ABSTRACT
Objective To investigate the mechanisms underlying allergic conjunctivitis caused by conjunctival epithelial cell damage, neutrophil migration and neutrophil extracellular traps (NETs) formation induced by crude extracts of Dermatophagoides farinae mite (CDM). Methods Human conjunctival epithelial cells were stimulated with 500, 1 000, 2 000, 4 000 ng/mL, and the expression levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and IL-8 were detected using quantitative real-time PCR (qPCR) assay and enzyme-linked immunosorbent assay (ELISA). The culture supernatant of human conjunctival epithelial cells was collected and co-cultured with neutrophils. Neutrophil migration was measured using Transwell migration assay, and the expression of NETs markers myeloperoxidase (MPO) and citrullinated histone H3 (CitH3) was quantified using immunofluorescence staining. Neutrophils were stimulated with phorbol 12-myristate 13-acetate (PMA), and then NETs were collected for treatment of human conjunctival epithelial cells. Cell apoptosis was detected using flow cytometry, and the levels of IL-6, TNF-α, IFN-γ and IL-8 were measured in the cell culture supernatant using ELISA. Results Treatment with CDM at concentrations of 2 000 ng/mL and 4 000 ng/mL up-regulated IL-6, TNF-α, IFN-γ and IL-8 expression in human conjunctival epithelial cells. Following treatment with CDM at concentrations of 2 000 ng/mL and 4 000 ng/mL, the culture supernatant of human conjunctival epithelial cells promoted neutrophil migration and induced increases in the staining intensity of MPO and CitH3. In addition, increased NETs triggered the apoptosis of human conjunctival epithelial cells and IL-6, TNF-α, IFN-γ and IL-8 secretion in the culture supernatant of human conjunctival epithelial cells. Conclusions CDM induces human conjunctival epithelial cell damages, thereby promoting neutrophil migration and NETs formation, while the release of NETs further aggravates human conjunctival epithelial cell damages.
ABSTRACT
OBJECTIVE@#To investigate the mechanisms underlying allergic conjunctivitis caused by conjunctival epithelial cell damage, neutrophil migration and neutrophil extracellular traps (NETs) formation induced by crude extracts of Dermatophagoides farinae mite (CDM).@*METHODS@#Human conjunctival epithelial cells were stimulated with 500, 1 000, 2 000, 4 000 ng/mL, and the expression levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and IL-8 were detected using quantitative real-time PCR (qPCR) assay and enzyme-linked immunosorbent assay (ELISA). The culture supernatant of human conjunctival epithelial cells was collected and co-cultured with neutrophils. Neutrophil migration was measured using Transwell migration assay, and the expression of NETs markers myeloperoxidase (MPO) and citrullinated histone H3 (CitH3) was quantified using immunofluorescence staining. Neutrophils were stimulated with phorbol 12-myristate 13-acetate (PMA), and then NETs were collected for treatment of human conjunctival epithelial cells. Cell apoptosis was detected using flow cytometry, and the levels of IL-6, TNF-α, IFN-γ and IL-8 were measured in the cell culture supernatant using ELISA.@*RESULTS@#Treatment with CDM at concentrations of 2 000 ng/mL and 4 000 ng/mL up-regulated IL-6, TNF-α, IFN-γ and IL-8 expression in human conjunctival epithelial cells. Following treatment with CDM at concentrations of 2 000 ng/mL and 4 000 ng/mL, the culture supernatant of human conjunctival epithelial cells promoted neutrophil migration and induced increases in the staining intensity of MPO and CitH3. In addition, increased NETs triggered the apoptosis of human conjunctival epithelial cells and IL-6, TNF-α, IFN-γ and IL-8 secretion in the culture supernatant of human conjunctival epithelial cells.@*CONCLUSIONS@#CDM induces human conjunctival epithelial cell damages, thereby promoting neutrophil migration and NETs formation, while the release of NETs further aggravates human conjunctival epithelial cell damages.
Subject(s)
Animals , Humans , Extracellular Traps , Neutrophils , Interleukin-8/metabolism , Dermatophagoides farinae , Interleukin-6/metabolism , Tumor Necrosis Factor-alpha/metabolism , Epithelial Cells , Interferon-gamma/metabolism , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
Objective:To establish a quantitative detection method for the main components of dust mite allergens Der p 1, Der p 2 specific immunoglobulin E (sIgE) by using the nano-magnetic particle chemiluminescence immunoassay.Methods:The performance indexes of the established method were evaluated after setting up and optimizing the chemiluminescence detection system and immune reaction conditions of sIgE for dust mite allergen. Serum sIgE levels of 50 suspected allergic patients with dust mite were determined by this chemiluminescence method. At the same time, this method was compared with the Phadia kit and the consistency was analyzed by Kappa test. Results:The optimal amount of magnetic beads was 25 μg, the optimal reaction buffer (pH=7.4) contained 0.1 mol/L Tris-HCl and 0.25%( W/ W) casein, the optimal coating solution contatined 20 mmol/L phosphate buffer (PB) and 1%( W/ W) bovine serum albumin (BSA), and the luminescence enhancement solution contained 0.05%( V/ V) Triton X-100. The two-step immunoreaction was adopted, and the detection could be completed with 20 μl sample at the optimal reaction temperature of 37℃. The limit of detection (LOD) of the established nano-magnetic particle chemiluminescence system in detecting Der p 1 and Der p 2 sIgE antibodies were both less than 0.01 kU/L, with the linear range of 0.2-100.0 kU/L, the precision of less than 7%, and the cross contamination rate of 0.19% and 0.21%. Compared with the Phadia system, the positive and negative coincidence rate of Der p 1 were 78.0%(32/41) and 9/9 with good consistency ( Kappa=0.65, P=0.008), and the positive and negative coincidence rate of Der P 2 were 93.3%(28/30) and 85.0%(17/20) with good consistency ( Kappa=0.79, P=0.003). Conclusion:The nano-magnetic particle chemiluminescence immunoassay is successfully established for detecting dust mite allergen sIgE, which has good detection performance and good consistency with Phadia system.
