Objective To establish the HepG2 cell lines which can stably express and replicate hepatitis 13 virus (HBV).Methods One point two X unit length of HBV genome was cloned intn SalⅠsite of the eukaryotic expression vector pREP10 to construct the recombinant plasmid pREP-HBV. Human hepatoblastoma cell HepG2 was transfected with pREP-HBV by Lipofectamine 2000 and seh,cted by bygromycin at the concentration of 250?g/mL.HBsAg and HBeAg were monitored by enzyme linked immunosorbent assay (ELISA)kits.H13V particles presemed in supernatant were ex- amincd by electronic microscopy.DNA isolated from intracellular HBV core particles was analyzed by Southbern blot using HBV-specific probe.Results The recombinant vector pREP HBV containing 1.2?unit length of HBV DNA was constructed successfully.After transfection of pREP-HBV to HepG2 cells and consistently cultured in hygromycin selective medium.5 drug-resistant cell lines, RHBV1-RHBV5.were established,and all of them could stably express HBsAgand HBeAg.South ern blot analysis revealed that HBV could replicate in all cell lines,as confirmed by the presence of replicateintermediatc DNA in intracellular HBV core particles.Clustered 42 nm Dane particles as well as 22-26 nm spherical H13sAg particles in condensed cuhure supernatant were visualized by elec tronic microsopic analysis.Conclusion HepG2 ceil lines in which HBV can replicate and express specific antigens are successfully established.Up to now,the cells have been passaged every three days for 50 times.

Background Granulocyte colony-stimulating factor(G-CSF)has been reported to have beneficial effect on cardiac dysfunction in post infarction and doxorubicin-induced cardiomyopathy.Objective To investigate the effects of G-CSF on cardiac remodeling in cardiac hypertrophy induced by angiotensin Ⅱ(Ang Ⅱ).Methods Thirty-six male wild type mice(WT)were allocated randomly to receive subcutaneously G-CSF(10 ?g/kg per day, n=9),or Ang Ⅱ(2.88 mg/kg per day,n=9),or Ang Ⅱ plus G-CSF(Ang Ⅱ 2.88 mg/kg+G-CSF 10 ?g/kg,n =9)for 4 weeks with untreated WT(n=9)as controls.Blood pressure and cardiac function were measured. Heart weight/body weight ratio,myocyte cross-sectional area and fibrosis area were determined.The mRNA ex- pression of osteopontin(OPN)in myocardium was detected by RT-PCR.The expressions of angiotensin converting enzyme(ACE),ACE2 and phosph-p70S6 kinase protein in myocardium were assessed by Western-Blot.Results Ang Ⅱ significantly elevated blood pressure(SBP,Ang Ⅱ:139.7?1.6 vs WT:108.7?2.3 mmHg,P0.05),but significantly attenuated the myocyte cross-sectional area(Ang Ⅱ+G-CSF:181.06?0.11 vs Ang Ⅱ:202.02?0.16 ?m~2,P

OBJECTIVERecent studies have shown that cytokines TNF-alpha and IL-6 play important roles in myocardial injury or dysfunction. Transcription nuclear factor (NF-kappa B) have been implicated in the regulation of a variety of cytokines in response to cellular defense. The authors observed the activity of NF-kappa B and cytokines TNF-alpha, IL-6 mRNA expression in myocardium to further investigate the mechanism of myocardial injury caused by infectious pneumonia. The therapeutic effect of exogenous adenosine was also studied by observing the influence on NF-kappa B and cytokines.

METHODSThirty rats were divided into three experimental groups at random, each group had 10 rats. The model of pneumonia was induced by the injection of Staphylococcus aureus into the trachea of rats. Adenosine-treated rats were given daily slow intravenous injection of adenosine at a dose of 150 microg/kg.min for 3 days from the second day. All rats were killed on the fifth day. Myocardial tissues were preserved in liquid nitrogen for examination. Pathological examination of myocardium was done and TNF-alpha and IL-6 mRNA expression was detected by reverse transcription polymerase chain reaction (RT-PCR). NF-kappa B activity was measured by electrophoretic mobility shift assay (EMSA).

