Increasing the production and secretion of endogenous opioid peptide by immune cell can significantly induce myocardial protective effects against ischemia-reperfusion injury. Gene therapy is promising to increase endogenous enkephalin (ENK). However, classical viral and plasmid vectors for gene delivery are hampered by immunogenicity, gene recombination, oncogene activation, the production of antibacterial antibody and changes in physiological gene expression. Minimalistic immunologically defined gene expression (MIDGE) can overcome all the deficients of viral and plasmid vectors. The exon of rat's preproenkephalin (PPENK) gene was amplified by PCR and the fragments were cloned into pEGFP-N1 plasmids. The recombined plasmids were digested with enzymes to obtain a linear vector contained promoter, preproenkephalin gene, RNA stable sequences and oligodesoxy nucleotides (ODNs) added to both ends of the gene vector to protect gene vector from exonuclease degradation. A nuclear localization sequence (NLS) was attached to an ODN to ensure the effective transport to the nucleus and transgene expression. Flow cytometry, laser confocal microscopy and Western blotting demonstrated that PPENK-MIDGE-NLS can transfect leukocyte of rat in vivo, increase the expression of proenkephalin (PENK) in tissue, and the transfection efficiency depends on gene vector's dosage. These results indicate that PPENK-MIDGE-NLS could be an innovative method to protect and treatment of myocardial ischemia-reperfusion injury.
Animals ; Cloning, Molecular ; Enkephalins ; genetics ; Gene Expression ; Genetic Therapy ; Genetic Vectors ; Leukocytes ; Plasmids ; Promoter Regions, Genetic ; Protein Precursors ; genetics ; Rats ; Transfection ; Transgenes

Aim To investigate the effect of CD44 anti-body-A3 D8 on the expression of IL-3 Rα and down-stream PI3K/Akt in NB4 cells. Methods The ex-pression of IL-3 Rα mRNA was detected by real-time quantitative RT-PCR, the IL-3Rα protein expression and changes of PI3 K/Akt signal pathway in NB4 cells treated with A3D8 were analyzed by Western blot. An-nexin-V-FITC/PI double staining flow cytometry was u-tilized to detect the apoptotic cells. The inhibitor of PI3 K/Akt signaling LY294002 combined with A3 D8 was used to inhibit the PI3K/Akt in NB4 cells. Re-sults After treated with A3 D8 , both the transcription-al level and translational level of IL-3 Rα were remark-ably reduced, and the PI3K/Akt pathway was inhibi-ted. LY294002 improved the inhibitory and apoptotic effects of A3D8 on NB4 cells. Conclusion CD44 antibody A3 D8 can downregulate the expression of IL-3Rα and inhibit the downstream PI3K/Akt pathway.

Objective To establish an animal model of sacral nerve root avulsion in rats and e-valuate its efficiency. Methods A total of 20 adult SD rats (either sex) were chosen at random to es-tablish the sacral nerve root avulsion model by avulsing the fight L4-6 nerve roots out of intervertebral fo-ramina without laminectomy. The left side was set as control group. The models were evaluated in aspects of survival rate, Basso-Beattie-Bresnahan (BBB) scores, somatosensory evoked potential (SEP), horse radish peroxidase (HRP) tracing, bilateral weight and cross section area (CSA) of muscle biceps femo-ris, fiber of triceps surae and anterior tibial muscle. Results Of all, 19 rats were survived but one died, with survival rate of 95.0%. The BBB score was (10.78+3.15) points in experimental group and 21 points in control group. The success rate of establishing animal model was 89.5% ,for there detected no SEP in bilateral cerebral cortex of the wounded extremity of 17 rats. HRP showed positive reaction in the spinal segment of L4-6 in two rats but negative in 17, with success rate of 89.5%. There was statistical difference in aspects of weight and muscle fiber CSA of double biceps femoris, triceps surae and anterior fibial muscle between experimental group and control group. Electron microscope found denervation chan-ges including muscle fiber atrophy, nucleus shifting to (center and muscle satellite cell. Conclusion A-vulsion of L4-6 sacral nerve root out of vertebral canal is a feasible and ideal method to establish the avui-sion model of sacral nerve root injury in rats.

