Objective:To investigate and compare the inhibitory effects on KDR gene expressinon of antisense oligonucleotides(asONs),short hairpin small interfering RNA(siRNA) targeting the mRNA of VEGFR2/KDR(vascular endothelial growth factor receptor 2/kinase insert domain-containing receptor)in MCF-7 cells.Methods:3 antisense oligonucleotides were designed.According to the target KDR mRNA accessible sites of antisense oligonucleotides,3 DNA vector-based pPUR/U6/KDR-siRNA for intracellular transcription KDR specific short hairpin small interfering RNA were constructed.The inhibitory effects on KDR protein expression of antisense oligonucleotides,and pPUR/U6/KDR-siRNA for stable transfection were evaluated using Western-blotting assay.The antisense oligonucleotides inhibitory effects on cell proliferation were determined with MTT assay,while the growth curve of MCF-7 cells stably transfected with pPUR/U6/KDR-siRNA was measured using cell counting.Results:3 antisense oligonucleotides significantly reduced the KDR protein expression,with the inhibitory rates being 66.16%,50.21% and 58.35% at 0.8?mol/L,respectively.They also decreased the proliferation of MCF-7 cells in a dose dependent manner.Compared to the cells transfected with vacant pPUR/U6 control vector,two of three pPUR/U6/KDR-siRNA plasmids caused effcient down-regulation of KDR protein expression in stable transfected MCF-7 cells.At the 15th day after stable transfection of pPUR/U6/KDR-siRNA,the inhibitory rates were 75.21% and 80.08%,respectively;and at the 30th day the inhibitory rates were 59.29% and 60.15%,respectively.The stable transfection of pPUR/U6/KDR-siRNA also slowed MCF-7 cells growth.Conclusion:It is an effective approach to design siRNA according to the antisense oligonucleotides target accessible sites.The endogenous short hairpin siRNA produced by DNA plasmid can more significantly inhibit KDR gene expression than the antisense oligonucleotides for a long time.

Hepatitis C, Chronic ; Humans

Nucleos(t)ide analogues (NAs) are effective inhibitors for HBV replication and have become the preferred antiviral regimen for most patients with chronic hepatitis B (CHB). HBeAg seroconversion is an important index used to evaluate the durability and efficacy of antiviral therapy in HBeAg-positive CHB patients. The search for biomarkers that can predict HBeAg clearance or seroconversion after NAs treatment plays an important role in the selection of antiviral drugs, the adjustment of treatment regimens, and the achievement of individualized treatment. This article reviews the value of related markers, including HBV DNA, HBV RNA, anti-HBc, and HBcrAg, in predicting HBeAg clearance or seroconversion in CHB patients treated with NAs.

Objective To analyze distribution and drug resistance of pathogenic bacteria isolated from blood specimens of inpa-tients,so as to guiding the principle of clinical use of antibacterials and improve clinical efficacy.Methods The results of bacterial culture and drug sensitivity test of 2 125 blood specimens,from November 2012 to November 2014,in the Rizhao Hospital of Tradi-tional Chinese Medicine were retrospectively analyzed.Results A total of 233 strains of pathogens were isolated(the positive rate was 10.96%),including 57 strains of gram-positive coccus(accounted for 24.46%)and 1 74 strains of gram-negative bacilli(accoun-ted for 74.68%).The coagulase-negative staphylococci and Staphylococcus aureus were most common in gram-positive coccus,the detection rate of methicillin-resistant coagulase-negative staphylococci (MRCNS)and methicillin-resistant Staphylococcus aureus (MRSA)were 84.2% and 40.0%,respectively.The rate of drug resistance of coagulase-negative staphylococci and Staphylococcus aureus to penicillin,erythromycin and clindamycin were no less than 80.0%.The Escherichia coli,Klebsiella pneumoniae and Pseud-omonas aeruginosa were most common in gram-negative bacilli,the detection rate of extended spectrum beta-lactamases(ESBLs) producing Escherichia coli and Klebsiella pneumoniae were 35.85% and 28.13%,respectively.The sensitive rate of Escherichia coli and Klebsiella pneumoniae to imipenem were both 100.0%.Conclusion Gram-negative bacilli is the most common pathogen in this hospital and multidrug resistance is observed.Therefore,cultures of blood specimen should be timely submitted in order to guiding the rational antimicrobial application in clinic.

