Objective: To investigate the enhancive effect of TNF-α on transfection efficiency of adenovi-rus in mononuclear cells of mouse and the enhancive capability of protection of bone marrow by MDR1. Meth-ods: Before the mononuclear cells of mouse were infected by recombinant adenovirus encoding human MDR1 gene, they had been pretreated by TNF-α. Transfection efficiency of adenovirus was monitored by fluo-rescence microscopy, immunohistochemistry and flow cytometry (FCM). mRNA and protein levels of MDR1 in the mononuclear cells of mouse were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot before and after treatment of TNF-α. Results: After treatment of TNF-α, tansfection rates of adenovirus were obviously increased in the treated group (26.26%) compared with the untreated group (11.96%). mRNA levels and protein levels of MDR1 were obviously increased in the treated group compared with the untreated group. Conclusion: TNF-α could enhance transfection efficiency of adenovirus in mononu-clear cells of mouse and enhance capability of protection of bone marrow by MDR1.

Objective To study the feature of neonatal infections and characteristics of antibiotic treatment in a tertiary children ' s hospital. Methods Clinical data including incidence of infection, primary disease,species of bacteria, complication and antibiotic utilization in hospitalized patients from Jan. 2010 to Dec. 2012 were retrospectively reviewed using their medical records. Results Among 1826 patients admitted to neonetal surgery ward, 542 infants ( 29. 7％) were with infection. The incidence of antibiotic resistance was 23. 51％. The top five infectious diseases were:perianal abscess, necrotizing enterocolitis, colicitis, omphalitis and subcutaneous gangrene. 12 cases of multi-resistant infection were cured by non-restricted antibiotics. 109 were cured by restricted antibiotics. And other 7 were cured by special antibiotics. No death nor multi-resistant nosocomial infection were found. Risk factors including multi-site infection, premature or low birth weight infants, liver, kidney or heart dysfunction,fever lasting more than 3 days after antibiotic therapy, septic shock, sepsis, digestive tract perforation and peritonitis,were vital in choosing specific antibiotics. Conclusions Infection is one of the most common diseases in neonatal surgery ward, with major pathogens sensitive to antibiotics. The clinical characteristics and drug sensitive test are conductive to the reasonable use of antibiotics. Special antibiotics can be used directly in patients with risk factors Clinical doses of antibiotics in neonates depend on the monitoring of drug concentration.

BACKGROUND: Compared to those original viruses systems, adeasy adenovirus, a recombinant adenoviral system widely used in recent years, based on viruses with a deletion of both El and E3, reported by T.C. He in 1998, is an improved one. It simplifies the generation and production of such viruses and expedite the process of generating and testing recombinant adenoviruses using homologous recombination in bacteria rather than in eukaryotic cells. Moreover, it can be conveniently followed with the aid of green fluorescent protein encoded by EGFP gene incorporated into the viral backbone.OBJECTIVE: To construct the recombinant adenovirus and to evaluate them by transfect them to mesenchymal stem cells (MSCs)and detect the expression of target gene hlGF-I at gene and protein levels.DESIGN: Repetitive measurement wail.SETTING: The Institute of Pediatric Research, Chongqing University of Medical Science.METHODS: The study was performed at the Institute of Pediatric Research, Chongqing University of Medical Science from November 2004 to March 2005. After the amplification of truncated hlGF-1 gene from pcDNA3.l-hlGF-I by polymerase chain reaction (PCR), the gene fragment was inserted into the shuttle plasmid pAdtrack-CMV for homologous recombination with backbone plasmid pAdeasy-I in bacteria BJ5183 to get adenovirus.Ad-hlGF-1. The high titer adenovirus supernatant was obtained by repeated transducing of HEK 293 cells by adenovirus harvested after confirmation of the adenovirus structure. As target cells,MSCs were infected with adenovirus earned target gene, hIGF-1, to determine the expression of hlGF-1 gene.MAIN OUTCOME MEASURES: ① The construction of recombinant adenovirus vector;② the expression of target gene hIGF-1 in HEK 293 cells and the proper multiplicity of infection (MOI); ③ hIGF-1 gene expression in MSCs.RESULTS: The adenovirus vector based on adeasy system was constructed successfully and the Ad-hlGF transducing was successfully or efficiently expressed in MSCs cells. The ideal expression of harvested recombinant adenovirus in MSCs was detected by fluorescence microscope, RT-PCR, immunocytochemistry, and Western Blot.CONCLUSION: Adenovirus vector is an effective vector tools for gene expression and wansfection of MSCs. MSCs transduced with Ad-hIGF-1 maybe another option to gene-modified seed cells for articular cartilage tissue engineering.

