OBJECTIVETo investigate the apoptosis regulation on hepatoma cells by HBx between genotype B and C.

METHODSGenotype B and C HBx gene fragments were amplified and inserted into green fluorescent protein (GFP) eukaryotic expression vector pEGFP-C1 to construct recombinant pGFP-XB and pGFP-XC. The pEGFP-C1, pGFP-XB and pGFP-XC were introduced into Bel-7402 cells by Fugene HD to obtain Bel-7402 cells expressing GFP. The transcription and expression of HBx gene were demonstrated by RT-PCR and Western Blot analysis. Bel-7402, Bel-7402/GFP, Bel-7402/GFP-XB, Bel-7402/GFP-XC cells were treated with adriamycin (2.5 microg/ml), and the apoptosis of the cells was determined by trypan blue exclusion, and flow cytometry analysis.

RESULTSRT-PCR and Western Blot analysis showed that HBx genes of genotypes B and C were transcribed and expressed in Bel-7402/GFP-XB, Bel-7402/GFP-XC cells. Trypan blue exclusion showed adriamycin induced time-dependent cell death in Bel-7402, Bel-7402/GFP cells while no significant cell death was observed in Bel-7402/GFP-XB, Bel-7402/GFP-XC cells. Flow cytometry analysis indicated that no significant differences of apoptosis rates of Bel-7402/GFP-XB (3.87%) and of Bel7402/GFP-XC (4.01%) were observed (P > 0.05), moreover, no significant differences of Bel-7402/ GFP-XB (3.87%), Be17402/GFP-XC (4.01%) and of the untreated cells. Apoptosis rates in Bel-7402/GFP-XB (3.87%), Bel-7402/GFP-XC (4.01%) cells were significantly lower than those in Bel-7402 (27.05%) and Bel-7402/GFP (29.14%) cells at 48 hours after the adriamycin treatment (P < 0.01).

CONCLUSIONSBel-7402 cell lines expressing GFP, GFP-XB and GFP-XC fusion proteins were successfully established. HBV X protein blocks adriamycin-induced apoptosis of Bel-7402 cells. There is no difference between HBx of genotype B and C in inhibiting apoptosis induced by adriamycin.

Antiviral Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Line ; Cell Line, Tumor ; Doxorubicin ; pharmacology ; Hepatitis B ; drug therapy ; physiopathology ; virology ; Hepatitis B virus ; classification ; drug effects ; genetics ; metabolism ; Humans ; Trans-Activators ; genetics ; metabolism

Objective To investigate the association of osteoporosis and coronary artery calcification in elderly patients with type 2 diabetes mellitus (T2DM).Methods A total of 82 elderly T2DM patients underwent dual-energy x-ray absorptiometry scanning (DXA) of lumbar spine and femur neck for getting bone mineral density (BMD),and dual-source computed tomography (DSCT) of coronary artery for calculating calcification score and total calcification score (TCS).All subjects were divided into two groups:osteoporosis group and non-osteoporosis group.The levels of serum calcium (Ca),parathyrin (PTH),phosphorus (P),alkaline phosphatase (AKP),triglyceride (TG),total cholesterol (TC),high density lipoprotein-cholesterol (HDL-C),low density lipoproteincholesterol (LDL-C) and glycosylated hemoglobin (HbA1c) were detected.Results Compared with non-osteoporosis group,the levels of serum Ca,PTH and TCS were higher [(2.32± 0.15)mmol/L vs.(2.04±0.20) mmol/L;(5.64±1.97) pmol/L vs.(5.01±1.93) pmol/L;(374.4±433.5) scores vs.(242.5±224.8) scores,t=5.790,5.331 and 2.248,all P<0.05] in osteoporosis group.Correlation analysis showed TCS was negatively associated with BMD of L2-4 and femur neck,while was positively associated with serum Ca and PTH (r=0.310,0.246,0.290,0.284 and 0.324,0.575 all P<0.05).Conclusions Osteoporosis is associated with coronary atherosclerosis.TCS could be considered as an index for judging the relationship between osteoporosis and coronary atherosclerosis.