RESULTS(1) The myocardium in pneumonia group showed significant pathological lesion when compared with control group (P < 0.01). The pathological lesion of myocardium in adenosine-treated group significantly decreased when compared to pneumonia group (P < 0.05). (2) Significant increase of TNF-alpha and IL-6 mRNA expression was observed in myocardium of pneumonic rats when compared with control group (2.27 +/- 0.27 vs. 1.05 +/- 0.16; 1.89 +/- 0.31 vs. 1.12 +/- 0.25: P < 0.01, respectively). NF-kappa B activity of myocardium in pneumonia group was significantly higher than that in control group (13,033 +/- 1286 vs. 383 +/- 15: P < 0.01). (3) TNF-alpha and IL-6 mRNA expression was significantly decreased in adenosine-treated group when compared with pneumonia group (1.25 +/- 0.18 vs. 2.27; 1.31 +/- 0.25 vs. 1.89 +/- 0.31, P < 0.01, respectively). Comparing to that in pneumonia group, NF-kappa B activity of myocardium in adenosine-treated group was significantly decreased (4 487 +/- 562 vs. 13033 +/- 1286, P < 0.01), but it was still significantly higher than that in control group (4487 +/- 562 vs.383 +/- 15, P < 0.01).

CONCLUSIONSIncreased activity of NF-kappa B and subsequent upregulation of TNF-alpha and IL-6 mRNA expression probably play a pivotal role in the mechanism of myocardial injury in rats with pneumonia. Exogenous adenosine can inhibit inflammatory change by lowering NF-kappa B activity and subsequent down-regulation of TNF-alpha and IL-6 expression. Our findings provide novel therapeutic evidence of adenosine in myocardial injury induced by pneumonia in clinic.

Adenosine ; pharmacology ; Animals ; Anti-Arrhythmia Agents ; pharmacology ; Cytokines ; genetics ; metabolism ; Disease Models, Animal ; Electrophoretic Mobility Shift Assay ; Female ; Gene Expression ; drug effects ; Interleukin-6 ; genetics ; metabolism ; Male ; Myocardium ; metabolism ; NF-kappa B ; genetics ; metabolism ; Pneumonia, Staphylococcal ; drug therapy ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

Objective To assess the liver histopathological characteristics of chronic hepatitis B (CHB) patients with alanine aminotransferase(ALT)lower than 2 times the upper limit of normal (ULN) through liver biopsy, and try to provide subjective evidence for clinical anti-viral treatment.Methods From October 2005 to August 2010, patients accepted liver biopsy in department of infectious disease, Sichuan provincial people's hospital were enrolled. The criteria for liver biopsy was as follow, (1) HBsAg-positive for more than 6 months, (2) HBeAg-positive patients with HBV DNA ≥103 copies/ml or HBeAg-negative patients with HBV DNA≥ 104copies/ml, (3) ALT was lower than 2 times ULN for more than 6 months,and without any hepatic protectants, (4) never accepted any antiviral treatment before, including IFN or nucleoside analogues, (5) willing to accept liver biopsy. Before liver biopsy, routine blood test, prothrombin time, liver function test, hepatitis B antigen and antibody test, HBV DNA quantification were examined. The biopsy position was located under routine ultrasound, liver biopsy were performed to assess the grading of inflammation and necrosis and the degree of fibrosis. The correlation between all the factors and liver inflammation and fibrosis were analyzed. Results Totally 383 cases (240 males and 143 females) met the diagnostic criteria, aged from 16 to 59 years old and the mean age was 28.0 years old. Cases of liver inflammation in G0, G1, G2, G3andG4 grade was 2 cases (0.5％), 165 cases (43.1％), 191 cases (49.9％), 25 cases (6.5 ％ ) and 0 cases (0 ％ ) respectively, cases≥G2 grade accounted 56.4 ％ of total. Meanwhile,stage of fibrosis in S0, S1, S2, S3 and S4 was 103 cases (26.9％), 265 cases (69. 2％), 13 cases (3.4％), 2 cases (0.5％) and 0 cases (0％) respectively, percentage of liver fibrosis in S2stage and over was only 3.9％. The occurrence of serious liver inflammation was associated with age, ALT levels, HBV DNA levels and HBeAg status (P＜0.05). There was no obvious association between HBV DNA level and liver fibrosis (P＞0.05). Conclusions There were obvious liver inflammation and different degree of liver fibrosis in CHB patients with alanine aminotransferase(ALT)lower than 2 times ULN. The degree of liver injury assessed by liver biopsys is recommended as an evaluation for the necessary of anti-viral therapy.

