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Objective To investigate the role and mechanism of macrophage activation induced by exosomes from high glucose-treated renal tubular epithelial cells.Methods (1) The supernatant of renal tubular epithelial cells which were cultured in normal glucose control group (5.5 mmol/L D-glucose) or high glucose group (30.0 mmol/L D-glucose) for 48 h were collected and ultracentrifuged to harvest exosomes.Exosomes were identified by transmission electron microscope and Western blotting.(2) Exosomes were labeled with the green lipophilic fluorescent dye PKH67 and cultured with THP-1 macrophage to investigate whether HK2-derived exosomes could be internalized by THP-1 macrophage.Observing the morphology microscopically and detecting the chemotaxis function of THP-1 macrophages in Transwell chamber after co-cultured with exosomes.The expression of inducible nitric oxide synthase (iNOS),tumor necrosis factor-α (TNF-α),Interleukin-1β (IL-1β),monocyte chemoattractant protein-1 (MCP-1) in cells and supernatants were separately detected by quantitative Real-time PCR (qRT-PCR) and enzyme linked immunosorbent assay (ELISA),and the expression of p-c-Jun NH2-terminal kinase (p-JNK),mitogen-activated protein kinase p-p38 (p-p38MAPK) and nuclear factor κB p65 (NF-κB p65) in THP-1 macrophages were detected by Western blot.Results (1) Vesicles that harvested by ultracentrifugation ranged in size from 30 nm to 100 nm and expressed exosomal marker CD63,TSG101 but absence of calnexin which is a marker of endoplasmic reticulum,suggesting that the exosomes were not contaminated with cells.(2) Results from laser scanning confocal microscope showed that each group of exosomes can be internalized by THP-1 macrophages.Compared with normal glucose exosomes group,high glucose exosomes had increased the expression of iNOS,TNF-α,IL-1β and MCP-1 in THP-1 macrophages (all P ＜ 0.01),moreover,p-JNK,p-p38 MAPK and NF-κB p65 proteins level also increased significantly (all P ＜ 0.01).Conclusions Exosomes from high glucose-treated HK2 cells can induce THP-1 macrophage activation and functional changes through MAPK/NF-κB pathway.
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Objective To evaluate clinical application value of polymerase chain reaction (PCR) detection for bacteria in peritoneal dialysis associated peritonitis (PDAP).Methods Peritoneal dialysis fluid specimens were collected from January 2014 to December 2014 in The First Affiliated Hospital of Anhui Medical University.Conventional bacterial culture and PCR detection were used respectively.According to the bacterial 16S rRNA gene, universal primers were devised and designed, based on reference, the specific primers of 17 kinds of experimental bacteria.Real-time fluorescent PCR (Real-time PCR, qPCR) amplification was implemented.The establishment of standard strain DNA extract was used as positive control;sterile double distilled water was used as negative control.Results (1) The traditional bacterial culture results showed that positive proportion was 26/40 in specimen of 40 cases, gram-positive strains accounting for 18/26.Main species were epidermis staphylococcus (5/26), hemolysis staphylococcus (4/26), escherichia coli (4/26), and streptococcus viridans (3/26).(2) The PCR detection results showed that total positive rate was 33/40 in 40 patients specimens, among which 2 cases of positive samples ended up with no specific strains being detected;the main bacteria strains in PCR were not different from ordinary culture results.(3) With bacterial culture as the gold standard, the detection sensitivity of PCR technology for PDAP pathogenic bacteria was 96.15％ and specificity was 42.86％;the detection positive rate was significantly higher than ordinary culture method.(4) PCR technology for detecting pathogenic bacteria could produce results within 4-6 hours, while reported positive results in the traditional bacterial culture would take (77.88±15.53) hours, which was significantly longer than PCR.Conclusion Compared with traditional bacteria culture method, PCR method is more sensitive, simple, and quick.Bacteria detection using PCR technique is of clinic applied value in PDRP.
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Objective To evaluate the effectiveness of thoracic electrical bioimpedance(TEB)in monitoring the cardiac function of peritoneal dialysis patients.Methods One hundred and one patients with continuous ambulatory peritoneal dialysis (CAPD) and 30 healthy persons (control group)were included in the study.Thoracic electrical bioimpedance (TEB) noninvasive hcmodynamic monitoring and echocardiography were taken to analyze the correlation between indexes.Results Echocardiography showed that left atrial diameter (LAD),left ventricular end diastolic diameter (LVDd),left ventricular end systolic diameter (LVDs),interventricular septal thickness （IVST),interventricular septal thickness (PAP),left ventricle weight index (LVMI) of CAPD group were higher than that of the control group (all P ＜ 0.05),early and late wave of mitral valve flow (E/A) of CAPD group was lower than that of control group (P ＜ 0.05).TEB monitoring showed that cardiac output (CO),stroke volume (SV),acceleration index (ACI),ejection fraction (EF),velocity index (Ⅵ) of CAPD group were significantly lower than that of control group (all P ＜ 0.01),systolic time ratio (STR),SVR,TFC of CAPD group were significantly higher than that of control group (P ＜ 0.01).Correlation analysis show that left ventricular ejection fraction (LVEF) was negatively correlated with BNP (r =-0.467,P ＜ 0.01),LVMI was positively correlated with BNP (r=0.416,P ＜ 0.01),PEP,STR and TFC were positively correlated with BNP (r =0.404,P ＜ 0.01; r =0.572,P ＜ 0.01; r=0.471,P ＜ 0.01),EF was negatively correlated with BNP (r =-0.664,P ＜ 0.01).Correlation analysis between echocardiogaphy and TEB monitoring index showed there was significant correlation between EF and LVEF (r =0.451,P ＜ 0.01),SVR and TFC were positively correlated with LVMI (r =0.232,P ＜ 0.05; r =0.284,P ＜ 0.05),SV was positively correlated with E/A (r =0.285,P ＜ 0.05),pre-ejection period (PEP) and STR were negatively correlated with LVEF (r =-0.389,P ＜ 0.01; r =-0.446,P ＜ 0.01),TFC was positively correlated with LAD (r=0.279,P ＜ 0.05).Conclusion TEB monitoring can accurately evaluate the cardiac function with the advantage of dynamic monitoring and simple operation.It can partly replace the echocardiography test.