In order to establish stable high expression cell lines, the eukaryotic expression vector pIRES2EGFP and recombinant plasmid pIRES2EGFP-TIM-3 were transfected into mammalian cells CHO by Lipofectamine. The transfected cells were cultivated under selective growth medium including G418 and green fluorescent protein (GFP) positive cells were sorted by FACS. Simultaneously, growing transfectants were selected only by G418 in the medium. The GFP expression in stably transfected cells was detected by FACS. Under selective growth conditions with G418, the percentage of GFP positive cells was reduced rapidly and GFP induction was low. In contrast, the percentages of GFP positive cells were increased gradually after FACS. By 3 rounds of GFP selection, the stable high expression cell lines were established. Furthermore, using FACS analysis GFP and the target protein TIM-3 co-expression in the stable transfectants cultured in nonselective medium was detected. Theses results demonstrated that the stably transfected cell lines that express high titer of recombinant protein can be simply and fleetly obtained by using GFP and selective growth medium.

Objective To investigate the correlation between serum procalcitonin (PCT) levels and infection sites,as well as between PCT and bacterial species in gram negative (G-) bacteria induced sepsis,so as to provide rationale for therapeutic strategy of using antibiotic in sepsis.Methods The data of patients with sepsis admitted in Emergency Department and ICU from January 2014 to June 2015 were retrospectively analyzed.The blood culture of G-bacteria and PCT detection were carried out simultaneously within 24 hours after admission.The clinical data was analyzed to find out the correlation between PCT levels and infection sites,as well as between PCT levels and pathogenic bacterial species.Results A total of 187 specimens (came from 162 patients) were enrolled in the study with a median age of 70 years old and a median sequential organ failure assessment (SOFA) score of 4.PCT levels were found to be associated with bacterial species.PCT level caused by Escherichia coli bacteremia infection was higher than that caused by Acinetobacter baumannii bacteremia and Burkholderia cepacia bacteremia infection (4.62 ng/mL vs.2.44 ng/mL;4.62 ng/mL vs.0.81 ng/mL;P ＜ 0.05).Receiver operating characteristic (ROC) analysis showed an area under the curve (AUC) for PCT was 0.61 to discriminate Escherichia coli infection from Acinetobacter baumannii infection and an AUC was 0.66 to discriminate Escherichia coli infection from Burkholderia cepacia infection.When the cutoff point of PCT was 30.32 ng/mL,it could predict Escherichia coli infection rather than Acinetobacter baumannii infection with 94.10％ specificity,90.00％ positive predictive value and positive likelihood ratio for 4.24.When the cutoff point of PCT was 8.01 ng/mL,it could predict Escherichia coli infection rather than Burkholderia cepacia infection with 85.70％ specificity,93.94％ positive predictive value,and positive likelihood ratio for 3.01.When PCT cutoff value reached 47.31 ng/mL,the specificity and positive predictive value were both 100.00％.PCT level caused by urinary tract infection was higher than that caused by pulmonary infection (11.58 ng/mL vs.2.07 ng/mL,P ＜ 0.05),and the AUC was 0.69.When the cutoff point of PCT was 32.11 ng/mL,it could predict Escherichia coli infection rather than Acinetobacter baumannii infection with 90.60％ specificity,86.18％ negative predictive value and positive likelihood ratio for 3.68.Conclusions PCT elevation in G-bacteria induced sepsis might be associated with infection sites and bacterial species.

AIM: To study the effect of S-methylisothiourea(SMT) on adriamycin(ADM)-induced changes of superoxide dismutase and alutathione peroxidase activities in myocardial tissue of rats.METHODS: Rats were treated with ADM by intraperioneal injection (10.0 mg/kg body weight),and then the ADM-treated rats were intervened by SMT at different dosages by intravenous injection(once a day for three days).The contents of malondialdehyde(MDA) and NO_2~-/ NO_3~-,the activities of manganes superoxied dismutase(MnSOD),copper-zinc superoxide dismutase(Cu-Zn SOD),glutathione peroxidase(GPx) and inducible nitric oxide synthase(iNOS) were determined by spectrophotometry in myocardial tissue.The activity of isoenzymic CK-MB of creatine kinase(CK) was determined by enzymic rate method in serum.The expression of MnSOD mRNA,Cu-ZnSOD mRNA,GPx mRNA and iNOS mRNA were detected by reverse transcription-ploymerase chain reaction.RESULTS: The contents of MDA,NO~-_2/ NO~-_3,and iNOS activity of myocardial tissue,the activity of CK-MB in serum in SMT(5.0,10.0,20.0 mg/kg) intervention groups were significantly lower than those in ADM-treated group(P