In order to find microRNA associated with HBV infection and to explore the mechanism of the infection, first of all, we found in our preliminary study that in HepG2 cells transfected with HBV expression plasmid, miR-122 expression was up-regulated, suggesting that miR-122 was related to the HBV infection. On this basis, in the present study, miR-122 and pCH9-HBV1.1 plasmid were cotransfected into HepG2 cells. Southern blot detection result showed that miR-122 can inhibit HBV replication. Using MiRanda computer software, HBx was predicted to be the target sequence of miR-122; Luciferase reporter gene system and Western blot detection of HBx protein expression changes were further used to verify the HBx expression regulated by miR-122. And finally, it can be speculated that miR-122 may affected HBV replication by regulating the expression of HBx.
Gene Expression Regulation, Viral ; drug effects ; Hep G2 Cells ; Hepatitis B virus ; drug effects ; genetics ; physiology ; Humans ; MicroRNAs ; genetics ; Trans-Activators ; genetics ; metabolism ; Transfection ; Virus Replication

Objective To investigate the creatinine level of wound drainage and the changes of serum creatinine after radical nephrectomy or partial nephrectomy in patients with renal cell carcinoma,to explore the feasibility of testing creatinine level to predict urine leakage after surgery and to compare the influence on rcnal function after different kinds of operations.Methods 65 patients data were analyzed,in which 31 patients had undergone partial nephrectomy and 34 had radical nephrectomy for renal cell carcinoma from March 2012 to July 2012.The level of creatinine in serum and wound drainage were detected within 24 hours after surgery.Also,the serum creatinine were redetected 3 months later.Results The creatinine level of wound drainage were significantly lower than that in serum in both groups [(99.94±21.10) μmol/L vs (114.61± 25.09) μmol/L,P =0.000].Urine leakage was observed in only one patient (2.9 ％) after partial nephrectomy,which his level of creatinine in serum and wound drainage was 107 μmol/L and 686μmol/L,respectively.The other 30 patients' creatinine level of wound drainage were also significantly lower than serum after partial nephrectomy [(92.90±26.21) μmol/L vs (99.83±28.77) μmol/L,P =0.021).Although the level of creatinine in the wound drainage was not significantly different between these two groups (P =0.239),the serum creatinine was statistical lower in partial nephrectomy group than that of radical nephrectomy group (P =0.035).Also,after three months,the partial nephrectomy group had a lower level of serum creatinine [(81.43±12.82) μmol/L vs (106.53±21.73) μmol/1,P =0.001].Conclusion Partial nephrectomy has advantages in protecting renal function when compared with radical nephrectomy.The level of creatinine in wound drainage is significantly lower than serum.The level of creatinine in wound drainage is a predictive indicator for diagnosing urine leakage.

Acute-on-chronic liver failure (ACLF) is the most complicated form of liver failure among various types of liver failure. There are certain differences in the conception, etiology, and pathogenesis of ACLF between different regions. In recent years, ACLF has attracted attention of hepatologists all over the world, and some important progress has been made, which contributes to academic communication on ACLF, an early consensus on the diagnosis and treatment of ACLF, and eventually an increased survival rate in patients. This paper reviews the differences in conception between the Eastern and Western parts of the world, pathogenesis, treatment, and prognostic judgment of ACLF.