This research established an HPLC method for determination of six C-Glycoside flavones of warer-soluble total flavonoids from Isodon lophanthoides var. gerardianus (Benth.) H. Hara, and studied the antitumor activity of the warer-soluble total flavonoids. The HPLC system consisted of Kromasil 100-5 C18 (4.6 mm x 250 mm, 5 microm) column and a solution system of methanol, acetonitrile and 0.5% formic acid gradient elution at a flow rate of 0. 8 mL x min(-1) and the wavelength of detector was at 334 nm. The column temperature was 25 degrees C. The antitumor activity of water-soluble flavonoids was assayed using HepG2 cell as the tested cell. The linear ranges of vicenin II, vicenin III, isoschaftoside, schaftoside, vitexin, 6, 8-di-C-a-L-arabinosylapigenin were 0.25-2.53, 0.12-1.20, 0.37-3.69, 0.16-1.63, 0.19-1.92, 0.14-1.42 microg, respectively. The average recoveries (n = 6) were 99.6% (RSD 0.87%), 100.2% (RSD 2.0%), 99.6% (RSD 1.8%), 97.9% (RSD 1.5%), 98.8% (RSD 1.2%), 98.6% (RSD 1.2%), respectively. After exposure in 24, 48, 72 h, the total flavonoids showed inhibitory effect on the proliferation of HepG2 cells with IC50 as the evaluation index, the IC50 values of 1.89, 1.71, 1.51 g x L(-1), respectively. The method is quick, simple and accurate with good re- producibility, and can be used for determination of vicenin II, vicenin III, isoschaftoside, schaftoside, vitexin, 6, 8-di-C-a-L-arabino- sylapigenin in the warer-soluble total flavonoids from L lophanthoides var. gerardianus. The warer-soluble total flavonoids from L lophanthoides have inhibitory effect on the proliferation of HepG2 cells.
Antineoplastic Agents, Phytogenic ; analysis ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; analysis ; pharmacology ; Flavones ; analysis ; pharmacology ; Humans ; Isodon ; chemistry ; Monosaccharides ; analysis ; pharmacology

OBJECTIVETo establish a non-invasive, repeatable and dynamic study method in endometriosis rat model using magnetic resonance imaging (MRI), in order to explore the magnetic resonance characteristics of the model.

METHODEndometrium tissues were transplanted into left abdominal walls of unmated adult female SD rats. After surgery, pathological changes were observed and MRI scanning was made for the ectopic lesions.

RESULTThe endometriosis rat model was successfully established and the ectopic lesions imaged strong hyperintense on DWI, hypointense on T1WI, hyperintense on T2WI with a clear border, without enhancement on CE-T1 WI.

CONCLUSIONThe lesions can be clearly observed in the MRI images on the endometriosis rat model established by this method, which facilitates repeat experiments and continuous observation studies.

Animals ; Endometriosis ; diagnosis ; diagnostic imaging ; pathology ; Female ; Humans ; Magnetic Resonance Imaging ; methods ; Radiography ; Rats ; Rats, Sprague-Dawley

OBJECTIVERecent studies showed that adenosine played important roles in vasodilation. This study aimed to investigate the effects of adenosine, its A1 and A2b receptor agonists on pulmonary artery hypertension (PAH) induced by chronic hypoxia in rats by continuously subcutaneous administration with an osmotic pump for 14 days, and to see if rennin angiotensin system and inducible nitric oxygen synthase (iNOS)/nitric oxide (NO) mediate the effects.

