OBJECTIVE ToexplorethepossiblemechanismoractiontargetsofT-2toxinembryo toxicity by observing the effect of T-2 toxin on mitochondrial function of differentiated murine e mbryonic stemcells(mESCs).METHODS Duringdifferentiationat24,72and120h,ESCswereexposedto T-2 toxin 0.5 μg·L-1 .Meanwhile,mESCs were pre-treated with antioxidant Trolox (200 μmol·L-1 )for 30 min and exposed to T-2 toxin (0.5 μg·L-1 )for 72 h.The mitochondrial ultrasture of differentiated mESCs was observed under a transi mission electrical microscope (TEM).The differentiated ESC mito-chondrial function,including respiratory control ratio (RCR),ATP synthase activity and mitochondrial membranepotential(MMP),wasmeasuredat144hafterdifferentiation.RESULTS Significant decrease of the mitochondrial number,deformation of mitochondrial structure,and lack of complete mito-chodrial crest were observed through TEM in the groups of T-2 toxin exposed for 72 and 1 20 h,respec-tively.Compared with the normal control group,RCR declined by 49.5% and 55.1%,ATP synthase activity decreased by 84.9% and 89.3%,and MMP decreased by 23.2% and 35.2% in T-2 toxin 0.5 μg·L-1 exposure 72 and 1 20 h group,respectively.However,the inhibition of mitochondrial function by T-2 toxin in differentiated mESCs recovered significantly in the presence of the antioxidant Trolox. CONCLUSION T-2toxininducesoxidativestressandinhibitsmESCsmitochondrialfunctionindifferenti-ated mESCs,and ROS-induced mitochondrial malfunction plays an i mportant role in T-2 toxin e mbryonic toxicity mechanis m.

Objective To construct four expression plasmids, pcDNA3.1-GFP/HPV6bE6, pcDNA3.1-GFP/HPV6bE7, pcDNA3.1-GFP/HPV11E6, pcDNA3.1-GFP/HPV11E7 and their transfected murine cell lines. Methods The Four recombinant expression plasmids comprising HPV6bE6,HPV6bE7,HPV11E6 and HPV11E7 linked with GFP, respectively, were constructed and transfected to B16 cells by lipofectamine kit. Positive clones were selected by G418 and observed by fluorescent microscopy and identified by RT-PCR. Results The four constructed recombinant plasmids were authenticated by restriction enzyme digestion and DNA sequencing. Under the fluorescent microscope, the green fluorescence could be observed in cytoplasm and nucleus of four transfected B16 cell lines. The RNA extracted from positively transfected clones resistant to G418 were analyzed by RT-PCR, which demonstrated the presence of four expected fragments. Conclusions The transfected murine cell lines B16 can express HPV6bE6,HPV6bE7,HPV11E6 and HPV11E7 gene. These transfected cell lines can be further transplanted to mice in order to investigate the biological properties and immunological mechanisms of these genes in vivo.

AIM To investigate the clinical therapeutic effects and pulmonary function effects of phentolamine combined with Dangshen Bufei Decoction (Astragali Radix,Codonopsis Radix,Atractylodis macrocephalae Rhizoma,etc.) in the treatment of asthmatic disease in infants.METHODS From August 2013 to April 2016,94cases of infants with asthmatic disease in our hospital for diagnosis and treatment were selected and randomly divided into observation group of 47 cases and control group of 47 cases.The control group was given phentolamine treatment,while the observation group was treated with phentolamine combined with Dangshen Bufei Decoction.Two groups were both treated for seven days.RESULTS The total effective rates in the observation group and the control group were 97.9％ and 85.1％,respectively,the total effective rate in the observation group was significantly higher than that in the control group (P ＜ 0.05).The total disappearance time of dyspnea,cough,fever and other symptoms in the observation group were significantly faster than those in the control group (P ＜ 0.05).The incidence rate of facial flushing,irritability,bradycardia,nausea,vomiting and other adverse reactions in the observation group was 17.0％,which was 19.1％ in the control group,there was no significant difference between the two groups (P ＞0.05).CD4 + values in the observation group and the control group after the treatment were significantly lower than those before the treatment (P ＜ 0.05),while CD8 + values were significantly higher than those before the treatment (P ＜ 0.05).After the treatment,CD4 + and CD8 + values in the observation group showed significant statistical differences as compared with those in the control group (P ＜ 0.05).FVC values in the observation group and the control group after the treatment were (83.55 ± 15.29)％ and (75.20 ± 11.49)％,respectively,which were significantly higher than those before the treatment [(68.24 ± 15.20)％ and (69.01 ± 14.03)％,respectively] (P ＜0.05),and FVC value in the observation group was also significantly higher than that in the control group (P ＜ 0.05).CONCLUSION Phentolamine combined with Dangshen Bufei Decoction has good safety in the treatment of asthmatic disease in infants,which can improve clinical symptoms and immune function,so as to promote the improvement of lung function and therapeutic effects.

