Objective To isolate,purify and identify exosomes secreted by mouse primary peritoneal macrophages.Methods Five male C57BL/6 mice were intraperitoneally injected with 3% mercaptoacetate broth respectively,and the primary peritoneal macrophages were obtained by lavage,and then the purity was analyzed by flow cytometry.The exosomes of mouse primary peritoneal macrophages were extracted by ExoQuick TC exosome kit,which were measured for the protein content with BCA kit,observed for the morphology by transmission electron microscopy,detected for the particle size and distribution with nanoparticle tracking analyzer,and determined for the expression of exosome-specific markers(CD9,CD63 and TSG101) by Western blot.Results About 5 × 10~6 peritoneal macrophages with the purity of(99.17±0.65)%were obtained from each mouse.Approximately 869 μg of exosomal protein was extracted from 5 mL of mouse primary peritoneal macrophage culture supernatant.The exosomes of mouse primary peritoneal macrophages were typical tea saucerlike vesicles with strong refraction under electron microscopy,and highly expressed the exosome-specific markers TSG101,CD63 and CD9.The particle size distribution was concentrated between 100 and 200 nm,with an average particle size of175.2 nm.Conclusion Intraperitoneal injection of mercaptoacetate broth can improve the yield of mouse primary peritoneal macrophages.ExoQuick TC.exosome kit can extract sufficient amount of exosomes with high purity from mouse primary peritoneal macrophages.

Objective To isolate,purify and identify exosomes secreted by mouse primary peritoneal macrophages.Methods Five male C57BL/6 mice were intraperitoneally injected with 3% mercaptoacetate broth respectively,and the primary peritoneal macrophages were obtained by lavage,and then the purity was analyzed by flow cytometry.The exosomes of mouse primary peritoneal macrophages were extracted by ExoQuick TC exosome kit,which were measured for the protein content with BCA kit,observed for the morphology by transmission electron microscopy,detected for the particle size and distribution with nanoparticle tracking analyzer,and determined for the expression of exosome-specific markers(CD9,CD63 and TSG101) by Western blot.Results About 5 × 10~6 peritoneal macrophages with the purity of(99.17±0.65)%were obtained from each mouse.Approximately 869 μg of exosomal protein was extracted from 5 mL of mouse primary peritoneal macrophage culture supernatant.The exosomes of mouse primary peritoneal macrophages were typical tea saucerlike vesicles with strong refraction under electron microscopy,and highly expressed the exosome-specific markers TSG101,CD63 and CD9.The particle size distribution was concentrated between 100 and 200 nm,with an average particle size of175.2 nm.Conclusion Intraperitoneal injection of mercaptoacetate broth can improve the yield of mouse primary peritoneal macrophages.ExoQuick TC.exosome kit can extract sufficient amount of exosomes with high purity from mouse primary peritoneal macrophages.

Objective To evaluate the efficacy of intra-aortic balloon pump (IABP) on elderly patients with acute myocardial infarction (AMI) combined with cardiogenic shock (CS).Methods Among 62 elderly patients with AMI plus CS undergoing percutaneous coronary intervention (PCI),32 patients received IABP before PCI (IABP group) and 30 patients did not (control group).Diastolic blood press ure(DBP),mean arterial pressure (MAP),heart rate,cardiac index (CI),and ejective fraction before and 2 weeks after PCI in the two groups were observed.The short-and long-term therapeutic effects were compared between the two groups.Results The DBP,MAP and CI were higher and heart rate was lower in IABP group than in control group within 24 h after IABP [(64.4± 11.1) mmHg vs.(37.8±15.1) mmHg,(71.4±8.3) mmHg vs.(43.0±10.5) mmHg,(2.98± 0.33) L· min-1 · m-2 vs.(1.99±0.29) L· min-1 · m-2,(90.7±18.7) /min vs.(130.2±50.1)/min,t=7.97,11.83,12.51,4.16,all P=0.000].Two weeks after IABP,LVEF was significantly improved in IABP and control groups as compared with pre-IABP [(46.4±7.2)％ vs.(35.2± 7.2) ％,(39.1±6.8) ％ vs.(33.8±6.7) ％,both P＜0.01],and heart function was improved more significantly in IABP group than in the control group (t=3.91,P=0.000).Death tolls during hospitalization and after leaving hospital,and recurrence of AMI had no significant differences between the two groups (2 cases vs.4 cases,3 cases vs.8 cases,6 cases vs.10 cases,x2 =0.89,3.17,1.72,P=0.346,0.075,0.190).Conclusions IABP can improve the cardiac function in elderly AMI patients with CS after PCI.

