Objective To investigate the expression of exogenous gene transferred by piggyBac (PB) transposon in various gynecological malignant cell lines and reveal its potential application of gene therapy in gynecological cancer.Methods Amplified herpes simplex virus thymidine kinase (HSV-tk) gene coding region by PCR and integrated it into PB expression vector, PB[Act-RFP]DS, for reconstructing PB[Act-RFP, HSV-tk]DS (pPB/TK).By using different transfection reagents: FuGENE HD, jetPEI, lipofectamine 2000, pPB/TK together with helper plasmid Act-PBase were cotransfected into four mostly common gynecological malignant tumor cell lines HeLa, JEG-3, SKOV3 and HEC-1B.The mRFP1 report gene expressions was observed and detected by fluorescence microscope and flow cytometry to analyze transfection efficiency.The expressions of HSV-tk and mRFP1 gene were detected by reverse transcription PCR (RT-PCR).The cytotoxic effect of various concentration of pro-drug ganciclovir (GCV) for transfected cells was detected by methyl thiazole tetrazolium assay.The transfected cells were positive sorted by flow cytometry and limiting diluted to obtain the stable transfected cell line.The insertion sites of foreign gene tranferred by PB transposon in genome were analyzed by inverse PCR.Results (1) Double digests analysis and sequences test demonstrated that pPB/TK vector was reconstructed successfully.(2) Using three different transfective reagents, PB trausposon transferred HSV-tk gene and mRFP1 gene into HeLa, HEC-1 B, SKOV3 and JEG-3 cell efficiently, and the transfection efficiency of pPB/TK for the same cell was different by using different transfective reagents; in Hela cell, the transfection efficiency of FuGENE HD [(78.7 ± 9.2) %]was higher than that of lipofectamine 2000 [(54.1 ± 11.4) %]and jetPEI [(46.5 ± 7.4) %, all P < 0.05] ; using the same transfective reagent, the transfection efficiency of pPB/TK was also different on various cell lines, using FuGENE HD, the transfeetion efficiency of pPB/TK on HeLa, JEG-3 and SKOV3 cell was (78.7 ± 9.2) %, (74.4 ± 8.9) % and (83.2 ± 9.7) % respectively, which all were higher than that on HEC-1B [(39.5 ± 8.7) %, P < 0.05] .(3) RT-PCR showed that there were the mRNA expression of HSV-tk and mRFP1 in all cell lines.(4) 50% inhibitory concentration of GCV for transfected cells, HeLa, JEG-3, SKOV3 and HEC-1B, was 1.29, 3.35, 0.09 and 13.28 μg/ml respectively.Inhibitory effect of GCV (10 μg/ml) on SKOV3 transfected with pPB/TK was (86 ± 9) %, which was superior to that transfected with pORF-HSVtk alone [(52 ± 12)%, P < 0.05] .(5) The insertion sites of PB transposon in the target cells genome were located at TTAA sites, mRFP1 expression still could be detected in three months after transfected.Conclusions PB transposon could transfer exogenous gene into various gynecological malignant cells, which could integrated into genome and obtain a long-term and stable expression.It is expected that PB transposon may supply a more efficient and safer transgene technology platform for gene therapy in gynecological cancer.

Objective:To evaluate the effectiveness of integrated pediatric training course of extracorporeal membrane oxygenation(ECMO)based on simulation teaching.Methods:The instructors of ECMO team of Children′s Hospital of Fudan University developed the teaching curriculum.Team members as a unit were recruited to study simulation-based courses, whose theoretical knowledge, skills and teamwork were assessed, and feedback from the trainees were gathered.Results:Since May 2018, 22 teams of pediatric specialized hospitals or general hospitals have taken part in our ECMO simulation, with totally 132 participants, including 45 ICU doctors(34.1%), 60 ICU nurses(45.5%), 23 thoracic surgeons(17.4%) and four anesthesiologists(3.0%). Twelve of them(9.1%)had a little ECMO experience.After training, the trainees had a high evaluation on each part of the course, with average score more than four, of which the theoretical score was lowest.Their self-evaluation on the theory, skills, teamwork and confidence in the implementation of ECMO had been significantly improved.Most(80.3%)of the trainees were confident to carry out ECMO in their local hospitals.The trainees′ baseline score of theoretical knowledge was 55.2±7.6, and increased to 67.1±7.3 after training( P<0.001). The average teamwork score of the 22 teams was 70.2±8.2, and the qualified rate was up to 86.4%.The most prominent skill problems in operation assessment were no albumin and blood priming(90.9%), non-skill problems were extremely anxious during the overall assessment(84.8%)of the participants, poor team work(74.2%), poor leadership(68.2%). For the feedback of the whole course, 97.0% and 94.7% of the trainees thought that integrated training and simulation teaching were the highlights of the course.At present, 13(59.1%)units have successfully developed ECMO technology after the course, and the number of children treated has been up to 83, with a survival rate of discharge of 54.2%.However, it was found that the skill level of some participants decreased about 2 weeks after the course. Conclusion:As a new attempt of ECMO teaching, this curriculum has achieved certain efficiency in both teaching and clinical practice.At the same time, our study also plays a role in promoting the development of ECMO in pediatrics.

