Objective To establish a stable HCV full-length genome replication cell model which is labeled with reporter gene and easyly to quantify intracellular HCV proteins and RNA level. Methodsneo gene was inserted into Luc-JC1 to make Luc-JC construct. Luc-JC RNA was obtained by in vitro transcription and then delivered into Huh7 cells by transfection. G418-resistant clones of Huh7 cells were obtained by selection. Clones of HCV full-length genome replication cell were confirmed by luciferase activity assay, Western blot and cleaveage of eYFP-MAVS by HCV NS3/4A protease. Then, HCV replication cell colonies were treated by different dose IFN-α in order to observe the change of luciferase activity, HCV protein and RNA level. Results At 3-4 weeks post-transfection, visible colonies were selected and stained by crystal violet. Luciferase activity and HCV NS3, NS5A protein were detected by luciferase activity assay and Western blot, respectively. Subcellular localization of eYFP-MAVS transferred from mitochondria to cytoplasms by cleavage of NS3/4A protease in cell colonies. Luciferase activity, HCV protein and RNA diminished obviously after IFN-α treatment. Conclusion A stable HCV full-length genome replication cell model labeled by reporter gene was successfully established and reporter activity can be used to indicate level of HCV proteins and RNA in cells. This cell model is a useful tool for the study on HCV pathogenesis and the screening of antiviral drugs.

Objective To ascertain the utility and difference of sonography with echo-tracking (ET) technique and pulse-Doppler to assess vascular stiffness in rats with hypercholesterolemia and atherosclerosis.Methods Sonography associated with ET technique and pulse-Doppler were used to measure stiffness parameter (β),arterial compliance (AC),distensibility coefficient (DC),one-point pulse wave velocity (PWVβ),resistence index(RI),peak systolic velocity(PSV),end-diastolic velocity(EDV) and EDV/PSV of the aorta in cholesterol-fed SD rats (group T1,n =10,for 4 weeks;group T2,n =10,for 12 weeks) and normal control rats(group C1,n =10;group C2,n =10).All parameters and blood biochemical markers[total cholesterol(TC),triglycerides(TG),low-density lipoprotein cholesterol(LDL-CH) and highdensity lipoprotein cholesterol (HDL-CH)] among groups were analyzed with ANOVE factor analysis.Correlation was analyzed with Pearson analysis.Light microscopic evaluation were used to demonstrate atherosclerotic changes in the aorta.Results The PWVβ value and PSV of the aorta between group T1 and T2 were significantly different (P =0.001,P ＜0.05).The β,PWVβ values of the aorta in group T1 and T2 were significantly higher than those of group C1 and C2 (P ＜0.05).AC and DC values of the aorta in group T1 and T2 were significantly lower than those of group C1 and C2 (P ＜0.05).Correlation analysis showsed that RI was positively correlated with systolic pressure(P ＜0.05).All parameters had correlated with each other among β,PWVβ,AC,DC,TG,TC,systolic pressure and diastolic pressure.DC and AC were negatively correlated with β and PWVβ,also DC was negatively correlated with TG.Light microscopy confirmed morphologic typical changes of aortic atherosclerosis in group T1 and T2.Conclusions Sonography with the ET method compared with pulse-Doppler is much more sensitive and it can be used to evaluate tissue elastic changes in arterial walls associated with atherosclerosis and hypercholesterolemia.PSV can reflect atherosclerosis of rat's abdominal aorta well,but pulse-Doppler is limited in the diagnosis of earlier atherosclerosis period.

The Wnt signaling pathway plays an important role in bone metabolism. Inducing the Wnt signaling pathway promotes bone formation while restraining it results in osteopenic states. Although the regulation of this signaling pathway may bring enormous therapeutic potential, it still requires cautious approach because of the risks of tumorigenesis. The role of the Wnt signaling pathway in bone metabolism and the molecular targets of therapeutic potential for osteoporosis are discussed in this review.

OBJECTIVETo investigate the potential role of the preventive nutritional support in patients with nutritional risk defined by nutrition risk screening 2002(NRS 2002) before radical resection of gastric cancer.

METHODSPatients with gastric cancer were evaluated by NRS 2002 preoperatively. Elective patients with nutritional risk were randomly assigned into 3 d preventive enteral nutrition(EN) group versus control group. The preventive nutrition regimen was 1000 ml Nutrison Multi Fibre(4184 kJ/L). The changes in body weight lost, serum albumin, immunoglobulin were recorded postoperatively.

RESULTSOne week after operation, the preventive EN group showed less decrease in body weight as compared to control group, which was statistically significant. The levels of serum albumin and IgA on day 1 and day 3 after operation in preventive EN group were significantly higher than those in control group.

CONCLUSIONBefore operation for gastric cancer, patients with nutritional risk defined by NRS 2002 may benefit from preventive enteral nutrition, which improves the patients' nutritional condition and enhances their immunologic function.