Objective To construct the vector that expresses the fusion protein of HIV-Tat protein and red fluorescent protein(mCherry) in mammalian cells,and observe by fluorescence microscopy the intracellular transduction and localization of recombinant protein in cells,in order to obtain a useful tool for the study of the uptake mechanism and intracellular localization of HIV-TAT.Methods With the designed primer coding mCherry sequence,the mCherry gene was amplified by PCR with the vector pmCherry-C2 as template,and inserted into vector pET14b-His-TAT to construct the expression vector pET14b-His-TAT-mCherry.The constructed vector was then transformed into E.coli BL21(DE3),which had been identified by PCR and double digested with restriction endonuclease,followed by sequencing.After IPTG induction,the recombinant protein of His-TAT-mCherry was lyzed and analyzed with SDS-PAGE.Purified His-TAT-mCherry recombinant protein was added to Hela cells and the fluorescence was observed to evaluate the transduction efficiency.Results The results of identification by PCR,digestion with restriction endonuclease and sequencing indicated that the vector His-TAT-mCherry was correctly constructed.His-TAT-mCherry fusion protein was expressed in mammalian Hela cell line and purified successfully,and the fusion protein showed cellular transduction activity.It was found by fluorescence microscopy that the red fluorescence protein located mainly over the cytoplasm,and also the membrane to some extent.Conclusion The expression vector is successfully constructed for HIV-TAT labeled with mCherry sequence.Effective expression and purification of this fusion protein is achieved.It has been observed that the constructed vector may be expressed in mammalian Hela cell under active condition.Thus,it might be useful in the study of uptake mechanism and intracellular localization of HIV-TAT.

OBJECTIVE To investigate clinical features and etiology of inhalation pneumonia.METHODS Totally 108 cases of inhalation pneumonia during from Jan 2000 to Dec 2005 were completely surveyed and analyzed. RESULTS There were underlying diseases and susceptible factors, and it was not typical in their clinical signs and symptoms.Totally 177 pathogens were isolated from sputa. There were 96 strains of Gram-negative bacilli (54.2%), 41 strains (23.2%) of Gram-positive cocci, and 40 strains (22.6%) of fungi. The 45 cases (41.7%) were with polyinfections, and 19 cases (17.6%) with double infections.CONCLUSIONS We should enhance diagnosis of inhalation pneumonia, make rational use of antibiotic, and take vigorous precautions against inhalation pneumonia.

Objective To evaluate the therapeutic effect of Xiao'er Zhixie Paste (XZP) by using the young rat model of chronic diarrhea,and to explore its mechanism.Methods Chronic diarrhea model in young rats was induced by ig senna.Rats were ig with Montmorillonite powder of 1.62 g/kg,XZP of low,medium,and high dose (2.03,4.05,and 8.10 g/kg) for treatment.Loose stools rate,loose stool grade and diarrhea index were determined 1 and 3 d after treatment respectively.The water content of small intestine was measured and blood was collected for testing serum succinate dehydrogenase (SDH),amylase,D-xylose by colorimetric determination,testing serum D-lactic acid,IL-1 β,and TNF-α by Elisa after administration.Results The rate of loose stools in XZP 4.05 and 8.10 g/kg dose group,and diarrhea index in 8.10 g/kg dose group significantly reduced after the first treatment.The loose stools rate of XZP 2.03,4.05,and 8.10 g/kg dose group,diarrhea index,serum D-lactic acid level in 4.05,8.10 g/kg group significantly reduced,and serum D-xylose level in 8.10 g/kg dose group significantly increased 3 d after treatment.However,XZP had no significant effect on SDH,amylase activity and IL-1β,TNF-α levels.Conclusion XZP has obvious therapeutic effect on chronic diarrhea in young rats,the mechanism is to increase improve the absorptive function and permeability of intestinal tract.

Objective To investigate the selection of flaps for the reconstruction of facial skin defects.Methods A total of 40 patients with facial skin defects were treated from February 2011 to February 2015 in our hospital,and they were given different surgery methods ac-cording to the different facial skin defect sizes.Minor defects were primarily treated by direct suturing;medium-sized defects were treated by local skin flap transposition or island skin flap;and larger defects were treated by expanded skin flap.Results All patients were followed up for 1 to 2 years.All the flaps survived with good color and texture match.All the scars healed well which were almost invisible.There was no obvious deformity in the donor and recipient sites.In addition,there was no lesion recurred.Conclusion Select suitable flaps for the recon-struction of facial skin defects according to the face defect area could get reliable blood supply of the flap,inapparent incision scar and a high level of satisfaction.