METHODFifty-six male SD rats were randomly assigned to seven groups. Each group included eight rats. They were normoxic group, hypoxic group, adenosine-treated group [adenosine was administered at a dose of 150 µg(kg·min) under the hypoxic condition], adenosine A1 receptor agonist CPA-treated group [CPA was administered at a dose of 20 µg/(kg·min) under the hypoxic condition], CPA plus selective adenosine A1 antagonist DPCPX-treated group [CPA and DPCPX were administered simultaneously under the hypoxic condition, the dose of CPA was the same as the above, and the dose of DPCPX was 25 µg/(kg·min)], adenosine A2b receptor agonist NECA-treated group [NECA was administered at a dose of 30 µg/(kg·min) under the hypoxic condition], NECA plus selective adenosine A2b receptor antagonist MRS-treated group[ NECA and MRS1754 were administered simultaneously under the hypoxic condition, the dose of NECA was the same as the above, and the dose of MRS1754 was 50 µg/(kg·min)]. Osmotic pumps containing adenosine or selective adenosine A1 receptor agonist (CPA), or nonselective but potent adenosine A2b receptor agonist (NECA) were placed subcutaneously 7 days after hypoxia and continuously administered the agents for 14 days.Mean pulmonary artery pressure (mPAP) was detected after administration of the agents. Then blood samples were taken from heart for measurement of renin activity, angiotensin II (AngII) and endothelin-1 (ET-1) concentration by radioimmunoassay, NO by measuring nitrate. Small pulmonary arteries were prepared for immunoreactivity staining of proliferating cell nuclear antigen (PCNA) and iNOS.

RESULT(1) Chronic hypoxia induced PAH [mPAP: (31.38 ± 3.42) mm Hg]. Adenosine or CPA or NECA administered for 14 days by subcutaneous route attenuated the mPAP [(21.17 ± 3.56) mm Hg, (22.88 ± 2.95) mm Hg, (19.81 ± 2.39) mm Hg, respectively], which showed significant difference when compared with hypoxia group (P < 0.05 respectively). (2) Plasma rennin activity and AngII level in hypoxia group [(2.51 ± 0.25) ng/(ml·h), (83.01 ± 9.38) pg/ml] were significantly higher than that in normoxic group (P < 0.05, respectively).(3) Adenosine treatment decreased the rennin activity and AngII level when compared with hypoxic group(P < 0.05, respectively);CPA and NECA attenuated respectively the rennin activity and AngII level of rats induced by chronic hypoxia (P < 0.05, respectively). (4) Adenosine administration for 14 days attenuated the wall thickness induced by chronic hypoxia (P < 0.05). CPA showed no effect on wall thickness, but NECA significantly attenuated the wall thickness (P < 0.05). (5) The number of iNOS staining positive cells in small pulmonary artery was higher in hypoxia group than in that in normoxic rats (23.75 ± 7.91 vs. 8.00 ± 2.20, P < 0.05). Adenosine or CPA, or NECA administration increased respectively the iNOS expression in rats treated with chronic hypoxia. Chronic hypoxia caused significant decrease of nitric oxide level. Adenosine treatment increased the nitric oxide level in rats treated with chronic hypoxia. CPA and NECA also increased respectively the nitric oxide level in rats treated with chronic hypoxia. Chronic hypoxia caused significant increase of ET-1 level. The ET-1 level in rats treated with adenosine, CPA or NCEA respectively were lower than that in chronic hypoxia rats (P < 0.05). (6) Adenosine treatment partially attenuated the number of PCNA-positively stained cells. NECA treatment also attenuated the PCNA expression, but CPA showed no effect.

CONCLUSIONAdenosine and its agonists CPA, NECA administered continually by subcutaneous route attenuate mPAP of rats induced by chronic hypoxia. CPA attenuates mPAP through reduction of RA/AngII activity and balance of NO/ET-1 level. NECA attenuates mPAP by inhibiting PCNA expression and proliferation of mooth muscle of pulmonary artery.