Adult ; Aged ; Enteral Nutrition ; Female ; Humans ; Male ; Middle Aged ; Nutrition Assessment ; Preoperative Care ; Stomach Neoplasms ; diet therapy

AIM To optimize the subcritical aqueous extraction for polysaccharides from the leaves of Punica granatum L.and to evaluate the in vitro antioxidant activity.METHODS With reaction pressure,solid-liquid ratio,extraction time and extraction temperature as influencing factors,yield of polysaccharides as an evalution index,the extraction was optimized by Box-Behnken method on the basis of single factor test.Then the scavenging effects of polysaccharides on hydroxyl free radical,superoxide anion and DPPH free radical were detected.RESULTS The optimal conditions were determined to be 5 MPa for reaction pressure,1 ∶ 27 for solid-liquid ratio,11 min for extraction time,and 155 ℃ for extraction temperature,the yield of polysaccharides was 1.809％.There was a dose-effect relationship between scavenging rate and polysaccharides' concentration.0.1 mg/mL Polysaccharides displayed the strongest scavenging effects on hydroxyl free radical,superoxide anion and DPPH free radical with the clearance rates of 57.36％,70.51％ and 58.02％,respectively.CONCLUSION This stable and reliable method can be used for the subcritical aqueous extraction for polysaccharides from P.granatum leaves with obvious in vitro antioxidant activity.

Objective:To methodologically establish the lentivirus granule packaging, concentration and infection against CD34~+ cells from umbilical blood. Methods:The lentivirus system of the 3~(rd) generation was used to produce the virus. Ultrafiltration and ultracentrifugation were employed to concentrate virus. Several treatments were used to improve virus infection including in vitro amplification culture, facilitation of rest cells into cell cycle, promotion of cell adhesion and immobilization during infection, and repeat infection methods. Results:CD34~+ cells were not obviously changed by checking the expression level of CD34 marker on the cell surface after 48 h culture. After two-step concentration, virus titer was increased up to 5.06×10~7/ml, and the infection rate against CD34~+ cells from umbilical blood was increased up to 37.7%.Conclusion:Lentivirus supernatant with over 10~7/ml titer can be obtained using the above methods. Efficient infection against CD34~+ cells from umbilical blood can be achieved.

Objective To establish the imatinib (STI-571)-resistant subline in vitro and investigate its biological characteristics. Methods Human glioblastoma multiform drug-resistant cell line (named U251AR) was established in vitro by successively increasing the concentration of imatinib in a cell culture medium. The 50％ inhibitory dose (IC50) values and the resistance indexes ([IC50U251/STI-571]/[IC50 U251]) for other chemotherapeutic agents were evaluated using cell counting kit-8 assays. Expressions of acquired multidrug resistance P-glycoprotein (MDR 1, ABCB 1; MDR3, ABCB4),breast cancer resistance protein (BCRP, ABCG2) and multidrug resistance-associated protein 1 (MRP1,ABCC1) were detected by QRT-PCR. Flow cytometry was employed to detect the protein expression of ABCG2. Results The U251AR was developed after culture for 12 months and similar morphologies of U251 and U251/STI-571 cells were determined. The resistance coefficient of U251AR cells to imatinib was 20.41 times more than that of the parent cells, and U251AR cells showed cross-resistance to many anti-tumor agents (P＜0.05). The resistance coefficients of U251AR cell line to doxorubicin and cisplatin were 5.06 and 10.28 times, respectively, more than those of U251 cells (P＜0.05). QRT-PCR indicated that the mRNA levels of MDR1, MRP1, BCRPandABCB4 (P-g4) in the U251/STI571 resistant cells were significantly higher than those in the U251 cells (P＜0.05). The protein expression of ABCG2 in U251AR cell line was significantly increased as compared with that in the parent cells (P＜0.05).Conclusion We have successfully established multidrug resistant cell line U251AR, and the drug resistance of U251/STI571 is associated with over-expressions of ABCC1, ABCB1, ABCB4, and ABCG2 mRNA, and ABCG2 protein.

OBJECTIVETo observe the protective effects of safflor Injection (SI) and extract of Ginkgo biloba (EGB) on lung ischemia-reperfusion injury (LIRI) and investigate its mechanism.

METHODSIn vivo rabbit model of LIRI was reconstructed. Forty rabbits were randomly and equally divided into four groups: sham-operation group (sham group), ischemia-reperfusion group (model group), ischemia-reperfusion plus SI group (safflor group) and ischemia-reperfusion plus EGB injection group (EGB group). Malondialdehyde (MDA) content, superoxide dismutase (SOD) and xanthine oxidase (XO) activity in serum were measured. The wet/dry weight ratio (W/D) of the lung tissue and activity of myeloperoxidase (MPO) were also tested. Ultrastructure change of the lung tissue was observed by the electron microscope. The expression of intercellular adhesion molecule-1 (ICAM-1) was measured by immunohistochemistry (IHC).