Objective To investigate the characteristics and the risk factors of pulmonary function in patients with chronic obstructive pulmonary disease (COPD) for a 3 year follow-up.Methods Subjects diagnosed as COPD were followed up for 3 years in the Management Center of Chronic Respiratory Disease at XINQIAO Hospital from September 2009 to June 2012.This was a retrospective study.Parameters related to respiratory function mainly first second forced expiratory volume (FEV1),COPD assessment test (CAT),6 minutes walking distance (6MWD) and acute exacerbation were recorded during follow-up.Results Although the majority of patients were treated with drugs such as inhaled corticosteroid combined with longterm bronchial dilatation during the three years,FEV1 decreased progressively.The average annual decline of FEV1 was(31.80 ± 61.99) ml,translating into a mean annual decline of(3.74 ± 6.18) ％.However,there were significant differences in changes of FEV1.Approximately,FEV1 in 78.3％ (47/60) patients decreased,only 21.7％ (13/60) patients kept stable FEV1.There was a correlation between decrease of FEV1,FEV1 ％ predicted and the exacerbation (r =0.298,0.361,0.273;P ＜ 0.05).Logistic regression showed that the positive bronchodilator reversibility and the initial FEV1 were the independent factors associated with significant changes in FEV1 (respectively,OR =5.54,95％ CI 1.55-19.73;OR =8.28,95％ CI 1.42-48.32).Conclusion The changes of pulmonary function in patients with COPD are heterogeneous,although most patients are treated in a standard way.Nearly 80％ patients still represent deterioration of pulmonary function.Decline of FEV1 is closely related to the initial pulmonary function and bronchodilator reversibility.

Objective To investigate the level of platelet activating factor (PAF) in acute myocardial infarction (AMI) in minipig model and patients, and to study the relationship between PAF and lethal arrhythmia referring to ventricular fibrillation and ventricular tachycardia. Method ( 1 ) The levels of PAF in minipig models ( n = 20) were measured by using ELISA before and 1h after occlusion of left anterior descending coronary artery with balloon at the junction of 1/3 middle and distal portion. The lethal arrythmia was recorded by using electrocardiography. (2) In patients with AMI (n = 72), the levels of PAF were measured on arrival, and 24 h,48 h and 72 h later. The lethal arrythmia, acute heart failure and cardiogenic shock were documented. Results ( 1 ) In minipigs with occlusion of coronary artery for one hour, the mean level of PAF increased from (4.66± 2.89)ng/mL to (6.00±2.82) ng/mL,and thus the increment in PAF was (1 .34± 1.40) ng/mL (P ＜ 0.05). In 13 minipigs with lethal anythmia after occlusion of coronary artery for one hour, the increment in mean level of PAF was ( 1.92 ± 1 .34) ng/mL, whereas the increment in mean level of PAF in other 7 minipigs without lethal arrythmia after occlusion of coronary artery for one hour was as low as (0.28 ± 0. 74 ) ng/mL ( P ＜ 0. 05 ). ( 2 ) In patients, the mean levels of PAF on arrival, 24 h,48 h,and 72 hous after admission were (0.47 ± 0.05) ng/mL,(2.38±0.12) ng/mL,(3.65±0.15) ng/mL and (3.02±0.10) ng/mL, respectively. Of 72 ACI patients, 40 (55%) had complication of lethal arrythnia, heart failure or cardiogenic shock and their mean level of PAF 48 h after admission was (4.72 ± 0.16) ng/mL, whereas mean level of PAF in other 32 (44.44%) without complications was (2.31 ±0.03) ng/mL ( P ＜0.05). Conclusions The level of PAF increased after acute myocardial infarction, and the minipigs and AMI patients complicated with lethal arrythmia had higher levels of PAF.