This paper introduces the designing concept of the ECG treadmill system and discusses the methods of its realization.
Amplifiers, Electronic ; Computer Simulation ; Computer Systems ; Coronary Disease ; diagnosis ; Electrocardiography ; instrumentation ; methods ; Equipment Design ; Exercise Test ; instrumentation ; methods ; Humans ; Microcomputers ; Signal Processing, Computer-Assisted ; Software

Objective:To explore the expression of long non-coding RNA LINC00630 in bladder cancer cell lines, and to explore the effect of interference with its expression in vitro on the proliferation and migration of bladder cancer cells.Methods:Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of LINC00630 in bladder cancer cell lines 5637, BIU-87, T24, J82 and normal bladder epithelial cell line SV-HUC-1. The bladder cancer cell line with the highest LINC00630 expression was selected for follow-up experiments, then the cell line infected with the control lentivirus was used as the control group, and the cell line infected with the lentivirus that could interfere with the expression of LINC00630 was used as the experimental group. qRT-PCR was used to detect the expression of LINC00630 in the two groups of cells. MTS method and cell scratch test were used to detect the proliferation and migration abilities of cells in the two groups. qRT-PCR was used to detect the expression of neuregulin 1 (NRG1) mRNA in the two groups of cells, and Western blot was used to detect the expressions of NRG1 protein, cell proliferation-related proteins (cyclin D3 and CDK2) and cell migration-related proteins (Vimentin and N-cadherin) in the two groups of cells.Results:Compared with SV-HUC-1 cells (1.05±0.17), the expression of LINC00630 was significantly increased in all bladder cancer cell lines (all P < 0.01), and the expression was highest in J82 cells (relative expression 5.83±0.42). Compared with J82 cells of the control group, the expression of LINC00630 in J82 cells of the experimental group decreased (0.18±0.02 vs. 1.00±0.05, t=14.36, P < 0.01); from day 2 of transfection, the cell proliferation activity of the experimental group was lower than that of the control group (all P < 0.05). The cell scratch closure rate of the experimental group was lower than that of the control group [(27.4±7.1)% vs. (66.0±5.4)%, t = 4.31, P < 0.01]. Therelative expression of NRG1 mRNA in the experimental group was lower than that in the control group (0.34±0.03 vs. 1.07±0.24, t = 2.99, P < 0.05). Compared with the control group, the expressions of NRG1 protein, cell proliferation-related proteins and cell migration-related proteins in the experimental group were reduced. Conclusions:LINC00630 is up-regulated in bladder cancer cell lines, and interference with LINC00630 may inhibit the proliferation and migration of J82 cells by down-regulating the expression of NRG1 gene. LINC00630 may be a new molecular target for the treatment of bladder cancer.

OBJECTIVETo investigate the sensitizing potential of Shuanghuanglian Injection (SHL) by comparing the popliteal lymph node (PLN) response in mice induced by SHL and chemicals.