Objective:To study the effect of pig interleukin 2(IL 2) eukaryon expression plasmid on cellular immune responses of BALB/c mice immuned with pcDNA PRRSV ORF5 DNA vaccine.Methods:BALB/c mice were immunized with pcDNA PRRSV ORF5 DNA vaccine and pig interleukin 2(IL 2) eukaryon expression plasmid by the routes of co injection and DNA vaccine injection alone respectively, with PBS and pcDNA3 1(+) as controls. Fluoresecence Activated Cell Sorter(FACS),T lymphocyte proliferation test(MTT) were used to detect the number of CD4 +、CD8 + and the T lymphocyte proliferation in peripheral blood of mice vaccinated.Results:ConA response of T lymphocytes in blood was higher in experiment group than the control group ( P

OBJECTIVE To explore the anti-inflammatory and antifungal action mechanism of taste-masked Lithospermum Saf-flower emulsion in vivo and in vitro.METHODS In vitro,the anti-inflammatory effect was detected by MTT assay,qPCR and ELISA.The anti-fungal effect of the product was investigated by broth dilution experiment,bactericidal kinetics,germ tube inhibition and XTT reduction test.In vivo,the effect was evaluated and the mechanism was investigated on the skin disease model of Candida al-bicans in mice.RESULTS Lithospermum in taste-masked Lithospermum Safflower emulsion had a significant inhibitory effect on the proliferation of RAW264.7 cells,and Safflower inhibited the production of IL-6 induced by LPS in a dose-dependent manner.Litho-spermum significantly inhibited the activity of Candida albicans,and its bactericidal mode is time-and concentration-dependent;Lithospermum significantly reduced the formation of Candida albicans germ tubes and destroyed its biofilm;Safflower had no direct kill-ing effect on Candida albicans,was not able to inhibit its biofilm formation,but significantly reduced the hyphal growth of Candida al-bicans and increased its ROS level.CONCLUSION The combination of Lithospermum and Safflower in the taste-masked Lithosper-mum Safflower emulsion can work synergistically to reduce inflammatory damage and treat Candida albicans infection of the skin.

Objective To examine the association between uric acid (UA)levels of patients with acute ischemic stroke at ad-mission and discharged outcome.Methods The acute ischemic stroke patients of Xinganmeng People’s Hospital in Inner Mongolia,from June 1,2009 to May 31,2012 were continuity included in the present study,the included analysis sample size were 3 440 cases.Poor discharged outcome was defined as the occurrence of disability or death.With reference to the Modi-fied Rankin's Scale (MRs)Stroke Scale,Scores were recorded in the questionnaires,score of 3 or more (MRs≥3)was de-fined as disability.The patients were all grouped by P20,P60,P90 of UA,binary logistic regression were used in studying of risk factors,calculated the odds ratios (Odds ratio,OR)and 95% confidence interval (95% Confident interval,95%CI).All tests were two-sided test and a significance level of 0.05.Results A total of 359 people occurred poor outcomes in the stud-y,accounting for 10.44%.Univariate logistic regression analysis of poor outcome occurred showed that relative to the lowest group(P20,UA≤222.6 mmol/L),the second and third group (UA:222.7 ~ 310.9 mmol/L and 311.0~419.7 mmol/L) OR (95% CI)were:0.70(0.53~0.91)(P <0.05)and 0.66(0.49~0.88)(P <0.05).After adjusted age,body tempera-ture,high blood pressure,hyperglycemia,history of stroke,high triglycerides,high LDL-C and smoking,relative to the low-est level group,the second and third group occurred poor outcoming OR (95% CI)were:0.70(0.53~0.93)(P <0.05)and 0.66(0.48~0.90)(P <0.05).Conclusion Higher levels of uric acid levels in patients with acute ischemic stroke may inde-pendently related with occurred poor discharged outcome.