ObjectiveToanalyzethepathogensofsuppurativeendophthalmitis,theirdistributionanddrug susceptibility.Provide valuable information for the clinical options in effective use of antimicrobial agents.Methods Collected 259 cases of endophthalmitis from the patients with aqueous humor and vitreous fluid for pathogens and drug sensitivitytestinordertocarryontheanalysisoftheresults.Results 113casesofbacterialculturepositivein259 cases of specimens,with a detection rate of 43.63%.6 cases of multiple bacteria infection,and 2 case of fungal infec-tion.The total separation of pathogens was 120 strains.89 strains were gram positive coccus,accounting for 74.17%, 11 were gram positive bacillus strains, accounting for 9.17%, 13 strains of gram-negative bacilli, accounted for 10.83%,gram-negative in 5strains(4.17%),fungus in 2strains(1.67%).Conclusion Main pathogenic bacteria suppurative endophthalmitis is gram-positive, the pathogen species distribution and antimicrobial susceptibility of change should be timely monitored and the rational using of antibiotics can reduce the generation of drug-resistant strains.

Objective:To investigate the effect of NS-398,a selective cyclooxygenase-2(COX-2) inhibitor,on the proliferation and invasion of human colon carcinoma cells in vitro,so as to determine the possibility of COX-2 as a new target for treatment of colon carcinoma.Methods: The expression of COX-2 in colorectal cancer cells(CW-2,COLO-320) was detected by RT-PCR and Western blotting.COLO-320 cell proliferation was measured by MTT after treatment with NS-398.Cell invasion ability was measured using migration and invasion chamber systems.Western blotting assay was used to examine the influence of NS-398 on MMP-2 expression.Results: Our results showed that CW-2,COLO-320 cells expressed COX-2 mRNA and protein.NS-398 inhibited the proliferation of COLO-320 cells in a time-and concentration-dependent manner.Invasion test showed that NS-398 inhibited the migration and invasion of COLO-320 cells.Western blotting revealed that NS-398 inhibited the expression of MMP-2 in COLO-320 cells.Conclusion: The selective COX-2 inhibitor NS-398 can inhibit COLO-320 cell proliferation and invasion,indicating COX-2 may serve as a new target for colon carcinoma treatment.

Objective To investigate the relationship between serum leptin and carotid intima-media thickness in patients with type 2 diabetes mellitus. Methods 35 patients with type 2 diabetes mellitus (DM) and 16 normal controls were studied. Fasting serum leptin, fasting glucose(FBG), HbAlc, C-peptide, lipide, systolic(SBP), diastolic blood pressure(DBP), body mass index (BMI) and waist circ-umference (WC) were measured in all subjects. The intima-media thickness (IMT) of common carotid artery was measured by high-resolu-tion ultrasound imaging. Results IMT of T2DM was positively correlated with leptin concentration. Conclusion Serum leptin concentra-tion is independently associated with the IMT of carotid artery, which suggest that leptin may be an independent risk factor of atherosclerosis.

Objective:To investigate the correlation between single nucleotide polymorphism ( SNP ) of rs7517847 and rs10489629 on IL-23R gene and susceptibility of ankylosing spondylitis in Jilin.Methods:IL-23R gene polymorphisms of 188 cases on ankylosing spondylitis were detected by polymerase chain reaction-restriction fragment length polymorphism ( PCR-RFLP ) , compared with those of 100 cases of healthy control group.Results:The frequency distribution differences ,between AS group and comparison group of separate genotype and the allele on two SNPs (rs7517847 and rs10489629) both showed statistical significance (P<0.05).Meanwhile, by the assumed genetic way , compared homozygous mutant GG of rs 7517847 with TG+TT, compared homozygous mutant AA of rs10489629 with GA+GG,such frequency distribution difference between the above two groups also showed statistical significance ( P<0.05).Conclusion:Polymorphisms about IL-23R gene of the rs7517847 and rs10489629 are both associated with susceptibility of AS in Jilin.The risk of AS will be increased if the person both with G/A allele and GG/AA genotype , which may be one of susceptive factors by AS.