Objective To observe the antioxidative dosage-effect relationship of grape seed proanthocyanidin extract (GSPE). Methods The mice were randomly divided into seven groups:blank group, model group, 5 mg/kg GSPE group, 15 mg/kg GSPE group, 45 mg/kg GSPE group, 135 mg/kg GSPE group and 405 mg/kg GSPE group. The mice in blank group were dealt with saline solution by intraperitoneal injection, the others were dealt with D-galactose (120 mg/kg) by intraperitoneal injection for seven weeks to make oxidative damage model. Meanwhile, the mice were given corresponding dose of the drug. Subsequently the level of malondialdehyde (MDA) and activity of superoxide dismutase (SOD) in the serum were measured to observe the antioxidative dosage-effect relationship of GSPE. Results The 45, 135, 405 mg/kg GSPE group reduced the MDA level, and the 15, 45, 135, 405 mg/kg GSPE group increased the SOD activity. Conclusion GSPE has significant antioxidant activity on mice dealt with D-galactose above the dose of 15 mg/kg, suggesting that the clinical use of GSPE should guarantee a certain dose to play a good antioxidant effect.

OBJECTIVETo identify mouse acute pneumonia model induced by influenza virus adapted strains (FM1 strain) using RT-PCR, gene clone and sequence analysis and pathological examination of lung tissues.

METHODSAcute pneumonia was induced by intranasal drip of FM1 strain. The lungs were collected after 3, 5 and 7 days. RT-PCR was used to detect the viral load. Amplified PCR products were cloned and sequenced. Pathological and histological changes to the lungs were observed.

RESULTSThere were no abnormalities in the alveoli, alveolar sacs and alveolar septa and no inflammatory cell infiltration was found in normal mice. In the model group, we found disappearance of alveoli, alveolar sacs, alveolar ducts and alveolar septa, thickening of the alveolar septal and bronchiolar walls, and infiltration of inflammatory cells after 3, 5 and 7 days of influenza virus (IV) infection. Compared with the normal group, pathological changes at various time points were significantly increased (P<0.01). Viral nucleic acid can be detected in the lung tissue of the model group at various time points, and the pathological changes of the lung tissue were positively correlated with viral load. Sequence analysis demonstrated that there was 99.1% consistency between RT-PCR products of lung tissues in the model group and the known IV cDNA sequence (P<0.01).

CONCLUSIONSGene clone and sequence analysis may be used to identify acute mouse pneumonia model induced by FM1 strain.

Acute Disease ; Animals ; Base Sequence ; DNA, Complementary ; chemistry ; Disease Models, Animal ; Female ; Influenza A Virus, H1N1 Subtype ; Lung ; pathology ; Male ; Mice ; Molecular Sequence Data ; Pneumonia, Viral ; etiology ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA

Background The integration of segregated pathways from the two eyes first appears in V1 neurons,where it not only plays a critical role in the generation of a three-dimensional visual representation.Abnormal visual experiences in critical period usually lead to amblyopia and binocular integration defects.Objective Present study was to investigate how neurons of kitten coordinate their activity patterns in response to synchronous dichoptic stimulus inputs in striate cortex.Methods Spike rate and local field potential(LFP) gamma band(20-90Hz) power of three kitten(1-1.2Kg,8-10 weeks old) to monocular and synchronous dichoptic presented gratings were assessed for 28 binocular neurons in V1 of kitten by in vivo extracellular record method under anaesthesia and paralysis.Ocular dominance index(ODI) and binocular integration index(BII) were assessed and the correlation between these two indexes were analyzed.Results In 28 cells with binocular characteristic,the absolute value of spike-ODI was significant larger than that of LFP-ODI(t=2.606,P=0.021).A positive linear correlation between the ocular preferences of spike and LFP was found(R2=0.513,F=27.423,P=0.003).In dichotic trails,binocular facilitation with BII for spike was 2.348±0.996,showing a significant reduce in comparison with BII for LFP(3.678±1.974)(t=2.671,P=0.019).Binocular integration index for two signals were greater when monocular responses of both eyes were similar(P=0.035 and P=0.124,respectively).Conclusion Both spike rate and gamma band power of LFP exhibited binocular facilitation to synchronous presented dichotic stimuli with significant facilitation induced by balanced monocular responses.Spiking activity and LFP reflect neural activities of different spatial scales and source components.

etabolic response to radiotherapy may predict the prognosis of paitents with locally advanced NPC. The prognosis is poor for patients with high FDG uptake before and after radiotherapy or SUV max-NSUV max-P .