Objective:To observe the killing effect of irradiated peripheral blood mononuclear cells (PBMCs) at low dose combined with apogossypolone (ApoG2) on cultured human prostate cancer PC-3 cells.Methods:Human PBMCs were irradated by gamma ray at 1 gray,the irradiation dose rate was 17 Gy/min.The experiment were divided into PC-3 tumor cell control group,PC-3 cells with irradiated and non-irradiated PBMCs co-culture groups,ApoG2 treatment group,irradiated PBMCs and ApoG2 co-treatment group.Acridine orange/ethidium bromide (AO/EB) staining and MTT method were used to observe the killing effect of PBMCs and/or ApoG2.Results:The killing activity of irradiated PBMCs group and ApoG2 treatment group were obviously increased and were higlaer than that of non-irradiated group (P＜0.05).The killing activity of combined group were much higher than that of irradiated group and ApoG2 treatment group (P ＜0.01 ).Conclusion:Irradiated PBMCs at low dose combined with ApoG2 can enhances the anti-tumor effects markedly.

Objective To analyze the pattern of antibiotic use and antibiotic resistance tendency of gastrointestinal surgery in a tertiary children′s hospital .Methods 2 625 patients(which account for 27 .52% of all the hospilitalized patients ,the resistant rate was 15 .70% ) detailed morbidity ,entity ,bacteria ,complication ,antibiotic utilization was retrospectively reviewed using the hospital medical records from 2010 to 2012 .Results 2 625 patients the percentages of the top five disease category were :appendicitis ac-counting for 40 .72% ,perianal abscess accounting for 21 .53% ,periappendiceal abscess accounting for 9 .30% ,necrotizing enterocol-itis accounting for 3 .73% ,omphalitis accounting for 2 .93% .The top three pathogen were :escherichia coli ,klebsiella pneumoniae subsp ,staphylococcus aureus respectively .255 multi-resistant bacteria of the superficial infection patients and 157 of the invasive in-fection patients .49 multi-resistant infections were cured by first or second generation of cephalosporins and penicillinase-fast peni-cillin ,and 346 were cured by third or forth generation of cephalosporins and penicillinase-fast penicillin ,and 17 were cured by car-bapenem or vancomycin .No dead or multi-resistant hospital infectious case was reviewed .Conclusion The sensitive rates of surgi-cal infected patient were 84 .3% ,and opportunistic pathogen infection was the main characteristics .To aware the clinical character-istics and drug sensitive test is conductive to the reasonable use of antibiotics of severe infections .The cases of superficial resistant infection or invasive non-resistant infection tend to use restricted antibiotics .The cases of invasive resistant infection tend to use special antibiotics .