Adenosine ; administration & dosage ; pharmacology ; Angiotensin II ; blood ; Animals ; Disease Models, Animal ; Endothelin-1 ; metabolism ; Hypertension, Pulmonary ; drug therapy ; etiology ; metabolism ; Hypoxia ; complications ; Male ; Nitric Oxide ; blood ; Nitric Oxide Synthase Type II ; metabolism ; Proliferating Cell Nuclear Antigen ; metabolism ; Pulmonary Artery ; drug effects ; physiopathology ; Purinergic P1 Receptor Agonists ; administration & dosage ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Renin ; blood

OBJECTIVETo investigate the effect of Ca(2+) mobilization on release and activation of matrix metalloproteinases (MMPs) in human hepatocellular carcinoma cells.

METHODSCa(2+) and chemicals which can induce or inhibit Ca(2+) mobilization were added into human SMMC-7721 hepatoma cells in vitro. SDS-PAGE protein electrophoresis and gelatin zymography analysis were carried out to detect the changes of release and activation of MMPs in the cell culture supernatant.

RESULTSAddition of CaCl(2) into culture system resulted in an enhanced secretion and activation of MMP-2 and MMP-9 in a dose-dependent manner. At a dose of 0.8 mmol/L CaCl(2), it maintained a stable high level of MMPs, especially of MMP-2 with (109.71 +/- 27.93)% elevation as compared to the cells without CaCl(2) addition (P < 0.001). SDS-PAGE analysis showed that most secreted proteins were MMPs (MMP-2 and MMP-9) when the cells cultured in media without serum. Thapsigargin (Tg, 4 micromol/L), an inducer of intracellular Ca(2+) stores depletion, significantly enhanced the release and activation of MMP-2 and MMP-9, compared to the control with (58.63 +/- 31.04)% elevation (P < 0.05), while the inducing effect of Tg on MMPs release and activation was significantly inhibited by S-nitro-N-acetylpenicillamine (SNAP, 200 micromol/L), an NO donor.

CONCLUSIONIntracellular Ca(2+) regulation pathways may play an important role in the process of release and activation of MMPs.

Calcium ; metabolism ; Carcinoma, Hepatocellular ; enzymology ; pathology ; Cell Line, Tumor ; Dose-Response Relationship, Drug ; Humans ; Liver Neoplasms ; enzymology ; pathology ; Matrix Metalloproteinase 2 ; metabolism ; secretion ; Matrix Metalloproteinase 9 ; metabolism ; secretion ; Nitric Oxide Donors ; pharmacology ; Penicillamine ; analogs & derivatives ; pharmacology ; Thapsigargin ; pharmacology

OBJECTIVETo compare the liver pathohistological and clinical features between chronic HBV carriers and chronic hepatitis B patients with mild elevated in ALT.

METHODS128 patients were divided into 3 groups according to the ALT: group A: ALT is less than or equal to 0.5*ULN, group B: 0.5*ULN less than ALT is less than or equal to 1*ULN, group C: 1*ULN less than ALT less than 2*ULN. The age, sex, serum HBV DNA, HBeAg status, expression of HBcAg in liver, thickness of spleen, breadth of portal vein ,blood stream speed of protal vein, right liver obliqua diameter, grade of liver inflammation and stage of liver fibrosis were compared in the three groups.

RESULTSAmong 128 patients, 57(44.5%) patients had G1 hepatitis and 71 (55.5%) had G2 hepatitis, no G0 hepatitis was found in these patients; 72 patients (56.3%) had S1 fibrosis, 30 (23.4%) patients had S2 fibrosis, and 26 (20.3%) patients did not have liver fibrosis. The liver inflammation in group C was more aggravated than that in group A (P less than 0.05). And there were significant differences in thickness of spleen and right liver obliqua diameter between group C and group A, as well as between group C and B (P all less than 0.01). With the aggravating of liver inflammation, the serum ALT, thickness of spleen, breadth of portal vein and expression of HBcAg in liver were increased obviously (P less than 0.05). With the aggravating of liver fibrosis, the thickness of spleen, breadth of portal vein, right liver obliqua diameter and HBeAg negative patients were increased obviously, while the blood stream speed of portal vein was decreased obviously (P less than 0.01).