Objective: To explore the correlation between pull-up ability and upper body composition of male college students in a college in Guangxi, and to provide scientific guidance for college students’ exercise. Methods: A total of 685 male college students were randomly selected from a college in Guangxi.Pull up tests were implemented according to the national physical health test standards. Measurements of muscle mass, fat mass, fat percentage, etc. of the upper limbs were conducted by using the Ogilvy Body Composition Meter (TANITA MC-180). Data entry and analysis were performed by using SPSS 23.0. Results: The pass rate of male college students in the region was 21.7%; There was a statistically significant difference in the fat mass and percentage of body fat between males with different pull-up ability (F=11.30，14.18，12.91，15.22，P<0.01).After controlling age, height, weight and BMI, partial correlation analysis showed that there was a negative correlation between the pull-up ability of male college students and the fat mass and limb fat rate of both upper limbs(r=-0.22, -0.33, -0.31, -0.38, P<0.01). Conclusion The ability of male students in Guangxi to pull up is needed to be improved. Pull-up exercises can reduce fat mass and fat percentage in the upper limbs and improve body composition.

Animal study plays a key role to evaluate the safety and effectivity of medical devices. This study aims to share evaluated consideration on the role, general principles, necessity, study design and quality management system of animal study, in order to improve the quality of animal study and reduce unnecessary study.
Animals ; Equipment and Supplies ; Models, Animal

OBJECTIVETo retrospectively explore the effectiveness of surgical intervention model for repairing the tuberculosis wound with sinus tract.

METHODSForty-three patients with tuberculosis wound with sinus tract who met the inclusion criteria were admitted to the 309th Hospital of PLA from January 2010 to October 2015. These patients were divided into test group (n=38) and control group (n=5) according to the different treatment and patient's consent. Patients in test group were treated as follows. Firstly, antituberculosis drugs were taken orally for at least 3 weeks, and the wounds were accurately assessed using magnetic resonance imaging combined with 3-dimensional reconstruction software. Then sinus tract and its surrounding devitalized tissue were completely excised, and vacuum sealing drainage (VSD) treatment with negative pressure value of -26.6 kPa was performed for 1 to 2 weeks (dressing change was performed per 7 days). Lastly, the wounds were covered through direct suture or grafting skin or flap. Patients in control group were firstly given antituberculosis drugs orally for at least 3 weeks, and then they were treated with routine dressing change in outpatient service every 3 days. After the former therapy, patients in both groups were given antituberculosis drugs by oral administration for at least 6 months and were followed up for 6 to 36 months. Detection of Bacillus tuberculosis, Acid-fast bacilli, and tuberculosis granuloma, wound healing time, and relapse of tuberculosis wound in patients of both groups were recorded. The rates of single sinus tract, two sinus tracts, and more than or equal to 3 sinus tracts of patients in test group were recorded. Data were processed with Fisher's exact test and Wilcoxon rank-sum test.

RESULTSBacillus tuberculosis was respectively detected in wounds of 5 patients in test group and 2 patients in control group. Acid-fast bacilli were positively expressed in wounds of 8 patients in test group and 3 patients in control group. A typical tuberculosis granuloma phenomenon was observed in the wounds of 27 patients in test group and 4 patients in control group. These differences in above-mentioned 3 indexes between two groups were not statistically significant (with P values respectively 0.238 4, 0.154 4, 1.000 0). The median of wound healing time of patients in test group was 19.6 d, which was significantly shorter than that in control group (94.4 d, χ(2)=12.986 0, P=0.000 3). There were 2 and 1 patients with recurrent tuberculosis wound in test group and control group respectively, without statistically significant difference (P=0.363 0). Among patients in test group, the rate of single sinus tract was 23.7%(9/38), the rate of two sinus tracts was 28.9%(11/38), and the rate of more than or equal to 3 sinus tracts was 47.4% (18/38).

