In the present study, the specifically knockdown models of P-gp or MRP2 were constructed by using a series of chemically synthesized small interfering RNA (siRNA) in vitro. The expression of P-gp and MRP2 was measured by real-time PCR and Western blot, and the function was evaluated by applying P-gp and MRP2 substrate, rhodamine and methotrexate. The results showed that MRP2 siRNA-3 or P-gp siRNA-2 significantly decreased the mRNA expression of MRP2 or P-gp, the inhibition ratio was 68% or 84%; MRP2 siRNA-3 or P-gp siRNA-2 at a dose of 80 nmol x L(-1) significantly reduced the protein expression of MRP2 or P-gp at 48 h after treatment, the inhibition ratio was 62% or 70%. Meanwhile, other transporters were not influenced by siRNA. When pretreatment with MRP2 siRNA-3 or P-gp siRNA-2, the efflux of methotrexate or rhodamine decreased significantly and the intra-cellular concentration increased. The results suggested that chemically synthesized siRNA could significantly inhibit the expression and function of MRP2 and P-gp, and the model of RNAi in vitro could be used to evaluate the role of efflux transporters in transportation of drugs.
ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; Gene Knockdown Techniques ; Multidrug Resistance-Associated Proteins ; genetics ; RNA Interference ; RNA, Small Interfering ; Real-Time Polymerase Chain Reaction

OBJECTIVETo study the effects of acute cold exposure on the inflammation and pathologic injuries in pulmonary of rats, and explore the mechanism induced by cold stress.

METHODSForty male Wistar rats were randomly divided into five groups(n = 8): control group (23 ± 2) °C 2.5 h, -25°C 0.5 h group, -25°C 1 h group, -25°C 2 h group and -25°C 2.5 h group. Rats were exposed to cold at -25°C and no wind by keeping them in a low temperature chamber except control group. Rectal temperatures of the rats were measured before and after cold exposure. The morphological changes of pulmonary were observed by the optics microscope. The levels of tumer necrosis factor-α(TNF- α), interleukin-6 (IL-6) and interleukin-β (IL-1β) in lung tissue homogenate were measured by ELISA.

RESULTSCompared to the control group, body core temperatures of the -25°C 1 h group, -25°C2 h group and -25°C 2.5 h group were decreased significantly, and the D-values of rectal temperature were increased before and after cold exposure (P < 0.05). The infiltration of inflammatory cells and alveolar edema fluid appeared in the lung tissue of the -25°C 2.5 h group. The concentrations of tumor necrosis factor-α (TNF α), interleukin-6 (IL-6) and inter- leukin-1β (IL-1β) in lung tissue homogenate were increased significantly in -25°C l h group, -25°C 2 h group and -25C° 2.5 h group (P < 0. 05).

CONCLUSIONThe infiltration of inflammatory cells and the increase in proinflammatory cytokine from pulmonary may lead to the lung tissue injury after acute cold exposure.

Animals ; Cold Temperature ; adverse effects ; Inflammation ; physiopathology ; Interleukin-1beta ; metabolism ; Interleukin-6 ; metabolism ; Lung ; metabolism ; physiopathology ; Male ; Rats ; Rats, Wistar ; Tumor Necrosis Factor-alpha ; metabolism

Adult ; Exons ; Female ; Gastrointestinal Stromal Tumors ; genetics ; metabolism ; pathology ; Gene Deletion ; Humans ; Proto-Oncogene Proteins c-kit ; genetics ; metabolism

OBJECTIVETo evaluate the effects of antiviral agents on intrahepatic HBV covalently closed circular DNA (cccDNA) in HBeAg-positive chronic hepatitis B patients.

METHODSSeventy-one HBeAg positive chronic hepatitis B patients were enrolled in this study. Lamivudine was administered to 35 patients for 48 weeks, sequential therapy with lamivudine-IFN alpha-2b to 24 of the 71 patients for 48 weeks, and interferon alpha (IFN alpha-2b) was administered to 12 for 24 weeks. All subjects were followed-up for 24 weeks. Serum HBV DNA, intrahepatic HBV DNA and cccDNA were measured quantitatively by PCR. HBV genotypes were analyzed by PCR-RFLP.