Hemolytic jaundice has various etiologies and clinical manifestations. Although some patients have hemolysis, they may have jaundice as the initial manifestation due to unobvious or mild anemia, and the disease may last for a long time with difficulties in treatment and can be misdiagnosed as unexplained chronic hepatitis and jaundice with unknown cause. In addition, it may be easily confused with congenital indirect bilirubinemia including Gilbert’s syndrome, which may lead to missed diagnosis and delays in diagnosis and treatment. Physicians in hepatology should master the knowledge of hemolytic jaundice and chronic hemolytic diseases. This article summarizes the clinical features of hemolytic jaundice and thoughts in differential diagnosis and briefly introduces the clinical features of common chronic hemolytic diseases, so as to provide a reference for clinical physicians.

Objective To analyze the role of a key intracellular signaling molecule GSK3β in hepatocyte apoptosis induced by endoplasmic reticulum stress (ERS).Methods Using mouse hepatoma cell lines(Hepa 1) as cell apoptosis model triggered by tunicamycin,an endoplasmic reticulum stress inducer.One hour before Hepa 1 apoptosis induced by tunicamycin,SB216763 specifically inhibited the activity of GSK3β.Living cells/apoptotic cells were detected using acetoxymethyl (AM)/propidium iodide (PI) staining; Furthermore,the measurement of lactate dehydrogenase(LDH) of cell culture supernatant to evaluate the apoptosis.We detect p-GSK3β,GSK3β,the ERS-related protein(GRP78,CHOP and caspase-12) and caspase-3,cleaved caspase-3 protein expression using Western blot.Results Endoplasmic reticulum stress induced by tunicamycin promotes GSK3β activity; Inhibition of GSK3β activity alleviates endoplasmic reticulum stress:the expression of GRP78,CHOP and caspase-12 expression are inhibited.At the same time,GSK3β activity inhibition significantly reduced the endoplasmic reticulum stress-induced apoptosis:compared to cell apoptosis model group,the intervention group of SB216763 showed that the level of LDH decreased significantly,and PI staining of apoptotic cells was also significant reduction.Western blot results showed that the inhibition of GSK 3 β activity reduced reactive cleaved caspase-3 protein.Conclusion GSK3β is an important signaling molecule in the apoptosis pathway induced by endoplasmic reticulum stress ;Endoplasmic reticulum stress promotes hepatocyte apoptosis by mediating GSK3β.

OBJECTIVETo investigate the protective effects of hepatic fibrosis against a lethal dose of acetaminophen (APAP) and its underlying mechanisms using a carbon tetrachloride (CCl4)-induced mouse model of fibrosis.

METHODSThe experimental model of hepatic fibrosis was established by intraperitoneal injection of CC14 (in mineral oil), twice a week for 6 weeks; mice given a 6-week course of mineral oil injections served as normal controls. At the end of fibrosis induction, the expmimental (Fib group) and control (Norm group) mice were challenged with APAP (1 g/kg). Sera and liver tissues were harvested for analyses.To assess tolerance of the normal and fibrotic mice to the lethal dose of APAP, the survival rate,serum alanine aminotransferase (sALT) levels and hepatic histopathological changes were compared before and after the acute APAP challenge.HMGB 1 expression was analyzed by immunohistochemistry.One-way ANOVA test and Newman-Keuls test were used in statistical analysis.

RESULTSThe fibrotic liver was tolerant to the lethal dose of APAP,as evidenced by:(1) significantly higher survival rate in the Fib ± APAP group (80% vs. Norm+APAP group: 0%); (2) markedly lower sALT levels in the Fib+APAP group (6 437 ± 1 913 U/L vs. 12 456 ± 3 441 U/L), P=0.022; (3) remarkably well-preserved liver architecture in the Fib+APAP group.Immunohistochemical analysis showed high HMGB1 expression and cytoplasmic translocation in the Norm+APAP group,which was absent in the Fib+APAP group.

CONCLUSIONSCCl4-induced liver fibrosis protects mice against lethal dose of APAP, Possibly by a mechanism involving inhibition of the cytoplasmic translocation of HMGB1.