CONCLUSIONAmong the chronic HBV infection patients whose ALT less than 2*ULN, there were 55.5% patients had G2 of liver inflammation and 23.4% patients had S2 of liver fibrosis. The serum ALT, thickness of spleen, breadth and blood stream speed of portal vein, right liver obliqua diameter and expression of HBcAg in liver are associated with pathohistological changes in these patients.

Adult ; Alanine Transaminase ; blood ; Biopsy, Needle ; Carrier State ; blood ; pathology ; virology ; DNA, Viral ; blood ; Female ; Hepatitis B Core Antigens ; metabolism ; Hepatitis B e Antigens ; blood ; Hepatitis B, Chronic ; blood ; pathology ; virology ; Humans ; Liver ; metabolism ; pathology ; virology ; Liver Cirrhosis ; pathology ; Male ; Polymerase Chain Reaction ; methods ; Retrospective Studies ; Virus Replication

OBJECTIVEAccurate and reliable assessment of renal function is important in the management of children with chronic kidney disease (CKD). Glomerular filtration rate (GFR) is the best index of assessing kidney function. For assessment of GFR, both gold standard tests and prediction equations have been used. The well-known 24-hour endogenous creatinine clearance (Ccr), the Schwartz formula and the Filler formula are increasingly used in daily clinical practice. However, there are few studies on the applicability of these prediction equations for estimating GFR in Chinese children with CKD. The aim of this study was to compare these prediction equations estimating GFR with an isotope clearance method [isotope glomerular filtration rate (rGFR)] in such patients.

METHODChildren aged 1-16 years who underwent isotope (99m)Tc-diethylenetriaminepentaacetic acid ((99m)Tc-DTPA) GFR testing (Gates' method) between the year of 2002 and 2005 were studied retrospectively. GFR was estimated using: (1) 24-hour Ccr, which was calculated using the standard formula: [urine creatinine (milligrammes per millilitre) × 24-hour urine volume/serum creatinine (milligrammes per millilitre) × 1440] × [1.73 (m(2))/body surface area (m(2))]; (2) the Schwartz formula, which is: eGFR (ml/min per 1.73 m(2)) = k × height (centimetres)/serum creatinine (micromoles per litre), where k is 62 in males at 13 years of age and older, 40 in infants, and 49 in all other children; and (3) the Filler formula, which is: logGFR = 1.962 + [1.123 × log(1/Cys C)], where cystatin C is measured in milligrammes per litre. Serum and urinary creatinine levels were detected by alkaline kinetic method. Serum cystatin C was analysed by particle-enhanced immunoturbidimetric assay. Bias and precision were evaluated.

RESULTThirty subjects (18 males and 12 females; mean age 9.4 years) fulfilling both inclusion criteria and exclusion criteria were included in this study. The mean (SD) rGFR was 81.57 (36.92) ml/min per 1.73 m(2); 18 subjects were in CKD stage I, 8 in CKD stage II, 8 in CKD stage III, and 1 in CKD stage IV. Only the mean 24 h Ccr-eGFR was slightly higher than rGFR (0.4 ml/min per 1.73 m(2) higher). Within 95% limits of agreement, the maximum absolute value of bias was about 50 ml/min per 1.73 m(2). Accuracy (estimated GFR values within ± 30% of rGFR) for all formulae was poor, ranging from 23.3% to 43.3%. All formulae overestimate or underestimate rGFR in different CKD stages.

CONCLUSIONIn Chinese children with CKD, there was a significant difference between measured GFR and estimated GFR using 24h Ccr, Schwartz formula and Filler formula. More suitable GFR predictive equations to assess glomerular function of such patients should be developed.

Adolescent ; Child ; Child, Preschool ; Female ; Glomerular Filtration Rate ; Humans ; Infant ; Kidney Failure, Chronic ; physiopathology ; Kidney Function Tests ; Male ; Renal Insufficiency, Chronic ; physiopathology