RESULTSForty-eight weeks of sequential lamivudine-IFN alpha-therapy and lamivudine monotherapy and 24 weeks of IFN alpha monotherapy reduced the intrahepatic HBV DNA to (4.7+/-1.1) log10, (4.6+/-1.5) log10 and (5.6+/-1.5) log10, and cccDNA to (3.4+/-1.3) log10, (3.8+/-1.1) log10 and (5.0+/-1.5) log10, significantly lower than therapy (P < 0.05). Seventeen of the 71 patients developed HBeAg seroconversion, and the reduction of cccDNA in the HBeAg seroconverted patients was significantly more than that of the HBeAg positive patients (P < 0.05). After 24 weeks of antiviral therapy withdrawal, 18 patients achieved sustained virological response, and the baseline intrahepatic cccDNA in the patients with sustained virological response was significantly lower than that of patients with virological rebound (P < 0.05). The change in intrahepatic cccDNA correlated positively with the reduction in intrahepatic HBV DNA (P < 0.05). The cccDNA levels correlated with the serum HBeAg titers at the end of the treatment (P < 0.01). Of the total 71 cases, HBV genotype C accounted for 85.9% (n = 61), and genotype B for 14.1% (n = 10). There was no significant difference in the changes of intrahepatic HBV DNA and cccDNA levels between HBV genotypes C and B (P >0.05).

CONCLUSIONSBoth 48 weeks of sequential lamivudine-IFN alpha and lamivudine monotherapy strongly reduced intrahepatic HBV DNA and cccDNA more than 24 weeks of IFN alpha monotherapy. Low baseline intrahepatic cccDNA levels might predict a good long-term efficacy of antiviral treatment. The reduction of intrahepatic cccDNA correlated positively with the changes of intrahepatic HBV DNA, and intrahepatic cccDNA levels correlated with serum HBeAg titers. HBV genotypes had no obvious influence on intrahepatic HBV DNA load or cccDNA load.

Adult ; Antiviral Agents ; pharmacology ; therapeutic use ; DNA, Circular ; DNA, Viral ; Female ; Hepatitis B e Antigens ; blood ; Hepatitis B virus ; drug effects ; genetics ; Hepatitis B, Chronic ; drug therapy ; virology ; Humans ; Interferon-alpha ; pharmacology ; therapeutic use ; Lamivudine ; pharmacology ; therapeutic use ; Male ; Middle Aged ; Recombinant Proteins ; Young Adult

Objective To evaluate the application of western blot in treponema pallidum antibody screening among blood donors.Methods Through standard blood liquidation of syphilis,the sensitivity and specificity of two kinds of TP -ELISA regents and one TPPA regent were evaluated.TP -WB was used to test 279 positive specimens with TP -ELISA,and the correlation between test results of the two methods and the distribution characteristics of WB bands were analyzed. Results The sensitivity of the two kinds of TP -ELISA reagent was 1 00.00%,while the specificity were 92.86% and 85.71 % respectively.At the same,the sensitivity of TPPA reagent was 88.46%,while the specificity was 1 00.00%.In 279 positive specimens with TP -ELISA method,WB confirmed positive was 21 6,positive rate was 77.42%;Including S /Co value >5 and two kinds of ELISA reagent testing both positive specimens were 205,the WB confirmation test positive rate was 1 00.00%,accounted for 99.91 % in 21 6 WB positive samples.Orderly Logistic regression analysis,the method of ELISA S /Co value >5 and double reagent is positive,had statistical correlation with test positive for WB,respectively(P <0.01 );21 6 TP -WB positive specimens WB banding distribution analysis,TP1 7 belt with syphilis antibody IgM,TP1 5 belts and IgG +IgMand IgG exist statistical correlation respectively(P <0.01 ).Conclusion ELISA method of double reagent positive and S /Co value >5 specimens of the basic can be diagnosed with syphilis,WB test with positive results for blood donation member state judgment has guiding significance.