METHODSSixty female C57BL/6J mice were equally and randomly divided into six groups, i.e. the blank control group (A) and five treated groups treated respectively with phenobarbital 1 mg/mouse (B), mercuric chloride ( HgCl2) 50 microg/mouse (C), D-penicillamine 2 mg/mouse (D), and SHL in low (1 mg/mouse) and high (5 mg/mouse) dosages (E and F) via subcutaneous injection into left pad of hind foot. Animals were sacrificed on the 8th day after injection, their bilateral PLNs were isolated and weighed respectively to calculate the PLN mass index (MI). Then the PLNs get from four mice in each group were fixed with 4% paraformaldehyde solution for histopathologic examination; the other six PLNs were prepared into single-cell suspensions to calculate cell index (CI) for comparing the changes of PLN in various groups.

RESULTSMI and CI in Group F reached to > or = 2 and > or = 5 (average) respectively, which was higher than those in Group A (P<0.05). Pathological examination showed that the left PLN in Group F enlarged, with remarkable germinal center and increased high endothelial venules proliferation.

CONCLUSIONSHL could induce significant PLN response in C57BL/6J mice, suggesting it has certain sensitizing potential.

Animals ; Drugs, Chinese Herbal ; adverse effects ; Female ; Hypersensitivity ; pathology ; Local Lymph Node Assay ; Lymph Nodes ; drug effects ; immunology ; pathology ; Mice ; Mice, Inbred C57BL

Objective To study the capability of high field MRI in demonstrating the post-mortem fetal brains at different gestational age(GA).Methods One hundred and eight post-mortem fetal brains of 14-40 weeks GA were evaluated by 3.0 T MRI. Eleven brains of 14 to 27 weeks GA with good 3.0 T MRI images were chosen and scanned by 7.0 T MRI. The developing sulci, layered structures of fetal cerebral cortex and basal nuclei were evaluated on MRI of different Tesla(3.0 T and 7.0 T)and their results analyzed. Results On T_1 WI of 3.0 T MRI, the layered structures of fetal cerebral cortex were present at 14 weeks GA, the sulci were more accurately identified after 16 weeks GA. The basal nuclei were clearly distinguishable after 20 weeks GA. and these structures were better visualized as the GA increased. On T_2WI of 7.0 T MRI, the sulei, layered structures of fetal cerebral cortex and basal nuclei were shown more clearly at the same GA when compared to 3.0 T, especially the sulci at the early developmental stages. Conclusions T_1 WI of 3.0 T MRI could show the developing structures of post-mortem fetal brain well, but the T_2 WI of 7.0 T MRI were comparatively better.

OBJECTIVE(1) To investigate the promoter methylation status of gene p16(INK4a) and gene RB in breast carcinoma and the adjacent non-neoplastic hyperplastic epithelial tissue. (2) To study the correlation of p16(INK4a) gene expression at protein level with the abnormal gene methylation, the clinical manifestation and the pathological parameters.

METHODSMethylation status of promoters of p16(INK4a) gene and RB gene was detected by using methylation specific PCR in 46 cases of breast cancer, 22 cases of the adjacent non-neoplastic hyperplastic epithelium tissue and 7 cases of normal breast tissue. In addition, the p16(INK4a) gene protein expression level was also detected using immunohistochemical technique(SP method) in 46 cases of breast cancer and 22 cases of the adjacent hyperplastic epithelial tissue.

RESULTSThe methylation rate of p16(INK4a) gene was 23.9% (11/46) in breast cancer, 18.2% (4/22) in the adjacent non-neoplastic hyperplastic epithelial tissue and 1/7 in normal breast tissue, respectively. The methylation rate of RB gene was relatively low, which was 10.8% (5/46), 9.1% (2/22) and 0(0/7) in the above 3 groups, respectively. Methylation rate of p16(INK4a) gene and RB gene was not significantly different among the breast cancer, the adjacent non-neoplastic hyperplastic tissue and the normal tissues (P > 0.05). However, the methylation status of p16(INK4a) gene was closely correlated with its protein expression level and the negative ER expression result of the breast cancer (P < 0.05), but not correlated with the size of the cancer, differentiation status, lymph node metastasis, and age. The methylation status of RB gene was correlated with lymph node metastasis, but not with the size, the differentiation status, ER expression of the breast cancer and the age of the patients.

CONCLUSIONSThe abnormal methylation of p16(INK4a) gene may not play a significant role in the early stage of breast cancinogenesis, but may play a role of in the progression of the cancer. RB gene methylation may also be a indicator in choice to identify the progression and prognosis of breast cancer.

Adult ; Aged ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Carcinoma, Ductal, Breast ; genetics ; metabolism ; pathology ; Carcinoma, Intraductal, Noninfiltrating ; genetics ; metabolism ; pathology ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; metabolism ; DNA Methylation ; Female ; Gene Expression Regulation, Neoplastic ; Genes, p16 ; Humans ; Lymphatic Metastasis ; Middle Aged ; Receptors, Estrogen ; metabolism ; Retinoblastoma Protein ; genetics ; metabolism

OBJECTIVETo study the effect of N-glycosylation on the modification of microvilli on the surface of rat liver epithelial cell WB-F344 and the growth of the cells in culture.

METHODSRecombinant adeno-associated virus (rAAV) expression vector pAGX (+) containing an antisense or a sense fragment of 6A8 cDNA encoding a human alpha-mannosidase was constructed. The recombinant vectors or the mock were transfected into WB-F344 cells by means of lipofectAmine. The transfected cells were selected in G418 medium and cloned by means of limiting dilution. Integration of the transfected DNA into host DNA was detected by neo PCR. Rat liver ER alpha-mannosidase activity in cell supernatant was measured by using P-nitrophenyl-alpha-D-mannopyranoside as a substrate. Microvilli on cell surface were observed upon a scan electron microscope. The growth curves of the cells in culture were drawn.

RESULTSThe cell clones transfected with antisense 6A8 showed reduction of ER alpha-mannosidase activity with various degrees. Clone AS1 and AS2 cell showed a pronounced reduction of the enzymatic activity. In the study on AS1 cells, Con A binding to the cells was found to be enhanced, cell growth in culture became slow from day 5. The microvilli on the cells were reduced and blunted.

CONCLUSIONSTransfection with antisense 6A8 resulted in reduction and blunting of microvilli on the surface of growing WB-F344 cells, which might be related to N-glycosylation modification.

Animals ; Cloning, Molecular ; Epithelial Cells ; cytology ; Glycosylation ; Liver ; cytology ; Microvilli ; Rats ; Transfection ; alpha-Mannosidase ; genetics ; metabolism

OBJECTIVETo investigate the inhibitory effect of 6A8 alpha-manosidase expression on the adhesiveness of CNE-2L2 cells to laminin and the lamellipodia on cell surface.

METHODS6A8 alpha-manosidase expression was detected by Western blotting. For assaying the adhesion of cells to laminin, cells were incubated in laminin-coated plate at 37 degrees C for 1 h, the adhered cells were stained with crystal purple dissolved in 0.1 mol/L Sodium Citrate/50% ethanol. Absorbance 540 nm was measured. Adhesion rate (R) was calculated according to formula R = AT/A100 x 100%. Here A100 represents 100% adhesion. lamellipodia on cell surface was observed upon a scanning electron microscopy.

RESULTSThe adhesion rate of two clones (AS1 and AS2) with inhibition of 6A8 alpha-manosidase expression to laminin was 0.447 +/- 0.096 and 0.533 +/- 0.065 respectively. The adhesion rate of three controls with normal expression of 6A8 alpha-manosidase to laminin was 0.78 +/- 0.035, 0.7 +/- 0.05 and 0.80 +/- 0.04 respectively. The difference was significant (P < 0.01). CNE-2L2 cells with normal expression of 6A8 alpha-manosidase was rich in lamellipodia on their surface. Lamellipodia nearly disappeared on the cells with inhibition of 6A8 alpha-manosidase expression.

CONCLUSIONSInhibition of 6A8 alpha-manosidase expression results in decrease of adhesion to laminin and reduction of lamellipodia of human nasopharyngeal carcinoma cell CNE-2L2.

Cell Adhesion ; drug effects ; Humans ; Laminin ; physiology ; Nasopharyngeal Neoplasms ; pathology ; Neoplasm Metastasis ; Neoplasm Proteins ; genetics ; physiology ; Pseudopodia ; physiology ; Tumor Cells, Cultured ; alpha-Mannosidase ; biosynthesis ; genetics