In order to establish stable high expression cell lines, the eukaryotic expression vector pIRES2EGFP and recombinant plasmid pIRES2EGFP-TIM-3 were transfected into mammalian cells CHO by Lipofectamine. The transfected cells were cultivated under selective growth medium including G418 and green fluorescent protein (GFP) positive cells were sorted by FACS. Simultaneously, growing transfectants were selected only by G418 in the medium. The GFP expression in stably transfected cells was detected by FACS. Under selective growth conditions with G418, the percentage of GFP positive cells was reduced rapidly and GFP induction was low. In contrast, the percentages of GFP positive cells were increased gradually after FACS. By 3 rounds of GFP selection, the stable high expression cell lines were established. Furthermore, using FACS analysis GFP and the target protein TIM-3 co-expression in the stable transfectants cultured in nonselective medium was detected. Theses results demonstrated that the stably transfected cell lines that express high titer of recombinant protein can be simply and fleetly obtained by using GFP and selective growth medium.

Objective:To investigate the expression of PDLIM3 in the ectopic and orthotopic endometrium of endometriosis(EMT)patients,as well as in healthy endometrium,and to explore its relationship with clinical signifi-cance.Methods:Bioinformatics analysis based on the Gene Expression Omnibus(GEO)database was conduc-ted,and a prospective cohort study was undertaken,involving 58 EMT patients treated at West China Second Hospital of Sichuan University and 27 patients with other benign gynecological diseases as the control group from December 2019 to July 2020.Ectopic and orthotopic endometrium samples were obtained from EMT patients,and normal control samples were obtained from patients with other benign gynecological diseases.Real-time quantita-tive polymerase chain reaction(qPCR),western blot,and immunohistochemistry were utilized to investigate PD-LIM3 expression in various tissue samples.Clinical parameters were analyzed to determine the correlation be-tween PDLIM3 and clinical features.Results:Compared to orthotopic endometrial tissues of EMT patients and normal endometrial tissues of control subjects,the mRNA(F =46.05,P<0.05)and protein(F =238.6,P<0.05)expression levels of PDLIM3 in ectopic endometrial lesions were significantly upregulated.The average optical density(AOD)of PDLIM3 protein obtained through immunohistochemical experiments in ectopic endometrial le-sions showed a positive correlation with the degree of dysmenorrhea(r =0.86,P<0.05)and CA 125 level(r = 0.41,P<0.05)in EMT patients.But no obvious correlation was found between the expression level of PDLIM3 and disease stage,menstrual volume,and CA199 level(P>0.05).Conclusions:The expression level of PDLIM3 in endometriosis lesions was significantly upregulated and correlated with the degree of dysmenorrhea and serum CA125 level.These findings suggest that PDLIM3 may play a significant role in the development and progression of endometriosis and could serve as a potential therapeutic target.

Objective To utilize the magnetic resonance neurography (MRN) as a feasible tool for measuring the anatomical parameters of lumbar spinal nerves, and further to evaluate the neuro-safety of interlaminar percutaneous endoscopic lumbar discectomy.Methods Thirty healthy adult volunteers without significant history of low back pain or lumbar deformity were selected in our hospital from September 2016 to December 2016. All subjects accepted MRN. The nerve roots of L2-S1 were measured at the starting point of dural sac, and the angles between nerve roots and dural sac were measured. The distances between L2-L5 nerve roots and the edge of ipsilateral dural sac were measured and analyzed statistically.Results All MRN showed a gradual increase in the origin of the nerve roots from L2 to S1. The origin of the root was found to be below the corresponding disc for the L2 to L4 roots. There were 70％ of the L5 roots originated below the L4/5 disc, 26.7％ at the L4/5 disc, and 3.3％ above the L4/5 disc; about 70％ of the S1 roots originated above the L5/S1 disc. There were no statistically significant differences in the angles between dural sac and both left and right nerve roots (P>0.05). The angels between the nerve root and the dural sac from L5 and S1 was smaller than those from L2, L3, and L4 (P<0.05); that from S1 was significantly smaller than that from L5 (P<0.05). The distance of the nerve root and the ipsilateral dural sac was significantly increased in each side from L2 to L5 (P<0.05). There was no statistically significant difference in the distances between the left and right nerve roots and the edge of the ipsilateral dural sac in the same segment (P>0.05).Conclusion MRN is a feasible tool to measure the anatomical parameters of the lumbar spinal nerve, and there is a safe neurological area of the percutaneous endoscopic lumbar discectomy through the interlaminar approach.

