume (MCV) < 80 fl, cell hemoglobin content (MCH) < 27 pg and hemoglobin A2 (HbA2) levels >3.3%, fetal hemoglobin (HbF) >2% for β-Thal screening indicators. The positive samples of screening indicators were detected and identified by PCR-reverse dot blot method for 18 common β-Thal mutations in Chinese populations, unknown mutations samples were subjected to DNA sequencing analysis of the β-globin gene.

RESULTTwenty-five cases of β-Thal carriers were observed from the 1726 samples, with 24 cases of β-Thal heterozygote and one case of double heterozygote. Therefore, the β-Thal carrier rate was 1.51%. After 1726 peripheral venous blood samples analyzed by hematological parameters, 164 positive cases of β-Thal screening indicators were found, with the positive rate being 9.50% (164/1726). A total of 6 different gene mutations were detected, the four most common mutations were as the following: CD41-42, IVS-II-654, CD17 and beta E. These four mutations as the major types in this area accounted for 88.00% of all the mutations. In addition, one rare mutation of 5 'UTR; + (43 -40) was found, and one case of the hemoglobin variant of Hb Zurich was reported in Chinese people for the first time.

CONCLUSIONChongqing is a high risk region of the β-Thal. Epidemiological Data from the research was useul for the genetic counseling and the prevention of β-Thal major.

Asian Continental Ancestry Group ; genetics ; Blood Chemical Analysis ; Child ; Child, Preschool ; China ; epidemiology ; Female ; Gene Frequency ; Genetic Counseling ; Hemoglobins ; analysis ; genetics ; Hemoglobins, Abnormal ; analysis ; genetics ; Heterozygote ; Humans ; Infant ; Male ; Mutation ; genetics ; Prevalence ; beta-Globins ; genetics ; beta-Thalassemia ; epidemiology ; ethnology ; genetics

OBJECTIVESevere acute respiratory syndrome (SARS) is an emerging infectious disease that first manifested in humans in November 2002. The SARS-associated coronavirus (SARS-CoV) has been identified as the causal agent, but the pathology and pathogenesis are still not quite clear.

METHODSPost-mortem lung samples from six patients who died from SARS from April to July 2003 were studied by light and electron microscopy, Masson trichromal staining and immunohistochemistry. Evidence of infection with the SARS-CoV was determined by reverse-transcription PCR (RT-PCR) , serological examination and electron microscopy.

RESULTSFour of six patients had serological and RT-PCR evidence of recent infection of SARS-CoV. Morphologic changes are summarized as follows: (1) Diffuse and bilateral lung consolidation was seen in all patients (6/6) with increasing lung weight. (2) Diffuse alveolar damage was universal (6/6) with hyaline membrane formation (6/6), intra-alveolar edema/hemorrhage (6/6), fibrin deposition (6/6), pneumocyte desquamation (6/6). A marked disruption in the integrity of the alveolar epithelium was confirmed by immunostaining for the epithelial marker AE1/AE3 (6/6). (3) Type II pneumocytes, with mild hyperplasia, atypia, cytomegaly with granular amphophilic cytoplasm and intracytoplasmic lipid accumulation (5/6). (4) Giant cells in the alveoli were seen in five of 6 patients (5/6) , most of which were positive for the epithelial marker AE1/AE3 (5/6), but some cells were positive for the macrophage marker CD68(2/6). (5) A pronounced increase of macrophages were seen in the alveoli and the interstitium of the lung (6/6), which was confirmed by histological study and immunohistochemistry. (6) Haemophagocytosis was present in five of the 6 patients(5/6). (7) Lung fibrosis was seen in five patients(5/6), with alveolar septa and interstitium thickening(5/6), intraalveolar organizing exudates (6/6) and pleura thickening (4/6). Proliferation of collagen was confirmed by Masson trichromal staining, most of which was type III collagen by immunostaining. The formation of distinctive fibroblast/myofibroblast foci was seen in five patients (5/6) by light microscopy and immunochemistry. (8) Squamous metaplasia of bronchial mucosa was seen in five patients(5/6). (9) Thrombi was seen in all patients(6/6). (10) Accompanying infection was present in two patients, one was bacteria, the other was fungus. In addition, electron microscopy revealed viral particles in the cytoplasm of alveolar epithelial cells and endothelial cells corresponding to coronavirus.

CONCLUSIONDirect injury of SARS-CoV on alveolar epithelium, prominent macrophage infiltration and distinctive fibroblast/myofibroblast proliferation may play major roles in the pathogenesis of SARS.

Adult ; Antibodies, Monoclonal ; metabolism ; Antigens, CD ; metabolism ; Antigens, Differentiation, Myelomonocytic ; metabolism ; Epithelium ; pathology ; Female ; Humans ; Keratins ; immunology ; Lung ; pathology ; ultrastructure ; virology ; Male ; Middle Aged ; Pulmonary Alveoli ; pathology ; Pulmonary Fibrosis ; etiology ; pathology ; SARS Virus ; isolation & purification ; Severe Acute Respiratory Syndrome ; complications ; metabolism ; pathology ; virology

OBJECTIVEBoth the 2, 6 linkage and its topology on target cells are critical for the recognition by human influenza virus. The binding preference of avian flu virus H5N1 HA to the 2, 3-linked sialylated glycans is considered the major factor limiting its efficient infection and transmission in humans. To monitor potential adaptation of H5N1 virus in human population, the surveillance of receptor-binding specificity was undertaken in China.

