Objective To construct series of reporter plasmids with truncated and deleted hTERT promoter.Methods Gene fragments of hTERT promoter was amplified by PCR and cloned into pGL3-Basic to construct luciferase reporter vectors.Dual luciferase assays were performed with cell lysates of HepG2 and COS-7 cells cotransfected with hTERT promoter reporter plasmids and pRL-TK.Results Series of luciferase reporter plasmids with truncated and deleted hTERT promoter were successfully constructed and respectively named pGL3B-895,pGL3B-371,pGL3B-DELS2,pGL3B-349,pGL3B-329,pGL3B-318,pGL3B-306.Dual luciferase reporter assays showed that all the reporter vectors have promoter activity both in HepG2 and COS-7.Conclusion Series of luciferase reporter plasmids with truncated and deleted hTERT promoter were successfully constructed,and their promoter activity were verified.These plasmids provide necessary experimental naterials for further investigation of regulation of hTERT during hepatocarcinoma development.

Aim To study the effect of 3-pyridin-3-yl-4-[(4-hydroxy-3-methoxy-benzylidene)amino]-5-me-thylsulfanyl-4H-[1,2,4] triazole (LH-37) on induction of differentiation and apoptosis of hepatocarcinoma BEL-7402 cells. Methods BEL-7402 cells were cultured in RPMI 1640 and treated by LH-37 at different doses. The proliferation of the cells and the inhibition effect of LH-37 on the cell proliferation were examined by MTT assay. The reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the changes of alpha fetoprotein(?-FP)mRNA and albumin(Alb). The concentration of ?-FP in the cells was detected by ELISA assay. Cell morphology was observed by fluorescence microscope techniques. The protein expressions of active caspase-3 in BEL-7402 cells were observed by immunohistochemical staining. Western blot was used to assay caspase-3 and caspase-9. Colorimetric method was used to assay activity of superoxide dismutase (SOD) and catalase (CAT). The apoptotic cells were assayed by flow cytometry (FCM) with annexin V-FITC conjugated and propidium iodide (PI) staining. Results The cell proliferation was inhibited by LH-37 at 10 ?mol?L-1~1 mmol?L-1 in dose dependent manners. After treated with 10 ?mol?L-1 or 1.0 ?mol?L-1 LH-37, the mRNA and protein expression of ?-FP were significantly reduced but mRNA expression of Alb was significantly increased. Treatment of BEL-7402 cells with different LH-37 concentrations for 48 hours increased the percentage of active caspase-3 positive cells and protein expression of caspase-3 and caspase-9. More apoptosis features of cells were observed in LH-37 ( 100 ?mol?L-1) treatment groups than in control group. LH-37 markedly promoted the viability of SOD and decreased CAT in BEL-7402 cells. Counclusion LH-37 might inhibit proliferation and induce differentiation and apoptosis of human hepatocarcinoma BEL-7402 cells, which might be related to the cytotoxicity of the intracellular hydrogen peroxide (H2O2).

Objective To explore the risk factom of hepatitis B virus infection and alcoholic liver disease in patients with decompensated cirrhosis complicated with liver failure.Methods One hundred and fifty-eight cases hepatitis B virus infection and alcoholic liver disease in patients with decompensated cirrhosis were selected.According to whether complicated with liver failure,the patients were divided into observation group with 62 cases (complicated with liver failure group) and control group with 96 cases (without liver failure group).The clinical data and results of 2 groups were analyzed to screen the risk factors of liver failure.Results Compared with control group,observation group in alanine aminotransferase,aspartate aminotransferase,cholinesterase,total bilirubin,and prothrombin time,activated partial thrombin time live enzymes,thrombin time,fibrinogen,serum creatinine,the differences were not statistically significant (P ＞ 0.05);in albumin ((28.02±7.36) g/L vs.(23.26±6.54) g/L,t =4.421,P =0.002),serum urea nitrogen ((8.84±4.71) mmol/L vs.(9.33±5.24) mmol/L,t =3.656,P=0.007),upper gastrointestinal bleeding(x2 =7.534,P=0.006),ascites (x2 =8.615,P =0.003),infection (x2 =10.321,P =0.001),hepatic encephalopathy (x2 =6.561,P =0.010),hepatorenal syndrome(x2 =4.952,P=0.026),the difference were statistically significant.(2)The results of logistic regression analysis showed that upper gastrointestinal bleeding (OR =1.020,95％ CI:1.003-1.036),hepatorenal syndrome(OR=2.872,95％CI:0.385-21.423) were risk factor of hepatitis B virus infection and alcoholic liver disease in patients with decompensated cirrhosis complicated with liver failure.Conclusion Upper gastrointestinal bleeding,hepatorenal syndrome are independent risk factors of hepatitis B virus infection and alcoholic liver disease in patients with decompensated cirrhosis complicated with liver failure.

Objective To investigate the effect of auricular acupuncture therapy (AAT) and standardized acupoints on sleep parameters among people with insomnia. Methods A single-blind,randomized pilot study where the treatment group AAT on active points and the control group received AAT on sham points during a 4-week treatment period. Participants were recruited from the Acupuncture Outpatient Clinics in the General Hospital of People' s Liberation Army. In all, 125 patients were included in the study, with 63 in treatment group and 62 in control group. Sleep parameters were obtained by using the Pittsburgh Sleep Quality Index (PSQI). Results (1)Significant improvement in the PSQI total score and seven components score including sleep quality,time on falling asleep, sleeping time, efficiency and disorder of sleep, hypnotic and daytime functional disorder after treatment in both treatment group and control group (P＜0.01). (2) After treatment, there was a statistically significant difference seen in the PSQI total score and six components score (P＜0.01) except for hypnotic between the two groups. There were statistically significant differences in the rank differential value of total score and seven components of two groups (P＜0.01). (3) There were statistically significant differences in the mean rank of PSQI seven components using Mann-Whitney test (P＜0.01). Conclusion Evidence was found to support the hypothesis that AAT played an effective role in improving the quantity and quality of sleep in those subjects with insomnia. A standardized AAT might help the treatment of insomnia, especially when combined with other treatments as psychological and behavioral therapies.

Background:Oryz-Aspergillus Enzyme and Pancreatin Tablet is a double-deck digestive enzyme pellet containing Oryz-Aspergillus enzyme and pancreatin that has been widely used for treatment of functional dyspepsia (FD)in clinical practice. However,there is no randomized controlled trial focusing on the efficacy of this agent versus other drugs used for treatment of FD such as prokinetics and proton pump inhibitors (PPI). Aims:To compare the efficacy and safety among Oryz-Aspergillus Enzyme and Pancreatin Tablet,Domperidone Tablet and Esomeprazole Magnesium Enteric-coated Tablet, three drugs with different mechanisms of action on FD,in Chinese population. Methods:A total of 82 Helicobacter pylori-negative outpatients fulfilling the diagnostic criteria of FD in Rome Ⅲ were recruited from Nov. 2015 to Jun. 2016 at the First Affiliated Hospital of Zhejiang Chinese Medical University. These patients were randomly allocated into 3 groups,and received Oryz-Aspergillus Enzyme and Pancreatin Tablet (group A),Domperidone Tablet (group B)and Esomeprazole Magnesium Enteric-coated Tablet (group C)orally for 4 weeks,respectively. The improvement of dyspeptic symptoms and adverse events were observed and recorded. Results:After 4 weeks treatment,the overall efficacies for global symptoms in group A,group B and group C were 93. 1%,88. 9% and 69. 2%,respectively,statistically significant difference was existed among the three groups (P < 0. 05). Domperidone Tablet was effective for postprandial fullness and early satiety;Esomeprazole Magnesium Enteric-coated Tablet was sensitive for epigastric pain,epigastric burning,and belching and regurgitation;the efficacies of Oryz-Aspergillus Enzyme and Pancreatin Tablet for all five dyspeptic symptoms were in between. No adverse events were observed during treatment course. Conclusions:Digestive enzymes,prokinetics and PPI have different sensitive symptoms and optimal indications for treatment of FD. The overall efficacy of Oryz-Aspergillus Enzyme and Pancreatin Tablet is superior to that of prokinetics and PPI.

Objective:To investigate the inhibitory effects of melanoma differentiation associated gene-7(mda-7/IL- 24)on lymphoma cell line Namalwa in vitro and in vivo.Methods:Using RT-PCR,the expression of mda-7/IL-24 was examined in 10 malignant hematopoietic cell lines,including Namalwa,Raji,K562,NB4,U937,Ramous,CEM,KG1a, HL60,J6-1,etc.The coding region of mda-7/IL-24 was cloned from LPS-treated human peripheral blood mononuclear cells(PBMC)by RT-PCR,and the eukaryotic expression vector pTarget-IL-24 was constructed.The recombinant vector, after sequenced,was transfected into Namalwa cell line via lipofectamine reagent.The stable expression transfectants were selected by G418.The expression of mda-7/IL-24 mRNA and protein was verified by RT-PCR and Western blotting.MTT assay,colony forming assay,apoptosis detection,and tumorigenesis in nude mice were used to assess the effects of mda- 7/IL-24 on tumor proliferation,growth characteristics,colony forming,apoptosis,and tumorigenesis.Results:Expression of mda-7/IL-24 mRNA was not found in any of the 10 malignant hematopoietic cell lines and the expression of mda-7/IL- 24 mRNA and protein was found in Namalwa cells transfected with recombinant plasmid pTarget-IL-24.Significant de- crease in tumor cell viability was observed in Namalwa cells stably transfected with mda-7/IL-24,compared with control cells transfected with empty plasmid pTarget(P

Objective To survey the survival status, functional status, marital status and the present situation of the survival patients with spinal cord injury 40 years after Tangshan earthquake. Methods From February to May, 2016, a total of 216 patients with spinal cord injury were surveyed with questionnaire, in which 139 cases lived centralized and 77 cases lived scattered. The questionnaire was self-designed and included eleven items and 51 questions, which related to ability of daily life, marital status, employment status and socioeconomic status and so on. Results A total of 960 (25.15%) patients with spinal cord injury survived 40 years after Tangshan earthquake. In 216 surveyed pa-tients, the employment rate was 9.3%, the married rate was 53.2%, and 44.9%earned less than 500 yuan every month. The incidence was 25.9%for pressure score, 50.50%for neuralgia (severe pain accounted for 23.51%), and 19.40%for urinary tract stones. 56%of patients could take their own basic self-care. Conclusion There were many problems such as high complication rate, low employment rate and poor economic condition in the spinal cord injury group 40 years after Tangshan earthquake.

OBJECTIVETo construct a recombinant lentiviral U6 plasmids for RNA interference (RNAi) of galectin-3 gene and select the optimal target sequence of galectin-3 gene for RNAi.

METHODSDouble-stranded oligo DNAs were designed and synthesized according to the sequence of galectin-3 gene, and ligated into linearized pGCL-GFP/U6 plasmid followed by transformation into competent DH5alpha cells. After PCR and sequence analysis for verification of the positive clones, the plasmid pGCL-GFP/U6 Gal-3shRNA-1 was extracted and transfected into CaCl2-treated 293T cells to obtain the viral vectors containing the RNAi sequence. MCF-7 cells were infected with pGCL-GFP/U6 Gal-3shDNA-1, and at the infection rate over 50%, the cells were harvested to extract the RNA. Real time-PCR was performed to determine the expression level of galectin-3 mRNA in the infected cells.

RESULTSThe recombinant vector was successfully constructed as confirmed by sequence analysis. High titer of the virus was obtained, and after infection of MCF-7 cells, RNAi targeting the 1# and 3# sequences in galectin-3 gene resulted in suppression of galectin-3 mRNA expression by 95% and 85%, respectively.

CONCLUSIONThe recombinant lentiviral U6 plasmid for RNAi of Galectin-3 gene has been successfully constructed, which provides the basis for further study of the role of galectin-3 gene in tumor cells.

Breast Neoplasms ; genetics ; pathology ; Cell Line ; Cell Line, Tumor ; Female ; Galectin 3 ; genetics ; Genetic Vectors ; genetics ; Humans ; Lentivirus ; genetics ; RNA Interference ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

Objective To investigate the association between arterial stiffness of the common carotid artery(CCA)and insulin resistance in hemodialysis patients. Methods Arterial stiffness index β of CCA was evaluated by an ultrasonic phase-lock Echo-tracking system in 80stable non-diabetic hemodialysis patients.Insulin resistance was detected by the homeostasis model assessment method(HOMA-IR).Plasma hemoglobin,serum albumin,total cholesterol,high density lipoprotein,low density lipoprotein,triglyceride,lipoprotein(a),ApoA1,ApoB,CRP,calcium,phosphorus and creatinine were determined by standard methods. Results The stiffness index β was 11.41±4.13 in patients with previous cardiovascular disease(CVD)and 9.75±3.63 in those without CVD(P＜0.05).The stiffness index β was positively correlated with HOMA-IR(r=0.321,P＜0.01),as well as with age(r=0.376,P＜0.01),pulse pressure(r=0.267,P＜0.05),and duration of hemodialysis(r=0.219,P＜0.05).In stepwise multiple regression analysis,HOMA-IR(β=0.228,P＜0.05)and age(β=0.308,P＜0.01)were identified as significant independent variables for stiffness index β of CCA. Conclusions Insulin resistance is associated with aaefial stiffness in nondiabetic hemodialysis patients.The increased arterial stiffness may be the link between insulin resistance and cardiovascular morbidity as well as mortality in hemodialysis patients.

BACKGROUNDSite A132Arg mutations potentially impair the affinity of isocitrate dehydrogenase 1 (IDH1) for its substrate isocitrate (ICT), consequently reducing the production of α-ketoglutarate and leading to tumor growth through the induction of the hypoxia-inducible factor-1 (HIF-1) pathway. However, given that the roles of other active sites in IDH1 substrate binding remain unclear, we aimed to investigate IDH1 mutation pattern and its influence on enzyme function.

METHODSFifteen IDH1 catalytic active site candidates were selected for in silico mutagenesis and protein homology modeling. Binding free energy of the IDH1/ICT complexes with single-site mutations was compared with that of the wild type. The affinity of 10 IDH1 catalytic active sites for the ICT substrate was further calculated.

RESULTSThe IDH1 active site included seven residues from chain A (A77Thr, A94Ser, A100Arg, A132Arg, A109Arg, A275Asp, and A279Asp) and three residues from chain B (B214Thr, B212Lys, and B252Asp) that constituted the substrate ICT-binding site. These residues were located within 0.5 nm of ICT, indicating a potential interaction with the substrate. IDH1 changes of binding free energy (ΔE) suggested that the A132Arg residue from chain A contributes three hydrogen bonds to the ICT α-carboxyl and β-carboxyl groups, while the other nine residues involved in ICT binding form only one or two hydrogen bonds. Amino acid substitutes at A132Arg, A109Arg, and B212Lys sites, had the greatest effect on enzyme affinity for its substrate.

CONCLUSIONSMutations at sites A132Arg, A109Arg, and B212Lys reduced IDH1 affinity for ICT, indicating these active sites may play a central role in substrate binding. Mutations at sites A77Thr, A94Ser, and A275Asp increased the affinity of IDH1 for ICT, which may enhance IDN1 catalytic activity. Mutant IDH1 proteins with higher catalytic activity than the wild-type IDH1 could potentially be used as a novel gene therapy for glioblastoma multiforme.

Catalytic Domain ; genetics ; Glioblastoma ; genetics ; Humans ; Isocitrate Dehydrogenase ; genetics ; metabolism ; Isocitrates ; metabolism ; Mutagenesis ; Mutation ; Protein Binding ; Structure-Activity Relationship