Objective:To explore a new technique for lung dose reduction in esophageal cancer radiotherapy based on the dose distribution characteristics of the half-field combined with intensity-modulated radiotherapy (IMRT) technique.Methods:A three-dimensional water tank was used to measure the dose distribution at the edge of the symmetrical field and half-field, which was then compared and analyzed. Twenty patients with the middle and lower thoracic esophageal cancer receiving radiotherapy with prescription doses of 50.4-60.0 Gy were selected. Based on the Varian Vital beam linear accelerator and Eclipse planning system, flattening filter (FF) technique symmetrical field and half-field beam design and the flattening filter-free (FFF) technique symmetrical field and half-field design were adopted to compare and analyze various dose data and treatment MU numbers for the target area and the endangered organs. The field settings were chosen in the front 1 and back 4 mode.Results:Compared with the symmetrical field plan, the half-field plan significantly improved the irradiated dose to the lung with a statistically significant difference ( P<0.05), and the half-field FFF was slightly better than the half-field FF mode. Compared with the total lung V 5Gy, V 20Gy, V 30Gy, and D mean dosimetric parameters (Gy), the FF symmetric field and FFF half-field were (49.64±5.39)% vs.(42.70±5.53)%, (15.99±3.93)% vs.(13.32±3.06)%, (9.24±2.77)% vs.(8.50±2.62)%, and (10.45±1.76)% vs.(9.50±1.53)%, respectively. There was a significant reduction in the volume dose values for all structures of the lung (all P<0.05). For other comparative data, the irradiated dose for the heart of the symmetrical field was better than that of the half-field mode ( P<0.05), and the differences in conformity index (CI) and homogeneity index (HI), number of treated MU, and spinal cord associated with the target area were not statistically significant (all P>0.05). Conclusions:During radiotherapy for esophageal cancer, target area coverage and dose volume data of the lung are the main parameters affecting the efficacy and side effects. IMRT treatment based on the half-field mode can give full play to the advantages of half-field and IMRT, and significantly improve the irradiated dose to the lung, which can offer an additional clinical option.

Objective To observe the effects of electroacupuncture on the learning and memory abilities of Alzheimer disease(AD)rats;To explore its potential mechanism based on proteomics.Methods Totally 36 SD male rats were randomly divided into sham-operation group,model group and heart meridian acupoints group,with 12 rats in each group.Aβ1-42 were injected into the bilateral hippocampus to establish AD rat model,the sham-operation group was injected with an equal volume of normal saline.The heart meridian acupoints group received electroacupuncture treatment,accompanied by a stimulation time of 20 minutes,rest for 1 day after 6 days of electroacupuncture for 7 consecutive weeks.The Morris water maze experiment was used to evaluate the learning and memory abilities of AD rats,tandem mass tag(TMT)labeling technology and bioinformatics analysis were used to screen core differentially expressed proteins in important typical pathways,and key differentially expressed protein was verified by parallel reaction monitoring(PRM).Results There was no statistically significant difference in swimming speed between each group of rats(P>0.05).Compared with the sham-operation group,the escape latency of Morris water maze experiment in the model group increased significantly(P<0.01);compared with the model group,the escape latency of heart meridian acupoints group was significantly shortened on the 2-4th day(P<0.01).A total of 209 differentially expressed proteins were identified in different groups using TMT labeling quantification,among which 12 proteins showed significant changes among the 3 groups.GO annotation involved biological processes such as metal ion transport,sodium ion transport,and sodium ion transmembrane transport,as well as cellular components such as synapses,presynapse,and synaptic vesicle,involving solute:sodium symporter activity,organic acid:sodium symporter activity,amino acid:cation symporter activity,amino acid:sodium symporter activity,and other molecular functions;KEGG analysis significantly enriched the synaptic vesicle pathway.The PRM validation results indicated that electroacupuncture at the heart meridian acupoints could reduce the expressions of sodium and chloride dependent GABA transporter protein 3(GAT3),which was consistent with the quantitative detection results of TMT labeling quantification.Conclusion Electroacupuncture at the heart meridian acupoints can improve the learning and memory abilities of AD rats,possibly by regulating the expression of synaptic transporter GAT3 on the synaptic vesicle pathway to exert neuroprotective effects.