Objective To investigate the clinical effect of nicorandil for treatment of patients with acute respiratory distress syndrome (ARDS).Methods A prospective randomized controlled trial was conducted. A total of 40 cases of patients with ARDS admitted to Department of Critical Care Medicine of Guizhou Provincial People's Hospital from October 2012 to October 2014 were enrolled, and they were randomly divided into two groups, 20 cases in each group. The two groups were treated with routine western medicine after admission. On this basis, the observation group was given nicorandil 10 mg, while the control group was given warm boiled water 10 mL, through gastric tubes 3 times a day, the therapeutic course being consecutive 5 days in both groups. The length of stay in intensive care unit (ICU), duration of mechanical ventilation after treatment, oxygenation index (OI), alveolo-arterial oxygen partial pressure difference (PA-aO2), positive end-expiratory pressure (PEEP), acute physiology and chronic health evaluation Ⅱ (APACHE Ⅱ) score, Glasgow coma score (GCS) before and after treatment, the predicted death rate (PDR) and 28-day mortality were compared between the two groups. The predicitive factors for 28-day mortality were screened by binary logistic analysis.Results The length of stay in ICU and duration of mechanical ventilation of control group were longer than those of observation group, but the difference was not statistically significant [ICU length of stay (day): 14.55±12.71 vs. 9.15±6.00, duration of mechanical ventilation (day): 13.25±12.27 vs. 7.75±5.32, bothP > 0.05]. After treatment, the GCS was higher than that before treatment in control group and observation group (11.95±3.98 vs. 10.75±4.89, 12.95±3.67 vs. 12.20±4.56), while APACHE Ⅱ score, PDR and PEEP were all lower than those before treatment [APACHE Ⅱ: 21.05±8.58 vs. 24.90±5.63, 18.70±11.21 vs. 26.65±7.67; PDR: (47.71±29.49)% vs. (61.00±23.29)%, (36.79±18.49)% vs. (56.12±18.16)%; PEEP (cmH2O, 1 cmH2O = 0.098 kPa): 4.40±3.14 vs. 5.75±2.59, 3.80±2.55 vs. 7.55±3.32], but there were no statistically significant differences between the two groups before and after treatment (allP > 0.05). After treatment, the OI was significantly higher and the PA-aO2 was significantly lower than those before treatment in the two groups, and the degrees of improvement of the observation group were more remarkable than those of the control group [OI (mmHg, 1 mmHg = 0.133 kPa): 224.72±85.12 vs. 141.37±45.82, PA-aO2 (mmHg): 132.60±46.64 vs. 204.30±121.2, bothP < 0.05]. The 28-day mortality of observation group was lower than that of control group, but no statistically significant difference was seen [15% (3/20) vs. 25% (5/20),χ2 = 0.156,P > 0.05). Binary logistic regression analyses showed that the PA-aO2 [odds ratio (OR) = 0.958,P = 0.013, 95% confidence interval (95%CI) = 0.927 - 0.991], APACHE Ⅱ score (OR = 0.882,P = 0.010, 95CI = 0.803 - 0.970), GCS (OR = 1.399, P = 0.004, 95%CI = 1.111 - 1.761) and PDR (OR = 0.907,P = 0.002, 95%CI = 0.853 - 0.965) after treatment were the independent predictors of 28-day mortality.Conclusion Nicorandil can significantly improve oxygenation, but cannot reduce 28-day mortality in patients with ARDS.

Objective To construct a recombinant Escherichia coli ( E. coli) with surface-dis-played lead specific binding protein PbrR and to further study intestinal colonization by the recombinant bac-teria in mice and gastrointestinal tolerance of the bacterial surface-displayed PbrR. Methods Chimeric pro-tein Lpp-OmpA coding sequence was chemically synthesized and inserted into the expression vector pET-21a to construct the outer membrane display vector pLOA. PbrR coding sequence was also obtained by chemical-ly synthesis and inserted into pLOA to generate the outer membrane display plasmid pLOA-pbrr. E. coli BL21 (DE3)pLysS was transformed with pLOA-pbrr and induced by IPTG. The expressed recombinant proteins were analyzed by 15% SDS-PAGE and Western blot assay. Lead adsorption capacity of the cell surface-dis-played PbrR in the simulated intestinal juice and tolerance of the recombinant E. coli to simulated gastric juice were analyzed, respectively. KM mice were orally given the induced recombinant bacteria by gastric lavage for 7 consecutive days and then were continually fed until day 30. The contents of recombinant bacte-ria in stool samples were detected by dilution plate method on day 7, 15 and 30. The recombinant protein with His tag was detected by immunoblotting on day 7 and 15. Results Based on Lpp-OmpA, the PbrR outer membrane display vector was successfully constructed. The recombinant fusion protein Lpp-OmpA-PbrR-His tag was highly expressed in E. coli. The recombinant E. coli strains displaying PbrR on their outer membrane accumulated a significant level of Pb2+ in simulated intestinal juice. Moreover, those strains showed a tolerance to gastric acid in vitro and could colonize in the intestinal tracts of mice via oral infection. The surface-displayed recombinant fusion protein showed a better tolerance to the environment of digestive tract. Conclusion The recombinant E. coli strain displaying PbrR on its surface showed a stronger capabili-ty of lead accumulation from simulated intestinal environment and could colonize in the intestinal tracts of mice. The surface-displayed recombinant PbrR also showed a good tolerance to digestive juice. This study paved the way for further researches on the selective elimination of lead by biosorption based on animal mod-els.