RESULTSIn the model group, MDA and XO increased and SOD decreased in serum compared with the sham group (P<0.01). The values of W/D, MPO and ICAM-1 of the model group were higher than those of the sham group (P<0.01), but those of the safflor group and EGB group were significantly lower than those of the model group (P<0.01). The IHC demonstrated that ICAM-1 expression in lung tissue of the model group was significantly higher than those of the safflor group (P<0.01). Compared with safflor group, in the EGB group MDA, XO, MPO decreased, SOD and ICAM-1 expression increased (P<0.05), but the change of W/D was not statistically significant (P>0.05).

CONCLUSIONSSI and EGB may attenuate LIRI through antioxidation, inhibition of neutrophil aggregation and down-regulation of ICAM-1 expression. But EGB had more effect on the antioxidation, while SI did better on regulating ICAM-1 expression.

Animals ; Female ; Ginkgo biloba ; chemistry ; Immunohistochemistry ; Injections ; Intercellular Adhesion Molecule-1 ; metabolism ; Lung ; blood supply ; pathology ; Male ; Malondialdehyde ; metabolism ; Plant Extracts ; administration & dosage ; pharmacology ; therapeutic use ; Protective Agents ; administration & dosage ; pharmacology ; therapeutic use ; Rabbits ; Reperfusion Injury ; blood ; drug therapy ; Safflower Oil ; administration & dosage ; pharmacology ; therapeutic use ; Superoxide Dismutase ; blood ; Xanthine Oxidase ; blood

OBJECTIVETo study the effect of hirudin on the function of human hyperplastic scar fibroblasts (HSFBs).

METHODSHSFBs were cultured in vitro. Hirudin solution in the concentration of 1, 10, and 50 kU/L was respectively added into DMEM culture medium to form 1, 10, and 50 kU/L hirudin groups, with 9 wells in each group. HSFBs cultured without hirudin were set up as control group. Cell inhibition rate, secretion level of TGF-beta1 from cells, and expression levels of mRNA of type I and III precollagen were determined at 24, 48, and 72 h after culture.

RESULTSInhibition rates of HSFBs growth was respectively (29.3 +/- 0.9)%, (30.1 +/- 0.3)%, and (45.2 +/- 1.9)% when cultured with 10 kU/L hirudin for 24, 48, and 72 hs, which were higher than those in control group [(0.0 +/- 0.0)%, P < 0.05]. There was statistically significant difference between control group and 1 and 50 kU/L hirudin groups in the inhibition rates of HSFBs at some time points (P < 0.05). Secretion level of TGF-beta1 of HSFBs in 1, 10, 50 kU/L hirudin groups was respectively (228.5 +/- 1.8), (210.5 +/- 11.1), and (168.5 +/- 14.1) pg/mL when cultured for 48 hs, of which the last 2 figures were significantly lower than that of control group [(265.0 +/- 1.5) pg/mL, P < 0.05]. Hirudin in the concentration of 10 and 50 kU/L could inhibit the expression of mRNA of type I and III precollagen in HSFBs.

CONCLUSIONSHirudin solution in the concentration of 10 and 50 kU/L can inhibit the proliferation of HSFBs and secretion of TGF-beta1 and collagen in certain degree.

Cells, Cultured ; Cicatrix, Hypertrophic ; pathology ; Fibroblasts ; cytology ; drug effects ; secretion ; Hirudins ; pharmacology ; Humans ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo study the analgesia mechanism of needle-knife lysis in spinal cord and other parts of central nervous system by comparing the changes of Leu-Enkephalin (L-ENK) content in different parts of centrium of rats undergone needle-knife lysis and electro-acupuncture respectively.

METHODSSixty healthy SD rats were randomly devided into normal control group, model group, needle-knife lysis (NKL) group and electro-acupuncture (EA) group. 4% papain solution mixed with 0.3 mol/L cysteine solutin in the ratio of 1:1, paused for 0.5 h,injected the mixture, 20 microl each time,into the left knee joint cavities of rats in model, NKL, EA groups at the 1st, 4th, 7th day. After 4 weeks in NKL group and EA group were treated with needle-knife lysis and electro-acupuncture, respectively. Three weeks after treatment, samples of spinal cord of the swollen part of rat waists and rat brains were taken from and the content of L-ENK of medulla oblongata, midbrain, pituitary gland, and hippocampus were measured.

RESULTSL-ENK content of model group increased higher than that of normal control group in spinal cord, hippocampus and midbrain (P < 0.01); there were no significant difference between normal control group and modle group on L-ENK in medulla oblongata and thalamus (P > 0.05). After intervening of NKL or EA, L-ENK content of NKL group increased higher in hippocampus than that of model group and EC group (P < 0.05); but L-ENK content of NKL group in midbrain was lower than that of model group (P < 0.05).

CONCLUSIONNeedle-knife lysis has characteristic of regulation for the L-ENK content in different parts of central nervous system of rats with knee osteoarthritis, and analgesic effect of needle-knife was possibly related with regulation of center L-ENK.

Acupuncture Therapy ; methods ; Animals ; Central Nervous System ; chemistry ; Electroacupuncture ; Enkephalin, Leucine ; analysis ; Female ; Male ; Osteoarthritis, Knee ; metabolism ; therapy ; Rats ; Rats, Sprague-Dawley