METHODSThe binding specificity of 32 human H5N1 virus strains isolated from 2003 to 2009 was tested by 2, 3-specific sialidase-treated chicken red blood cell (CRBC) agglutination assay and a solid-phase direct binding assay with synthetic sialylglycopolymers.

RESULTSDual binding preference to 2, 3 and 2, 6-glycans were found in two strains: A/Guangdong/1/06 (A/GD/1/06) and A/Guangxi/1/08 (A/GX/1/08). Though minor effect of short-2, 6-binding was detected in A/GX/1/08 at a low virus titer, both showed high affinity to the oligosaccharide at a high load. Notably both are of the long-2, 6-recognition, with the same topology as that of human H1N1 and H3N2 viruses.

CONCLUSIONThe findings suggest that human H5N1 virus in China likely acquired the potential human-adaptation ability. Further research and surveillance on receptor-binding specificity of H5N1 viruses are required.

Adaptation, Biological ; Animals ; Chickens ; China ; epidemiology ; Hemagglutination Tests ; Humans ; Influenza A Virus, H1N1 Subtype ; metabolism ; Influenza, Human ; epidemiology ; Polysaccharides ; metabolism ; Receptors, Cell Surface ; metabolism ; Receptors, Virus ; metabolism ; Sialic Acids ; metabolism

OBJECTIVEAnalyze the proliferation of different host H1N1 subtype influenza viruses in A549 and BEAS-2B cells.

METHODSHuman, avain and swine three hosts of the H1N1 influenza viruses infected A549 and BEAS-2B cells and analyze the characteristics of different periods after inocubation. Determine the receptor binding specificity of influenza virus by hemagglutination (HA) test with RBCs with two types of receptor. And the receptors on surfaces of A549 and BEAS-2B cells were tested by flow cytometry.

RESULTSThe Cell Pathologic Effect (CPE) is obvious after 24 h inoculation in A549 cells by all the H1N1 influenza viruses, moreover, the peak hemagglutinin (HA) and 50% tissue culture cell infected dose (TCID50) titers was observed after 36 h of culturing in A549 cells. Otherwise, the CPE is not typical from 24 h-120 h inoculated by the same viruses and the HA, TCID50 titers were keep low all the periods in the BEAS-2B cell after inoculation. The receptor-binding preference of H1N1 viruses used in the study was screened by HA assay and some were found with 2-6-receptor binding affinity. Both SA a-2, 3Gal and SA a-2, 6Gal receptors were detected on A549 and BEAS-2B, furthermore, receptor density on A549 cells was significantly higher than that of BEAS-2B cells.

CONCLUSIONA549 cells were susceptible to human, avian and swine H1N1 influenza viruses infection and permissively for viral replication. However, BEASE-2B cells with similar receptor pattern and epithelium-derived propriety as A549 cells were unsusceptible to their infection and replication. Possible host factors involved in effective viral infection and replication were needed further study.

Animals ; Cell Line ; Cell Line, Tumor ; Chickens ; Dogs ; Humans ; Influenza A Virus, H1N1 Subtype ; physiology ; Virus Replication ; physiology

OBJECTIVETo analyse the correlation between the virus isolation and the specimen collection of the H5N1 human high pathogenic avain influenza cases in Mainland China.

METHODSThe specimens were collected in Mainland China from 2005.10 to 2009.3 and the H5N1 viruses were isolated by passage in embryonated chicken eggs.

RESULTSMost specimens were obtained within 14 days after disease onset. For the specimens collected within 7 days, the isolation rate was relatively high and the difference of the positive rate between different years was lower than those specimens collected after 7 days. Most of the samples in our study were collected from the upper or lower respiratory tract with few from blood, feces, et al. The isolation rate of lower respiratory specimens was higher and the difference of the positive rate between different years was relatively lower than those from upper respiratory specimens.

CONCLUSIONWe suggest that the samples should be collected from lower respiratory tract during the acute phase to get the higher isolation rate.

Animals ; Chick Embryo ; China ; epidemiology ; Humans ; Influenza A Virus, H5N1 Subtype ; classification ; genetics ; isolation & purification ; Influenza, Human ; epidemiology ; virology ; Respiratory System ; virology

A new flu caused by a novel influenza A(H1N1) virus has spread over the United States, Mexico and more than 40 other countries. And because of the immediate global concern, WHO has announced that the current level of influenza pandemic alert is raised to phase 5, indicating approaching of an influenza pandemic. As patients suffering from the influenza A (H1N1) have the similar symptoms as patients with seasonal influenza, differential detection and identification of the influenza virus have to depend on specific laboratory tests. We have successfully developed a RT-PCR based method for detection of the influenza A (H1N1) virus, and had applied the method to detection of clinical samples.
Humans ; Influenza A Virus, H1N1 Subtype ; genetics ; isolation & purification ; Influenza, Human ; virology ; RNA, Viral ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; methods