Objective To investigate the effect of the compound glycy-rrhizin on hepatitis related factors of hepatitis B in serum of model rat. Methods Sixty specific pathogen free animal rats were selected and divided into 6 groups, normal group, model group, low dose group, mid-dle dose group, high dose group and control group.Normal group and model group were gavaged with the volume of saline 3 mL daily, the experimental group were gavaged with 3 concentrations ( 10, 20, 30 mg? kg-1 ) of compound glycyrrhizin in 3 mL, the control group were gavaged with concentrations 23.3 mg? mL-1 of lamivudine solution 3 mL daily.The CD4 + T lymphocyte gene expression were detected by polymerase chain reaction method.The T lymphocyte subsets and activin a ( ACT-a) level were detected by Flow cytometry.The tumor free factorα( TNF-α) were detected by radioimmunoassay .Results Compared with the control group,CD4 +and CD8 +Tlymphocyte subsets, TNF-αand ACT -a of the high dose group and middle dose group were significantly lower ( P <0.05 ) .Conclusion Compound glycyrrhizin can inhibit the synthesis of tumor factor, and reduce the ACT-a levels and improve CD4 +and CD8 +T lymphocyte level.

Objective To explore the influencing factors of job satisfaction, social support and dimission intention of nursing staff. Methods Chose 295 cases of medical staff randomly in 2 provincial hospitals in Dalian. Collected data with self-designed questionnaire and analyze influencing factors. Results The influencing factors of job satisfaction included culture level, marriage status, working age, ideal commitment and emotional exhaustion; The influencing factors of social support included culture level, children, individual sense of achievement, standared commitment, economy commitment, society commitment and emotional estrangement;The influencing factors of dimission intention were ideal commitment, night-watch, emotional exhaustion and sentiment estrangement. Conclusions The influencing factors of job satisfaction, social support and dimission intention were defined, we should comprehensively improve physical and mental health of nursing staff aimed at these factors.

OBJECTIVE: To investigate the effect of interleukin-6 (IL-6) on the chemosensitivity of drug-resistant multiple myeloma (MM) cell lines to bortezomib (BTZ) and its mechanism. METHODS: Peripheral blood samples were collected from patients with BTZ-resistant MM before and after treatment. Human MM cell lines KM3 and KM3/BTZ were cultured in vitro. ELISA was used to detect the content of IL-6 in peripheral blood of MM patients, KM3 and KM3/BTZ cells. CCK-8 assay was used to detect the drug sensitivity of KM3 and KM3 / BTZ cells to BTZ. KM3 / BTZ cells were divided into KM3/BTZ control group (normal culture for 48 h), IL-6 neutralizing antibody Anti-IL-6 group (500 ng/ml Anti-IL-6 treated for 48 h), BTZ group (300 ng/ml BTZ treated for 48 h), BTZ + Anti-IL-6 group (300 ng/ml BTZ and 500 ng/ml Anti-IL-6 treated for 48 h). The proliferation activity of KM3 / BTZ cells was detected by CCK-8 assay. The cell cycle distribution of KM3/BTZ cells was detected by flow cytometry. The apoptosis of KM3/BTZ cells was detected by Annexin V-FITC/PI double staining. The mRNA expression levels of IL-6, Notch1, signal transducer and activator of transcription 3 (STAT3) in KM3/BTZ cells were detected by real-time fluorescent quantitative PCR (qRT-PCR), and the protein expression levels of IL-6, Notch1, STAT3 in KM3/BTZ cells were detected by Western blot. RESULTS: The level of IL-6 in peripheral blood of patients with BTZ-resistant MM after treatment was significantly higher than that before treatment (P<0.05). The level of IL-6 in KM3/BTZ cells was significantly higher than that in KM3 cells (P<0.05). The sensitivity of KM3/BTZ cells to BTZ was significantly lower than that of KM3 cells (P<0.05), and the resistance index (RI) was 19.62. Anti-IL-6 and BTZ could inhibit the proliferation of KM3 / BTZ cells, block cell cycle, and induce apoptosis (P<0.05). Compared with single drug treatment, the combined effect of Anti-IL-6 and BTZ was more obvious on KM3/BTZ cells (P<0.05), and significantly down regulated the mRNA and protein expression of IL-6, Notch1 and STAT3 in KM3/BTZ cells (P<0.05). CONCLUSION Antagonizing IL-6 can increase the chemosensitivity of MM cells to BTZ, and IL-6 may reduce the sensitivity of MM cells to BTZ through STAT3/Notch signaling pathway.
Antibodies, Neutralizing/therapeutic use* ; Apoptosis ; Bortezomib/therapeutic use* ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Interleukin-6/metabolism* ; Multiple Myeloma/drug therapy* ; RNA, Messenger ; STAT3 Transcription Factor/metabolism* ; Signal Transduction ; Sincalide/therapeutic use*

OBJECTIVETo evaluate the effect of antiviral agents on intrahepatic HBV DNA and histology in HBeAg-positive chronic hepatitis B patients.