Objective To randomly compare the therapeutic effect and safety between Levetiracetam (LEV) and Phenobarbital (PB) in the treatment of neonatal seizures.Methods A total of 61 infants with acute convulsion were randomly divided into 2 groups:LEV group (n =30) and PB group (n =31) during January 2013 to December 2014 in Urumqi Children's Hospital.All neonates received routine management including etiology treatment and adverse drug reaction monitoring.In the LEV group,subjects received oral formulation of LEV with initial loading dose 30 mg/kg,followed by 15 mg/kg twice a day.If the seizures were not controlled completely,PB treatment was added until seizures were completed controlled.If seizures were controlled quickly,the dose of PB was gradually reduced and LEV was used as monotherapy.The subjects in PB group received intramuscular or intravenous injection of PB with 10 mg/kg as the first dose,then 5 mg/(kg · d) oral PB was administered,if seizures were not controlled,LEV treatment was added,then dose of PB was gradually reduced until seizures were controlled completely,and then patients were switched to LEV monotherapy gradually.The drug adverse reactions were observed.Results (1) After LEV or PB monotherapy,66.7％ (20/30 cases) and 54.8％ (17/31 cases) of the subjects obtained sustainable seizure free respectively.Although,there was a higher control ratio in LEV group,but no significant difference was observed between the 2 groups (P ＞0.05).(2) LEV group (16/30 cases,53.33％) had higher rapid seizure control ratio with seizure controlled within 24 h after first dosage administration than that of PB group (8/31 cases,25.80％),and there was significant difference (x2 =4.841,P =0.028).Further more,if adding the cases who had to change to use another comparative one (LEV or PB) due to their seizures failure control with the first one treated,LEV group (21/44 cases,47.72％) still had higher rapid seizure control ratio in total patients than that of PB group(10/41 cases,24.39％),and there was significant difference (x2 =4.988,P =0.026).(3) Eight cases who changed to LEV after PB as the first treatment drug failed obtained sustainable seizure free.(4) One case in PB group with transient urinary retention was observed but the symptom disappeared 36 h after PB withdrawal,and no significant drug adverse reaction was observed in LEV group.Conclusion LEV is more rapid and safe for seizure control of neonates than PB.

Animals ; Cardiovascular Diseases ; etiology ; Cerebrovascular Disorders ; etiology ; Homocysteine ; metabolism ; Humans ; Methylenetetrahydrofolate Reductase (NADPH2) ; genetics ; Polymorphism, Genetic

Objective:To explore the early warning value of carbapenem-resistant Klebsiella pneumoniae (CRKP) positivity in liver transplantation recipients with rectal swabs, examine the risk factors of CRKP bloodstream infection and provide the relevant treatments.Methods:From June 2018 to December 2019 in Organ Transplantation Research Institute Affiliated Tongji Hospital Tongji Medical College Huazhong University of Science & Technology, 148 cases of liver transplantation recipients with positive CRKP rectal swabbing were recruited. Clinical data were retrospectively analyzed. And the risk factors of CRKP bloodstream infections were examined for intervention and non-intervention groups to observe the effect of interventions of CRKP bloodstream infections.Results:Among them, 23 cases (15.5%) were positive for CRKP and 5 cases (21.7%) were infected with CRKP bloodstream. Rectal swab culture was negative in 125 cases and no bloodstream infection occurred. Long-term use of broad-spectrum antimicrobial agents, severe basic diseases (severe hepatitis), postoperative delayed graft liver function recovery, acute renal failure requiring renal replacement therapy (RRT) and postoperative anti-thymocyte globulin (ATG) induction were risk factors. In intervention group, there were 2 cases (11.1%) of 18 patients with positive CRKP in rectal swab culture in late stage. Among 5 CRKP-positive recipients without intervention, 3 cases (60%) developed later CRKP bloodstream infection. The incidence of bloodstream infection was significantly lower in intervention group than that in non-intervention group ( P<0.05). Conclusions:Rectal swab culture for liver transplantation recipients provides early warning for CRKP bloodstream infection. Interventions for CRKP positive high-risk recipients with rectal swab culture may reduce the occurrence of CRKP bloodstream infection and lower the risk of CRKP bloodstream infection in liver transplantation recipients.