ABSTRACT
Objective:To observe the effects of four prostaglandin E2 (PGE2) receptors (EP 1-4R) on the activation of inflammasomes and cell damage in human retinal microvascular endothelial cells (hRMEC) in a high glucose environment. Methods:The hRMEC were divided into normal group and high glucose group, and they were cultured in Dulbecco modified Eagle medium containing 5.5 and 30.0 mmol/L glucose, respectively. Flow cytometry was used to observe the apoptosis rate of the high glucose group and the normal group; enzyme chain immunosorbent assay (ELISA) was used to detect the level of PGE2 in the culture supernatant of hRMEC cells. Western blot was used to detect the protein expression of cyclooxyganese (COX2) and EP 1-4R in hRMEC. Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of EP 1-4R mRNA in hRMEC. After 72 h of culture, the cells in the high glucose group were divided into control group, PGE2 group, EP 1-4R agonist group, PGE2+EP 1-4R inhibitor group, and dimethylsulfoxide group. According to the group, each group was given the corresponding agonist or inhibitor to continue the culture for 24 h. QRT-PCR was used to detect the expression of nucleotide-binding oligomerization structure-like receptor protein (NLRP3) and pro-interleukin (IL)-1β mRNA in each group of cells. ELISA was used to detect the content of IL-1β and lactic dehydrogenase (LDH) in the cell culture supernatant. Western blot was used to detect the expression of cleaved Caspase-1 in each group of cells. At the same time, hRMEC in a high glucose environment was given IL-1β stimulation for 24 h, and the activity of LDH in the supernatant of the cell culture medium was detected. Results:The apoptotic rate, COX2 protein expression, and PGE2 protein content in hRMEC in the high glucose group were significantly higher than those in the normal group, and they were time-dependent. Compared with the normal group, the expression levels of EP 1R, EP 2R, EP 4R protein and mRNA in hRMEC in the high glucose group were higher than those in the normal group ( P<0.05). Compared with the control group, PGE2 group ( t=4.627, P<0.01), EP 1-4R agonist group ( t=3.889, 3.583, 2.445, 3.216; P<0.05) hRMEC NLRP3 mRNA expression level was significantly increased; the expression level of pro-IL-1β mRNA increased, however the difference was not statistically significant (PGE2 group: t=1.807, P>0.05; EP 1-4R agonist group: t=1.807, 1.477, 0.302, 1.926, P>0.05). Compared with the PGE2 group, the expression of NLRP3 mRNA in hRMEC in the PGE2+EP 2R inhibitor group was significantly reduced ( t=2.812, P<0.05); the expression of pro-IL-1β mRNA in hRMEC in the PGE2+EP 3R inhibitor group was significantly increased ( t=4.113, P<0.01). The protein content of IL-1β in the cell culture supernatant of the PGE2 group, EP 1R agonist group and EP 2R agonist group was significantly higher than that of the control group ( t=5.155, 4.136, 4.817; P<0.01). Compared with PGE2 group, the protein content of IL-1β in the cell culture supernatant of the PGE2+EP 2R inhibitor group and the PGE2+EP 4R inhibitor group were significantly lower than that of the PGE2 group ( t=1.964, 4.765; P<0.05). The expression of cleaved Caspase-1 in hRMEC in the PGE2 group and EP 2R agonist group was significantly higher than that in the control group ( t=5.332, 4.889; P<0.05). The expression of cleaved Caspase-1 in hRMEC in the PGE2+EP 2R inhibitor group was significantly lower than that of the PGE2 group ( t=6.699, P<0.01). The LDH activity in the cell culture supernatant of the PGE2 group and the EP 2R agonist group was significantly higher than that of the control group ( t=4.908, 4.225; P<0.05). The activity of LDH in the cell culture supernatant of the PGE2+EP 2R inhibitor group was significantly lower than that of the PGE2 group ( t=5.301, P<0.01). Compared with the control group, the LDH activity in the culture supernatant of hRMEC cells in the high glucose environment was significantly increased ( t=3.499, P<0.05). Conclusions:The four receptors of PGE2 can activate NLRP3 and its effector molecules to varying degrees. EP 2R mainly mediates hRMEC damage under high glucose environment.