Objective To investigate the plantar pressure distribution of the injuried limbs and the healthy side after the operation for Pilon fracture. Methods 31 patients with Pilon fractures were tested with Footscan USB2 system, including the maximum force and impulse of 10 zones of the feet 1 year after surgery. Results Compared with the contralateral feet, the maximum force reduced under the the first metatarsal bone, medial heel, and lateral heel (P<0.05), and it increased under the fourth, and fifth metatarsal bone (P<0.05) of the injured feet. The impulse reduced under the the first metatarsal bone, medial heel, and lateral heel (P<0.05), and increased under the fifth metatarsal bone and midfoot (P<0.05). Conclusion The load decreased on the heel and medial forefoot, and increased on the lateral forefoot of the injured limbs after Pilon fracture, while the lateral forefoot and midfoot tend to be injured.

Objective To compare and analyze the results of patients' blood sugar value tested by Terumo glucometer and Hitachi 7180 biochemical analyzer, and to analyze their correlation. Methods Fifty blood samples with different blood sugar values. Terumo glucometer was used to test blood sugar for three times and an average value was calculat-ed;full-automatic Hitachi 7180 biochemical analyzer was used to test blood sugar for three times by serum centrifuga-tion of 3000 rpm, and an average value was calculated. SPSS software was used for analysis. Results Precision of Teru-mo glucometer CV% was less than 5%, and the relative bias accorded with related requirements. Compared with the test results by Hitachi 7180 full-automatic biochemical analyzer, results of blood sugar by Terumo glucometer were all within the allowed range. Standard deviation of sample average of 50 samples tested by Terumo glucometer was (7.51±0.69), and the standard deviation of sample average of samples tested by Hitachi 7180 biochemical analyzer was (7.37±0.66). Results of blood sugar tested by Terumo glucometer and Hitachi 7180 biochemical analyzer were not statistically different (t=0.15, P>0.05); correlation coefficient R2 of Terumo glucometer and Hitachi 7180 biochemical analyzer was 0.9739. Conclusion Correlation between glucometer and full-automatic biochemical analyzer in testing blood sugar is favorable, which can be used for blood sugar testing for diabetic patients, but the blood sugar value tested by biochemi-cal analyzer must be taken as diagnostic basis.

There is growing interest in biodiesel and this results in the accumulation of glycerol. The exploitation and application of glycerol has attracted more and more attention. In the current study, glycerol was biotransformed to produce 3-hydroxypropionaldehyde by genetic engineering bacteria. It is known that 3-hydroxypopionaldehyde has been widely used as an important intermediate for chemicals, effective antimicrobial agent, and fix agent for tissues. A pair of primers was designed on the basis of the sequence of both NH2-terminus and the amino acid sequence of glycerol dehydratase reported by NCBI, and a fragment about 1.6 kb was obtained by PCR amplification using the total genome DNA of Lactobacillus reuteri as template, then the fragment was cloned to the pMD18-T vector and sequenced. Two specific primers were designed according to the obtained sequence, and a fragment with length of 1674 bp was amplified using PCR with these two specific primers. Consequently, the resulting products were digested with EcoR I and Hind III and ligated using T4 DNA ligase to the pET28b vector digested with the same enzymes. The recombinant plasmid, named pET28b-dhaB, was transformed into E. coli BL21. The positive clones were induced with IPTG and the expression products were further analyzed by SDS-PAGE, indicating that protein with a molecule weight of around 65 kD was obtained. Furthermore, the glycerol dehydratase activity was evaluated and compared with the wild type strain as well.
Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Glyceraldehyde ; analogs & derivatives ; chemistry ; metabolism ; Hydro-Lyases ; biosynthesis ; genetics ; Lactobacillus reuteri ; enzymology ; genetics ; Propane ; chemistry ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics