OBJECTIVETo study the effect of Hepatitis B virus X (HBx) gene transfection on expression of human telomerase reverse transcriptase (hTERT) mRNA in human bile duct carcinoma cell lines QBC939 and to elucidate the significance of cis-activation of hTERT mRNA by HBx gene on the carcinogenesis of bile duct.

METHODSQBC939 were cultured in vitro and co-transfected with eukaryotic expression vector containing the HBx coding region and cloning vector containing enhanced green fluorescent protein (EGFP) coding sequence using liposome-mediated gene transduction technique. Thirty six hours after transfection, EGFP expression, the indicator of successful transfection in cells, was determined. Flow cytometry was applied to determine the transfection efficiency. Cells were harvested and total RNA was extracted with TRI(ZOL) Reagent. The expression of hTERT mRNA in QBC939 was assayed by Reverse Transcription Polymerase Chain Reaction. The expression of HBx protein in QBC939 was detected by immunocytochemistry staining and western blotting.

RESULTSThe transfection efficiency was 29.6% for both HBx expression vector and vector control group. The expression of hTERT mRNA was significantly increased when transfected with HBx expression vector than that transfected with OPTI-MEM medium and vector only. The expression of HBx protein could only be found in the cells when transfected with HBx expression vector by immunocytochemistry staining and western blotting.

CONCLUSIONHBx gene transfection may up-regulate the transcriptional expression of hTERT mRNA in bile duct carcinoma cells. The cis-activation of hTERT gene by HBx gene is primary mechanism for carcinogenesis of biliary epithelia after HBV infection.

Bile Duct Neoplasms ; genetics ; metabolism ; Cell Line, Tumor ; DNA-Binding Proteins ; metabolism ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; Humans ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Telomerase ; metabolism ; Trans-Activators ; genetics ; Transfection

A highly sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for the determination of actinoside E in rat plasma. The analytes were extracted by ethyl acetate and an analogue of actinoside F was used as the internal standard. The mobile phase consisted of methanol-water (50: 50, V/V) containing 0.1% formic acid was delivered at a flow rate of 0.3 mL·min(-1) to a Zorbax SB-C18 column (100 mm × 2.1 mm, 3.5 μm). The detection was performed by electrospray ionization mass spectrometry in the negative multiple reaction monitoring mode with a chromatograph run time of 3.0 min. Calibration curves of actinoside E were linear in the range of 0.5-2 500 ng·mL(-1). In this range, intra- and inter-day precision ranged from 1.7% to 7.5% and 2.0% to 8.9%, respectively. The accuracy ranged from 95.7% to 108.6%, and extraction recovery from 83.2% to 85.5%. This method was successfully applied to a pharmacokinetic study of actinoside E in rats after intravenous (5 mg·kg(-1)) and oral (100 mg·kg(-1)) administration, and the results showed that actinoside E was poorly absorbed with an absolute bioavailability being approximately 0.27%.
Actinidia ; chemistry ; Animals ; Chromatography, High Pressure Liquid ; methods ; Glycosides ; blood ; pharmacokinetics ; Kaempferols ; blood ; pharmacokinetics ; Male ; Plant Extracts ; blood ; pharmacokinetics ; Rats ; Rats, Sprague-Dawley ; Sensitivity and Specificity ; Tandem Mass Spectrometry ; methods

OBJECTIVETo study the relationship between nucleotide excision repair gene ERCC1 and resistance to cisplatin in ovarian cancer.

METHODSThe expression of gene ERCC1 in 58 ovarian cancer tissues and 4 cell lines were examined and its relationship with resistance to cisplatin were analyzed, the changes of sensitivity to cisplatin were observed after interference of ERCC1 gene with small interfering RNA (siRNA) in ovarian cancer cell lines.

RESULTSIn 58 ovarian cancer tissues, the positive rate of ERCC1 protein in chemoresistant cases (57.89%) was higher than that in chemo-sensitive cases (28.21%, P = 0.029). The mRNA levels of ERCC1 gene in ovarian cancer cell lines ES-2, SKOV3, COC1, COC1/DDP were related to cisplatin IC50 values (r = 0.932, P <0.05). The sensitivity of cell lines ES-2, SKOV3, COC1/DDP cells to cisplatin was increased by 53.88, 5.07, and 3.75 times, respectively, after RNA interfering ERCC1 gene.

CONCLUSIONERCC1 gene is associated with the resistance to cisplatin and the sensitivity to cisplatin can be enhanced by RNA interfering ERCC1 in ovarian cancer.

Adult ; Antineoplastic Agents ; pharmacology ; Cell Line, Tumor ; Cell Survival ; drug effects ; Cisplatin ; pharmacology ; DNA Repair ; DNA-Binding Proteins ; genetics ; metabolism ; Drug Resistance, Neoplasm ; Endonucleases ; genetics ; metabolism ; Female ; Humans ; Inhibitory Concentration 50 ; Middle Aged ; Ovarian Neoplasms ; genetics ; metabolism ; pathology ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Transfection ; Young Adult

Objective To investigate the clinical effect of S-1 combined with oxaliplatin (SOX regimen) in treatment of the patients with advanced pancreatic cancer. Methods A total of 106 patients with advanced pancreatic cancer in Qingdao Fuwai Hospital from April 2015 to June 2017 were randomly divided into the treatment group (53 cases) and the control group (53 cases) according to the random number table method. Patients in the control group were treated with S-1 combined with cisplatin treatment, and patients in the treatment group were treated with SOX regimen. The cell proportion of CD3+, CD4+, CD8+and CD4+/CD8+before treatment and after 2 cycles of treatment were detected by using flow cytometry of both groups. The clinical curative effects, immunity and adverse reactions of both groups were compared by usingχ2 test and t test. Kaplan-Meier method was used to make the survival analysis. Results After two cycles of treatment, there were 4 cases of complete remission (CR), 23 cases of partial remission (PR), 17 cases of stable disease (SD), 9 cases of progression disease (PD) in the treatment group, and 0 case of CR, 18 cases of PR, 20 cases of SD, 15 cases of PD in the control group. The rate of CR+PR in the treatment group was higher than that in the control group [50.94％(27/53) vs. 33.96％(18/53)], and there was a statistical difference (χ2=5.936, P<0.05). There was a significant difference in the cell proportion of CD4+and ratio of CD4+/CD8+between the two groups before and after treatment [the treatment group: (27.31±2.48)％ vs. (37.05±2.53)％, χ2= 6.491,P< 0.01; 0.91 ±0.23 vs. 1.53 ±0.50, χ2 = 5.913, P< 0.01; the control group: (27.43 ±2.47)％ vs. (30.32 ± 2.41)％,χ2= 11.214, P<0.01; 0.90±0.22 vs. 1.22±0.34,χ2=7.992, P<0.01]. After 2 cycles of treatment, the cell proportion of CD4+and ratio of CD4+/CD8+of the treatment group were higher than those of the control group, and there were statistical differences (χ2=5.309, P<0.01;χ2= 7.112, P< 0.01). The incidence rate of side effects had no significant difference in both groups after two cycles of treatment [22.64％ (12/53) vs. 18.87％ (10/53), χ2= 1.924, P> 0.05]. The progression-free survival time in the treatment group was longer than that in the control group (P<0.05). Conclusions SOX regimen has a favorable effect on the patients with advanced pancreatic cancer. It can help to improve the immunity and prolong the survival time of the patients.

Objective To study the etiological detection on samples from severe hand-footmouth disease (HFMD) cases and the genetic characteristics of enterovirus type 71 (EV71) isolates lrom severe patients in Beijing,2010.Methods Real-time RT-PCR was used to detect EV71 and Coxsackievirus A16 (CoxA16) and RD cells were used to separate virus strains from samples.Homogeneity of EV71 isolated strains were also analyzed. Results Four hundred and fourty-two severe cases were detected and 253 were positive,taking up 57.24％ of the total (253/442).The overall positive detection rate on EV71 was 54.55％ (138/253),with CoxA16 as 5.93％(15/253),and with other enterovirus group was 39.53％(100/253).The nucleotide homogencity of VP1 within these 12 strains was 97.2％ 100.0％,and with Beijing strains in 2007-2010,Shandong strains in 2007 and Anhui Fuyang strains in 2008 and the Guangdong strains in 2008 as 94.0％-99.9％.Conclusion Severe HFMD cases were most oftenly caused by EV71 but less caused by CoxA16 or other cnterovirus.The HFMD in 2010 in Beijing was mainly caused by EV71 subgenotype C4a with 4 transmission chains.Twclve isolated EV71 strains had high homogeneity with strains isolated from severe cases in Anhui Fuyang in 2007.

OBJECTIVESelecting variable-number tandem-repeat (VNTR) loci for different serogroups of Shigella spp to explore and establish multilocus variable-number tandem-repeat analysis (MLVA) method, in order to study the molecular characteristic of the isolated strains.

METHODSOf the Shigella strains found by dysentery surveillance in Beijing from 2001 to 2009, 180 strains were selected for this study, according to the number and serotypes of the surveillant strains, at the ratio of 15%; including 50 strains of Shigella sonnei and 130 strains of Shigella flexneri. After screening the polymorphism of the 18 VNTR loci, 10 VNTR loci (sh1-sh10) were retained and constructed three groups of multi-PCR methods to detect all he 180 strains and analyze MLVA molecular subtypes using capillary segments.

RESULTSA range of 2 to 11 alleles were found on the 10 VNTR loci among the 180 Shigella strains, with a diversity index value between 0.158 and 0.766. The 10 loci showed diversity in different serogroups, such as only one allele found in sh6 of Shigella flexneri, sh2 and sh3 of Shigella sonnei individually. The isolated 180 strains were divided into 84 MLVA subtypes, with a resolution ratio D value at 0.967 (95%CI: 0.956 - 0.978). The 130 strains of Shigella flexneri were divided into 63 subtypes, named as TF001-TF063; among which TF001, TF002 and TF 005 were the dominant subtypes, accounting to 17, 16 and 15 strains respectively. The 50 strains of Shigella sonnei were divided into 21 subtypes, named as TS001-TS021; among which TS002 (14 strains) and TS001 (7 strains) were the dominant subtypes.

CONCLUSIONMLVA subtyping method including 10 VNTR loci was preliminarily developed. The MLVA cluster analysis revealed that the subtypes of Shigella strains isolated in Beijing were diverse, and suggested the possibility of multiple-clone source.

Alleles ; Bacterial Typing Techniques ; methods ; China ; DNA, Bacterial ; genetics ; Genotype ; Minisatellite Repeats ; Polymorphism, Genetic ; Shigella ; classification ; genetics ; isolation & purification

BACKGROUNDSrc homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) is a kind of intracellular protein tyrosine phosphatase. Studies have revealed its roles in various disease, however, whether SHP-2 involves in renal fibrosis remains unclear. The aim of this study was to explore the roles of myeloid cells SHP-2 in renal interstitial fibrosis.

METHODSMyeloid cells SHP-2 gene was conditionally knocked-out (CKO) in mice using loxP-Cre system, and renal interstitial fibrosis was induced by unilateral ureter obstruction (UUO). The total collagen deposition in the renal interstitium was assessed using picrosirius red stain. F4/80 immunostaing was used to evaluate macrophage infiltration in renal tubular interstitium. Quantitative real-time polymerase chain reaction and enzyme linked immunosorbent assay were used to analyze the production of cytokines in the kidney. Transferase-mediated dUTP nick-end labeling stain was used to assess the apoptotic renal tubular epithelial cells.

RESULTSSrc homology 2 domain-containing protein tyrosine phosphatase-2 gene CKO in myeloid cells significantly reduced collagen deposition in the renal interstitium after UUO. Macrophage infiltration was evidently decreased in renal tubular interstitium of SHP-2 CKO mice. Meanwhile, the production of pro-inflammatory cytokines was significantly suppressed in SHP-2 CKO mice. However, no significant difference was observed in the number of apoptotic renal tubular epithelial cells between wild-type and SHP-2 CKO mice.

CONCLUSIONSOur observations suggested that SHP-2 in myeloid cells plays a pivotal role in the pathogenesis of renal fibrosis, and that silencing of SHP-2 gene in myeloid cells may protect renal from inflammatory damage and prevent renal fibrosis after renal injury.

Animals ; Enzyme-Linked Immunosorbent Assay ; Female ; Fibrosis ; enzymology ; pathology ; Immunohistochemistry ; Kidney Diseases ; enzymology ; pathology ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Myeloid Cells ; metabolism ; Protein Tyrosine Phosphatase, Non-Receptor Type 11 ; genetics ; metabolism ; Ureteral Obstruction ; enzymology ; pathology

Objective To explore the surgical technique of applying the pedicle composite tissue flap based on superficial palmar branch of the radial artery to repair the soft tissue defect of thumb and evaluate the clinical ef-fect. Methods From February,2013 to March, 2016, 5 cases of the soft tissue and tendon defect of thumb were treated with the pedicle composite tissue flap based on superficial palmar branch of the radial artery. The flap was de-signed at wrist not exceeding the wrist rasceta and the donor site was sutured directly. The size of the harvested flaps was between 3.0 cm ×2.2 cm to 4.2 cm ×3.2 cm, and the sensation of thumb or the flap was reconstructed via median nerve cutaneous branch. The Extensor pollicislongus muscle tendon defect was repaired via palm tendon carried by composite tissue flap. Postoperative follow-up was done termly. Results All transfering flaps survived and all cases were followed-up for 4 to 11 months. The donor site got primary healing with a linear scar. The appearance and tex-ture of the flap was satisfactory. The two-point discrimination ranged from 8 to 11 mm. The appearance of thumb re-covered well and the digit joint had a good motion. Conclusion The pedicle composite tissue flap based on superfi-cial palmar branch of the radial artery is easy to harvest and its vascular anatomy is constant, which is masked and a small incision for the donor site. When necessary, palm tendon or median nerve cutaneous branch can be contained in the flap to form a composite transplant. It is an ideal method for repair of thumb soft tissue defect.

To evaluate the surgical technique and clinical effect of applying Flow-through flap pedicled with superficial palmar branch of radial artery for bridging finger replantation complex defect of soft tissue and vessel. Methods From February, 2013 to March, 2018, 9 cases of severed fingers composited defect of soft tissue and vessel were treated with Flow-through flap pedicled with superficial palmar branch of radial artery.The flap was designed from the proximal end of rasceta and the donor sites were sutured directly. The size of flaps was 3.0 cm ×1.5 cm-4.0 cm×2.2 cm. The superficial branch of the radial artery in the flap was used to bridge the finger artery. And the vein of proximal and distal ends in the finger was bridged by the subcutaneous vein. The proper palmar digi-tal nerve defect was bridged by palm skin graft of median nerve. The appearance, feeling and joint function of fingers was followed-up regularly after operation. Results All transfering flaps survived and all cases were followed-up for 7 to 33 months. The donor sites got primary healing with straight scars. The appearance and texture of the flaps were satisfactory. Two-point discrimination ranged from 8 to 11 mm. The pain sensation, warmth sensation and touch sen-sation of the flaps got better. And the appearance and functions of severed fingers recovered well. Conclusion The Flow-through flap pedicled with superficial palmar branch of radial artery is easy to harvest and anastomose, which is masked and a small incision for the donor site. It is an ideal method for bridging severed fingers and repairing of fin-ger wound.

BACKGROUNDIn-hospital medical complications are associated with poorer clinical outcomes for stroke patients after disease onset. However, few studies from China have reported the effect of these complications on the mortality of patients with acute ischemic stroke. In this prospective work, the China National Stroke Registry Study, we investigated the effect of medical complications on the case fatality of patients with acute ischemic stroke.

METHODSFrom September 2007 to August 2008, we prospectively obtained the data of patients with acute stroke from 132 clinical centers in China. Medical complications, case fatality and other information recorded at baseline, during hospitalisation, and at 3, 6, and 12 months after stroke onset. Multivariable Logistic regression was performed to analyze the effect of medical complications on the case fatality of patients with acute ischemic stroke.

RESULTSThere were 39 741 patients screened, 14 526 patients with acute ischemic stroke recruited, and 11 560 ischemic stroke patients without missing data identified during the 12-month follow-up. Of the 11 560 ischemic patients, 15.8% (1826) had in-hospital medical complications. The most common complication was pneumonia (1373; 11.9% of patients), followed by urinary tract infection and gastrointestinal bleeding. In comparison with patients without complications, stroke patients with complications had a significantly higher risk of death during their hospitalization, and at 3, 6 and 12 months post-stroke. Having any one in-hospital medical complication was an independent risk factor for death in patients with acute ischemic stroke during hospital period (adjusted OR = 6.946; 95%CI 5.181 to 9.314), at 3 months (adjusted OR = 3.843; 95%CI 3.221 to 4.584), 6 months (adjusted OR = 3.492; 95%CI 2.970 to 4.106), and 12 months (adjusted OR = 3.511; 95%CI 3.021 to 4.080). Having multiple complications strongly increased the death risk of patients.

CONCLUSIONShort-term and long-term outcomes of acute stroke patients are affected by in-hospital medical complications.

Aged ; China ; Female ; Gastrointestinal Hemorrhage ; complications ; Hospital Mortality ; Hospitalization ; statistics & numerical data ; Humans ; Male ; Middle Aged ; Pneumonia ; complications ; Prospective Studies ; Registries ; statistics & numerical data ; Stroke ; mortality ; Urinary Tract Infections ; complications