CONCLUSIONSRepairing the tuberculosis wound with sinus tract in surgical intervention model of antituberculosis therapy+ accurate wound assessment+ debridement+ VSD treatment+ surgical repair is beneficial to making the optimal operation plan under the premise of knowing location of sinus tract, which can reduce surgical risk.

Debridement ; Humans ; Magnetic Resonance Imaging ; Negative-Pressure Wound Therapy ; Paranasal Sinuses ; pathology ; surgery ; Retrospective Studies ; Skin Transplantation ; Surgical Flaps ; Treatment Outcome ; Tuberculosis ; drug therapy ; surgery ; Wound Healing

OBJECTIVETo prepare and characterize rabbit polyclonal antibodies against Toxoplasma gondii vacuolar proton pyrophosphatase type I (TgVP1).

METHODS AND RESULTSTwo synthesized peptides TgVP1-1 and TgVP1-2 as the haptens were conjugated with KLH to immunize rabbits. Indirect ELISA showed that the titers of rabbit anti-TgVP1-1 polyclonal antibody and rabbit anti-TgVP1-2 polyclonal antibody reached 1:128 000. Western blotting results revealed that both purified polyclonal antibodies could specifically bind to a purified 85 kD T. gondii protein predicted as TgVP1. The protein detected by these two polyclonal antibodies was distributed in the cytoplasm of T. gondii tachyzoite, and this distribution pattern was consistent with that of acidocalcisome.

CONCLUSIONThe peptide-based method of antibody generation is efficient and the obtained TgVP1 polyclonal antibodies possess a high specificity to facilitate further study of T. gondii acidocalcisome and the diagnosis of toxoplasmosis.

Animals ; Antibodies ; immunology ; Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Protozoan Proteins ; immunology ; Pyrophosphatases ; immunology ; Rabbits ; Toxoplasma ; enzymology

Objective To investigate the efficacy of Madopar on vascular parkinsonism with paroxysmal atrial fibrillation(VPD+ PAF)in elderly patients and its effect on the autonomic nervous system. Methods We recruited 50 patients aged over 60 with VPD + PAF into this randomized controlled trial.Based on sinus rhythm recorded by 24 hour ambulatory electrocardiogram ,individuals with an average heart rate below 60 times per minute were assigned into a sinus bradycardia group (n＝15)and those with a normal average heart rate were randomly assigned into a normal rate group (n＝20)or a control group (n＝ 15)by drawing lots.The control group was given routine care for 12 weeks ,while the other two groups were treated with Madopar plus routine care for 12 weeks. The three groups were examined using UPDRS-3 ,dynamic electrocardiogram ,heart rate variability , echocardiography ,and plasma acetylcholine(ACH)levels 1 day before treatment ,4 weeks and 12 weeks after treatment ,and atrial fibrillation frequency 2 weeks after treatment. Results UPDRS-3 scores decreased significantly in the sinus bradycardia group and the normal rate group after treatment for 4 weeks and 12 weeks ,compared with those before treatment (P＜0.05) ;UPDRS-3 scores in the control group did not show statistically significant changes at week 4 after treatment but decreased significantly at week 12(P＜0.05).UPDRS-3 scores in the sinus bradycardia group and the normal rate group were lower than those in the control group after treatment for 4 weeks and 12 weeks(P＜0.05). The total effectiveness rates in the sinus bradycardia group (86.7%,n＝13)and the normal rate group(85.0%,n＝17)were higher than the rate in the control group(53.3%,n＝8)(P＜0.05).Ach levels in the sinus bradycardia group and the normal rate group significantly decreased after treatment for 4 weeks and 12 weeks ,compared with before-treatment levels ,but no significant difference was observed between before-and after-treatment levels in the control group. Frequencies of AF in the sinus bradycardia group(5.6 ± 3.5)and the normal rate group(5.2 ± 4.1)were significantly lower than in the control group(8.4 ± 5.2)(P＜0.05). Conclusions Madopar can depress vagus nerve activity ,relieve VPD+ PAF symptoms ,and reduce the frequency of AF ;Heart rate variability and ACH level are potential predictors of clinical efficacy of Madopar in VPD patients.