ABSTRACT
Objective@#To establish a nanometer magnetic particle chemiluminescence methodfor the detection of specific IgE antibodies to Tyrophagus putrefaciens allergen.@*Methods@#According to the routine operation steps of chemiluminescence, chemiluminescence reaction system and suitable immune reaction conditions for detection of specific IgE of Tyrophagus putrescentiae allergen are established. Considering the performance of the chemiluminescence method established in this paper, the American Somerfly Phadia method wasused as the gold standard to evaluate the test performance of the established chemiluminescence method.@*Results@#In thechemiluminescence reaction system, Luminol concentration in luminescent substrate A solution is 0.4 mg/ml, Urea hydrogen peroxidein luminescent substrate B solutionis 0.2 mg/ml.The sensitivity of this luminescent reaction system to horseradish peroxidase (HRP) is very high, the minimum detectable HRP is 0.01 ng/ml.In the immune response, the room temperature light-shielding reaction was detected for 5 min after the addition of substrates A and B, and the detection values were effective within 5-30 min. 37 ℃ is optimum luminescence reaction temperature. Choose the range of 0.35-100 IU/ml as the standard curve. According to the results of 120 clinical trials, using Phadia (immunofluorescence method) as the gold standard, the chemiluminescence method established in this study has significant significance for the detection of Tyrophagus putrescentiae allergen.@*Conclusion@#A nanometer magnetic particle chemiluminescence method was successfully established for the detection of specific IgE antibodies to Tyrophagus putrescentiae allergen.
ABSTRACT
Objective To establish a nanometer magnetic particle chemiluminescence methodfor the detection of specific IgE antibodies to Tyrophagus putrefaciens allergen.Methods According to the routine operation steps of chemiluminescence, chemiluminescence reaction system and suitable immune reaction conditions for detection of specific IgE of Tyrophagus putrescentiae allergen are established. Considering the performance of the chemiluminescence method established in this paper, the American Somerfly Phadia method wasused as the gold standard to evaluate the test performance of the established chemiluminescence method. Results In thechemiluminescence reaction system, Luminol concentration in luminescent substrate A solution is 0.4 mg/ml, Urea hydrogen peroxidein luminescent substrate B solutionis 0.2 mg/ml.The sensitivity of this luminescent reaction system to horseradish peroxidase (HRP) is very high, the minimum detectable HRP is 0.01 ng/ml. In the immune response, the room temperature light-shielding reaction was detected for 5 min after the addition of substrates A and B, and the detection values were effective within 5-30 min. 37 ℃ is optimum luminescence reaction temperature. Choose the range of 0.35-100 IU/ml as the standard curve. According to the results of 120 clinical trials, using Phadia (immunofluorescence method) as the gold standard, the chemiluminescence method established in this study has significant significance for the detection of Tyrophagus putrescentiae allergen.Conclusion A nanometer magnetic particle chemiluminescence method was successfully established for the detection of specific IgE antibodies to Tyrophagus putrescentiae allergen.
ABSTRACT
Objective To study the effects of positive urine glucose on maternal pregnancy.Methods A total of 1 338 pregnant women were tested urine glucose.On the basis of their urine-glucose level,two groups were devided:experimental,68 cases of posi-tive urine-glucose gravidas,control group,199 cases of negative urine-glucose gravidas.Analyze on the outcome of pregnancy of the two groups.Results Compared between positive group and control group,the incidence of died(abnormal)fetus,gestational hyper-tension,abnormal amniotic fluid,fetal distress,and fetal macrosomia were not statistically different(P >0.05),but the incidence of premature rupture of membranes was statistically different (P <0.05 ).Conclusion Positive glucose urine of gravidas might in-crease the risk of premature rupture of membranes,positive urine glucose detected during pregnancy should be highly valued.
ABSTRACT
Objective To explore the status and risk factors of the dyslipidemia among health examination population of Lanzhou for providing the intervention measures .Methods According to the stratified cluster random sampling method ,4 505 health exami-nation individuals were recruited for the study from 5 hospitals in the Lanzhou region through questionnaire ,biochemical analysis . Results Prevalence of dyslipidemia of the population was 45 .79% ,high TG was the main type .The level of serum TC ,TG ,HDL-C and LDL-C were (5 .27 ± 1 .08) ,(1 .74 ± 1 .38) ,(1 .41 ± 0 .43) and (2 .83 ± 0 .82)mmol/L .The prevalence was 53 .49% in male , and 34 .93% in female .The prevalence was higher among the group of 35- <45 years old for male and 55- <65 years old for fe-male .The level of HDL-C was low among young people .There was aggregation of risk factors among the participants with dyslipi-demia .Non-conditional logistic regression analysis showed that the risk factors were age (OR=1 .701) ,overweight (OR=5 .560) , abdominal obesity(OR=2 .398) ,smoking(OR=0 .545) ,intake of greasy diet(OR=5 .313) ,sleep quality(OR=2 .005) and diastolic blood pressure(OR=3 .061) .Conclusion Lipid disorders becomes a serious problem in the health examination population ,measures such as rational diet ,weight control ,sleep improvement ,pressure control and quiting smoking must be taken .