Humanin(HN,its analogue [Gly14]-Humanin,HNG)was originally identified as an endogenous peptide that protects neuronal cells from apoptosis induced by various types of Alzheimer's disease-related insults.But the relative low content of this peptide in its natural sources limits its further characterization.An expression vector pET32a/HNG was corstructed and transformed it into E.coli BL21 trxB(DE3).HNG was expressed as a fusion protein in the soluble fraction and was purified by nickel affinity chromatography.Subsequently,the purified fusion protein was cleaved by enterokinase and was further purified by reverse-phase HPLC.A 23 mg recombinant HNG(rHNG)from 1 L bacterial culture was purified.The molecular weight of rHNG determined by ESI-MS was 2876.5 Da which was the expected size for correctly processed peptide.The N-terminal amino acid sequence of rHNG determined by Edman degradation method is identical to the theoretical sequence.Neuroprotective bioassay studies of rHNG exhibited its potential neuroprotective effect comparable to that of the natural HNG peptide.

Objective To determine the expression of HPV16/18,31/33 DNA and p53,p21~(WAF1) and MDM2 proteins in invasive squamous cell carcinoma of cervix (ISCC)and to indicate the significance of them in the occurrence and development of ISCC.Methods Using tissue microarray,in situ hybridization (ISH) and immunohistochemical method,we detected the expression of HPV16/18,31/33 and investigate the expres- sion of p53,p21~(WAF1),MDM2 and proteins in ISCC,CIN and NCE.Results was analysed by SPSS vision 12.5. Results The positive expression rate of HPV16/18,p53,p21~(WAF1),MDM2 in ISCC was markedly higher than in CIN and NCE.We found the difference between HPV31/33 and lymph node transfer.Significant relation- ship was observed between p53 protein expression and histological grade and lymph node metastasis of the cancer.There was positive correlation between the expression of p21~(WAF1) protein and the depth of invasion(P

Objective To investigate the relationship between the expression of GATA6 mRNA and the occu-rrence of congenital heart disease(CHD). Methods A total of 60 cases of CHD (CHD group)were diagnosed at Obstetrical Department,Rizhao City Maternal and Child Health Hospital,and 60 cases of normal pregnancy women (control group)were collected in March 2013 to May 2017. The expression levels of mRNA of GATA6 in the peripheral blood of pregnant women in 2 groups were detected by using real-time quantitative PCR (qRT-PCR)technique,and the relevant information of 2 groups of mothers before and during pregnancy was collected so as to investigate the rela-tionship between mRNA and GATA6 in CHD. Results There were no significant differences in age,body mass index (BMI),passive smoking,alcohol consumption,pregnancy medication,pregnancy-induced hypertension,high -density lipoproteincholesterol (HDL-C),total cholesterol (TC),triacylglycerol (TG)between pregnant women in the congenital heart disease group and the control group (all P > 0. 05). The CHD group showed a statistical difference significance in the rate of taking folic acid(81. 67％)and gestational diabetes incidence(8. 33％),serum level of LDL-C[(3. 59 ± 0. 46)mmol/ L]compared with the healthy control group [96. 67％,0,(3. 20 ± 0. 44)mmol/ L] (all P < 0. 05). The expression level of GATA6 mRNA in peripheral blood of patients with congenital heart disease (0. 014 ± 0. 005)was significantly lower than that of the control group (0. 129 ± 0. 031),and the difference was statis-tically significant (t = 28. 386,P = 0. 000). Logistic regression analysis showed that the level of in GATA6 mRNA ex-pression increased in the peripheral blood and folic acid intake were protective factors for CHD (all P < 0. 05)during pregnancy;while fetal gestational diabetes,elevated levels of LDL-C are risk factors for congenital heart disease (all P < 0. 05)fetus. Conclusion Down regulation of GATA6 mRNA expression in the maternal peripheral blood is one of the independent risk factors for fetal congenital heart disease.