Objective: To establish a method for the determination of manganese in urine by graphite furnace atomic absorption spectrometry (AAS) without the use of matrix modifier. Methods: The urine samples were 5 times diluted with 1% nitric acid then directly determined by AAS. Zeeman was used for background correction. Results: The linear range for determination of manganese in urine was 5~60 μg/L (urine) . The correlation coefficient was greater than 0.995 with the detection limit of 1.5 μg/L and with the lower limit of quantification of 5.0 μg/L. The relative standard deviations (RSDs) of within-run precision was between 1.1%~4.3%, the RSDs of between-run precision was between 3.3%~7.0%. The average recovery was 102.6%. The samples can be stored for 14 days at room temperature, 4℃, -8 ℃ and -35 ℃. Conclusion The method is feasible for determination of manganese in urine.

OBJECTIVETo find the novel gene related to the multi-drug resistance in leukemia and explore the molecular mechanism of multi-drug resistance.

METHODSThe subtracted HL-60/VCR cDNA library was generated through the suppression subtractive hybridization using the wild HL-60 cells' cDNA as target and HL-60/ ATRA cells' as driver. A novel expression sequence tag (EST) sequence, which differentially expressed in HL-60/ ATRA cell, was screened by cDNA chip. Then a novel human gene, HV126 was assembled by the EST assembly tools. Bioinformatical databases and softwares were used to analyze and predict its function. Reverse transcription-PCR (RT-PCR) was used to detect the expression of HV-126 gene in leukemia cells before and after chemotherapy.

RESULTSThe full open reading frames (ORFs) of the novel EST assembled by overlapping dbEST sequences included a 1991 bp nucleic sequence, which was named HV126. The deduced amino acid sequence consisted of 365 amino acids. The sequence of the novel gene exhibited 43% homology to a known gene, which is a possible member of the death domain-flood family implicated in apoptosis and inflammation. The expression of HV126 was proved to be related to the drug sensitivity in leukemia cells by RT-PCR.

CONCLUSIONHV126, the novel gene, might have roles in regulating multi-drug resistance in leukemia. Further laboratory research should be done on cloning and making clear the gene function.

Antineoplastic Agents ; pharmacology ; Base Sequence ; Chromosome Mapping ; Chromosomes, Human, Pair 14 ; genetics ; Cloning, Molecular ; DNA, Complementary ; chemistry ; genetics ; Drug Resistance, Multiple ; genetics ; Gene Expression Regulation, Leukemic ; drug effects ; HL-60 Cells ; Humans ; Leukemia ; genetics ; metabolism ; pathology ; Molecular Sequence Data ; Multidrug Resistance-Associated Proteins ; genetics ; Oligonucleotide Array Sequence Analysis ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA

Objective To analyze the arrhythmia after treatment with recombinant human interleukin 11 (rhIL-11) because of down-regulating platelet in elderly patients with myelodysplastic syndromes (MDS), and to investigate the possible mechanism of arrhythmia induced by in MDS patients. Methods The data of 2 MDS patients with arrhythmia after rhIL-11 therapy were analyzed retrospectively. The patients'hemoglobin, electrocardiogram (ECG), myocardial enzymes, cardiac troponin Ⅰ (cTnⅠ), N-terminal pro brain natriuretic peptide (NT-proBNP) changes, as well as cardiac ultrasonography and Holter monitoring during arrhythmia were dynamically observed before and after use of rhIL-11, at the time of arrhythmia and restoring sinus rhythm after the withdrawal of rhIL-11. Results Before the use of rhIL-11, blood platelet count of patient 1 and patient 2 was 2×109/L and 3×109/L respectively. Arrhythmias occurred in the two patients at 11st and 14th days respectively. ECG showed atrial fibrillation with rapid ventricular rate, and dynamic ECG monitoring showed that syncope was caused by sinus arrest due to cardiac cardiogenic syncope. Heart ultrasound prompted ejection fraction (EF) values in the normal range. Creatine kinase, creatine kinase isoenzymes, aspartate transaminase, lactate dehydrogenase, and cTnⅠ had no obvious increase or decrease after rhIL-11 treatment, but NT-proBNP was increased significantly. After discontinuation of rhIL-11 and diuretic treatment, no syncope occurred. ECG restored sinus rhythm, and NT-proBNP was decreased significantly. Conclusion rhIL-11 in elderly MDS patients may induce arrhythmia, which can be restored after drug withdrawal, limited sodium diet and diuretic treatment, but much attention should be paid to the heart-related symptoms and signs, dynamic monitoring of NT-proBNP and timely treatment.