METHODSThirty-five patients were treated with lamivudine, 16 with interferon alfa (INF-alpha), 24 with sequential Lamivudine and INF-alpha. The total duration of therapy was 12 months. Intrahepatic HBV DNA was measured quantitatively by real-time polymerase chain reaction.

RESULTSThere was significant change in all parameters of the groups of patients at the end of treatment (P < 0.05). The patients treated with sequential treatment had slightly higher HBeAg seroconversion rate (38.1%) than that of the other patients (P=0.1352). The baseline levels of intrahepatic HBV DNA in the patients with HBeAg seroconversion or undetectable serum HBV DNA were significantly lower than that of the other patients (P < 0.05).

CONCLUSIONAntiviral agents could effectively inhibit intrahepatic HBV DNA and improve hepatic histology. The patients with low baseline intrahepatic HBV DNA level may achieve better antiviral efficacy. Sequential treatment might produce high HBeAg seroconversion rate.

Adolescent ; Adult ; Antiviral Agents ; pharmacology ; therapeutic use ; DNA, Viral ; blood ; metabolism ; Drug Therapy, Combination ; Female ; Hepatitis B e Antigens ; immunology ; metabolism ; Hepatitis B, Chronic ; drug therapy ; immunology ; pathology ; virology ; Humans ; Interferon-alpha ; pharmacology ; therapeutic use ; Lamivudine ; pharmacology ; therapeutic use ; Liver ; drug effects ; metabolism ; pathology ; virology ; Male ; Middle Aged ; Time Factors

OBJECTIVETo establish a new method to detect HBV cccDNA quantitatively and to apply it to detect cccDNA in liver needle biopsy specimens of chronic hepatitis B patients.

METHODSThe sequences of HBV DNA genotypes A through G were analyzed. According to the different sequence structure of cccDNA and rcDNA, primes and probe were designed in highly conservative region outside the nick of cccDNA in order to amplify cccDNA but not rcDNA. The best conditions of this method were found after testing experiments. Also we checked its specificity and sensitivity and reproducibility. The products of PCR were sequenced in order to ascertain if it was the right region expected. To amplify with standard plasmid ranged from 10(2) to 10(10) copies/ml to measure the sensitivity and amplify in parallel with standard plasmid of 10(6) copies/ml for 30 replicates so as to measure its reproducibility. DNA was extracted from 32 needle liver biopsy specimens of chronic hepatitis B patients. The cccDNA was quantitatively detected with this method. The data of cccDNA obtained before and after therapy and their relationship with total HBV DNA were analyzed. RESULTS Results of sequencing showed that the PCR product was from the right region. The sensitivity was 10(3)-10(10) copies/ml. The Ct value was 29.69+/-0.31 and the coefficient of variability was 1.04 percent calculated from the data of 30 PCR reactions with standard plasmid. The percentage of decrease in serum HBV DNA, total HBV DNA in liver and cccDNA in liver were 0.49+/-0.17, 0.22+/-0.18 and 0.16+/-0.28 respectively. There is 47 percent-98 percent cccDNA in total HBV DNA in liver and the mean is 81.5 percent.

CONCLUSIONThe method is good because of the simple and convenient operation, the high specificity, the wide linear detection range and the fine reproducibility. Therefore it can be used for both scientific research and clinical purpose. Lamividine can significantly inhibit serum HBV DNA by, but its inhibitory effect on cccDNA in liver was rather weak.

DNA, Circular ; genetics ; DNA, Viral ; blood ; genetics ; Hepatitis B ; diagnosis ; virology ; Hepatitis B virus ; genetics ; isolation & purification ; Humans ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity