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Human enterovirus 71 (EV71) is one of the major causative agent of hand,foot and mouth disease (HFMD) in infant.Clinical studies find that EV71 infection can cause a variety of clinical manifestations,from mild HFMD to fatal neurogenic pulmonary edema and even death,but the reasons remain unclear.The capsid protein VP1 of EV71 plays a key role in the processes of viral recognizing,binding and entering into the targeted cells and viral particles assembling.VP1 variation is a major determinant to EV71 fitness and immunogenicity.This study reviews the research progress of the structure,functions and associated antiviral vaccines and drugs of VP1,which further provides a theoretical basis for developing new and more effective antiviral vaccine and drugs.
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Objective@#To investigate the effect of curcumin on the apoptosis and autophagy of human gastric cancer cells with different degree of differentiation.@*Methods@#Gastric cancer cell lines BGC-823 and MKN-28 were treated with curcumin at different concentrations. The effect of curcumin on cell proliferation was measured by MTT assay. Apoptosis was assessed by flow cytometry. Autophagy status was analyzed by acridine orange staining. The expression levels of apoptotic and autophagy-related proteins were detected by Western blot.@*Results@#The cell viability of BGC-823 and MKN-28 was inhibited by curcumin in a time- and dose-dependent manner. At 48 h after treatment, the IC50 value of BGC-823 (15.18 μmol/L) was close to that of MKN-28 (15.84 μmol/L), and the difference was not statistically significant (P=0.513). Meanwhile, flow cytometry showed that curcumin induced the apoptosis of gastric cancer cells in a dose-dependent manner. Western blot results showed that the expression of pro-apoptotic proteins bax, active-caspase-3 and active-caspase-9 was significantly increased in BGC-823 and MKN-28 cells, whereas that of the anti-apoptotic protein bcl-2 was strikingly reduced. In addition, the formation of acidic vesicular organelles in cytoplasm, conversion of LC3-Ⅰ to LC3-Ⅱ and increased levels of autophagy-related proteins Beclin1, Atg7 and Atg5-Atg12 were observed in curcumin-treated cells. Moreover, activation of PI3K/Akt/mTOR signaling pathway was also significantly suppressed after curcumin treatment. Blocking autophagy by adding the autophagy inhibitor 3-methyladenine (3-MA) significantly promoted the apoptotic cell death induced by curcumin.@*Conclusions@#Curcumin induces apoptosis and protective autophagy in human gastric cancer cells in vitro. Curcumin combined with autophagy inhibitor may provide a more effective strategy for its clinical application.
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Objective To investigate the diagnostic values of serum free fatty acids (FFA) and urine podocalyxin (PCX) in the patients with type 2 diabetic nephropathy(DN).Methods A total of 120 patients with type 2 diabetes mellitus (DM) were enrolled,and they were divided into three groups,normal albumin group(NA),microalbuminuria group(MA) and heavy albuminuria group(HA),based on the urinary albumin-creatinine ratio(UACR).In addition,40 healthy persons participated in medical examination were selected as the control group.Serum FFA levels were detected by a biochemical analyzer and urine PCX levels by ELISA.The correlation between them was analyzed by Pearson correlation,and their diagnostic values in type 2 DN were evaluated by the receiver operating characteristic curve (ROC).Results Serum FFA levels in the NA,MA,HA and control groups were (0.61 ± 0.14),(0.81 ± 0.13),(0.95 ± 0.18) and (0.49 ± 0.11) mmol/L,respectively,and urine PCX levels were (1.86 ± 0.45),(4.47 ± 1.48),(6.72 ± 1.40)and(1.38 ±0.24) ng/mL,respectively.The levels of serum FFA and urine PCX in type 2 DM patients were significantly higher than those in the control group(P ＜ 0.05),and increased with the progression of type 2 DM.Pearson correlation analysis showed that serum FFA levels were positively correlated with urine PCX levels(r =0.73,P ＜ 0.05).The sensitivity of serum FFA,urine PCX and combined detection in the diagnosis of type 2 DN were 74.1％,80.6％ and 89.5％,respectively,and the latter significantly higher than the former two (P ＜ 0.05).Conclusion Serum FFA and urine PCX levels increase significantly in diabetic patients with renal injury,and both of them have important clinical values in the diagnosis of type 2 DN.
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Objective To investigate the expression of helper T cells (Th)17/regulatory T cells (Treg) balance associated factors in rheumatoid arthritis (RA) patients and their correlation with serum midkine (MK).Methods A total of 60 RA patients were divided into active RA patients (n=32) and inactive RA patients group (n=28).MK level in sera was detected by enzyme linked immunosorbent assay (ELISA) in 60 patients with RA and 30 healthy controls (HCs).The fraction of CD4+CD25+FOXP3+ Treg cells and IL-17+CD4+ Th17 cells in RA patients and healthy controls were determined by flow cytometry (FCM), and the expreasion of Foxp3, RORγt, Signal transducer and activator of transcription (STAT) 3 and STAT5 mRNA were detected with real-time polymerase chain reaction (PCR).Results were evaluated using ANOVA followed by q tests for comparisons of Th17 population between active RA patients, inactive RA patients and HCs, t test was used for comparing of Foxp3, RORγt, STAT3, STAT5 mRNA between RA group and HCs.The correlations between serum MK concentration and peripheral Treg cells, Th17 cells, Foxp3, RORγt, STAT3, STAT5 mRNA were analyzed by Pearson's correlation analysis.Results The percentages of Treg cells from active RA patients, inactive RA patients and HCs were significantly different (F=129.6, P＜0.01), the percentages of Treg cells of active RA patients [(1.41±1.05)％] were lower than that of the inactive RA patients [(3.6±1.6)％;q =7.92, P＜0.05] and healthy group [(7.7±1.7)％;q=22.45, P＜0.05], and there was significant difference between healthy group and inactive RA group (q=14.53, P＜0.05).The percentages of Th17 cells of the three groups were also significantly different (F=36.3, P＜0.01),the percentage of Th17 cells of active RA patients [(1.84±1.01)％] was significantly higher than that of inactive RA patients [(0.71±0.28)％;q=9.59, P＜0.05] and healthy group (0.53±0.16)％ [(P＜0.05;q=1 1.10, P＜0.05], there was no significant difference between the inactive RA group and healthy group (q=1.51, P＞0.05).The expression of RORγt and STAT3 mRNA in RA patients was higher than that of healthy controls (t=5.84, P＜0.01;t=4.52, P＜0.01).The expression of Foxp3 and STAT5 mRNA in RA patients were lower than healthy controls (t=6.01, P＜0.01;t=2.18, P＜0.05).Serum MK values were correlated with STAT5 (r=-0.55, P＜0.01), but not with Foxp3, RORγt, STAT3 mRNA or the percentage of Treg/Th17 cells.Conclusion Serum MK expression and the percentage of Th17 cells increase, while the percentage of Treg cells decrease in RA patients.Serum MK values are negatively correlated with STAT5 mRNA which is associated with Th17/Treg balance.This may be important in the pathogenesis of RA.
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Objective To evaluate the detection of membrane neutrophilic alkaline phosphatase ( mNAP) by flow cytometry in diagnosis of bloodstream infection .Methods A total of 298 patients with suspected bloodstream infections admitted in the First People ’ s Hospital of Lianyungang during June 2013 and October 2014 were enrolled;80 healthy subjects in physical examination center were also enrolled as the control group.Bloodstream infection was diagnosed by blood culture and mNAP was detected by flow cytometry.Serum levels of procalcitonin (PCT) and C-reactive protein (CRP) were detected by electro-chemiluminescence (ECL) and immune scatter turbidimetry , respectively.The value of mNAP, PCT and CRP in diagnosing bloodstream infection was determined by receiver operating characteristic ( ROC) curve. Results Among 298 patients, 109 were confirmed with bloodstream infections , including 43 patients with Gram-positive bacterial infections and 66 with Gram-negative bacterial infections .The median levels of CRP , PCT and mNAP in bloodstream infection group were 138.71 mg/L, 7.04 ng/mL and 13 929 AB/c, which were significantly higher than those in healthy control group (1.50 mg/L, 0.12 ng/mL, 1 831 AB/c;U=5.00, 48.50 and 65.01, P<0.01).The expression of mNAP in Gram-positive bacterial infection group was 9 598 ( 6 064-11 643 ) AB/c, which was significantly lower than that in Gram-negative bacterial infection group [16 512 (11 654-22 001) AB/c] (U=250.00, P<0.01).ROC curve analysis showed that, the areas under the curve (AUCs) of mNAP, PCT and CRP in diagnosing bloodstream infection were 0.987, 0.962 and 0.901.When 4 578AB/c, 0.90 ng/mL and 13.50mg/L were taken as optimal cut-off values, the sensitivities of mNAP, PCT and CRP in diagnosis of bloodstream infection were 95.8%, 93.0%and 90.3%; the specificities were 97.8%, 95.6% and 85.5%, respectively.Conclusion Among mNAP, PCT and CRP, mNAP is of the highest value in diagnosing bloodstream infection , and may be used as a biomarker for clinical diagnosis of bloodstream infection .
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Objective To establish a flow cytometric method for the detection of alkaline phosphatase (ALP) expression on the membrane of neutrophils (mNAP).Methods EDTA-K2 anticoagulant venous bloods were collected.Expression of mNAP in peripheral blood was measured by flow cytometry using a phycoerythrin (PE)-labeled anti-ALP monoclonal antibody.BD QuantiBRITE PE was used to generate a calibration curve for PE fluorescence and ratios of PE to anti-ALP antibody and detect the bound ALP antibodies per cell (antibodies bound per cell,AB/c).Preanalytical handling including anticoagulants (EDTA-K2,citrate,and heparin),storage temperature,storage time,and plasma ALP were optimized and measured the precision.The expression levels of mNAP from 481 healthy controls were measured to establish a clinical reference range.The mNAP levels of 84 patients with severe infection and local infection,39 patients with virus infection were determined by this method.Results For preanalytical handling,application of PBS washing can effectively eliminate the interference of plasma ALP.The mNAP levels were not influenced by different anticoagulants and storage conditions (stored for 12 h either at room temperature or 4 ℃).This method had preferable reproducibility (CV in batch were 2.01％-3.33％,average 2.67％ ; CV between batch were 5.80％-6.00％,average 5.90％).The median (quartiles) of mNAP in health controls were 1 758 (1 378-2 310) AB / c for men and 1 897 (1 369-3 249) AB / c for women.There was no significant difference between genders (U =27 140,P =0.243 8).The clinical reference ranges (2.5 percentile to 97.5 percentile) of mALP was 920.5-3 493.0AB / c.The expression levels of mNAP of patients with bacterial infections (13 532,9 756-16 869 AB / c) were significantly higher than those of patients with virus infection(1 143,536-2 012 AB / c) and healthy controls (1879,1399-2497 AB / c) (H=221.5,P＜0.01).Conclusion BD QuantiBRITE PE kit can be used to standardize flow cytometer settings and quantitatively detect molecules per cell.The flow cytometric method for detection of mNAP has important clinical application for differentiating bacterial and virus infection.
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Objective To establish a method for detecting the concentration of paraquat (PQ) and creatinine(Cr) in urine simultaneously by capillary electrophoresis.Methods Experimental methodological study.8 acute PQ poisoned patients who were treated in the First People's Hospital of Lianyungang from January 2011 to June 2012 were collected.2 were male,and 6 were female.The separation were carried out using a 25 mmol/L pH1.97 glycine-HCl buffer(containing 40 mmol/L NaCl) in a fused-sillica capillary tube of 47 cm ×75μm I.D.by capillary zone electrophoresis.Urine had been injected by pressure for 4 s after samples were centrifuged and diluted for 10 times with H2O.The detection were monitored by a diode-array detector at 200 nm while samples were separated at a voltage of 20 kV.A systemic methodological evaluation of this method was carried out (The linear range,detection limit,repeatability test,recovery test and interference test).And the method was used to detect the concentration of PQ and Cr in PQ poisoned patients' urine.Results The peaks of PQ and Cr appeared within 5 min.The linear ranges of PQ and Cr were 2-1000,10-5000 μmol/L,respectively,with the correlation coefficients of 0.9997 and 0.9999 (P ＜0.01).The detection limits were 1.0 μmol/L for PQ and 5.0 μmol/L for Cr.The mean within-day(n =10) CVs of peak area for PQ and Cr were 2.84％ and 1.72％,while the mean inter-day(n =10) CVs of peak area were 3.62％ and 3.06％.The average recovery rate of PQ and Cr were 88.6％ and 90.2％ respectively.Diquat(DQ) didn't interfere with the assay.The range of PQ/Cr(μmol/mmol) for 8 cases was 8.9-2215.Conclusions A method was established successfully for the rapid determination of PQ and Cr in urine by capillary electrophoresis.The CE method devised here for direct measurement of urinary PQ and Cr from PQ poisoned patients is simple,fast,automatic and with good repeatability.It is an ideal method for rapid detection of urinary PQ in PQ poisoned patients.
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Objective To investigate the relation between the apotosis of B cells in the peripheral blood (PB) and the expression of interleukin (IL)-17 in patients with rheumatoid arthritis (RA).Methods The proportions of apoptosis of B cells in the PB of 80 patients with RA and 80 healthy controls were measured by flow cytometry.B cells in the PB of 20 RA and 20 healthy individuals were isolated by MACS and Western blotting was used to detect the Bcl-2 and Caspase-3 protein levels.IL-17 levels were detected by enzyme-linked immunosorbent assay (ELISA).T-test and linear regression were used to analyze the data.Results The proportions of apoptosis of B cells in the PB of patients with RA and healthy controls were (14±6)％ and (24±9)％ respectively.The rate of apoptosis of B cells in patients with RA was significantly less than healthy controls (t=2.737,P=0.021).The Bcl-2 protein level of B cells in the PB of patients with RA group was significantly higher than that of control group (26±10,12±6,P＜0.01).Conversely,the Caspase-3 protein level of B cells in the PB of patients with RA group was significantly lower than that of the control group (16±7,31±12,P＜0.01).ELISA detected elevated level of serum IL-17 in the patients with RA as compared with controls [(69±19),(27±10) pg/ml,t=4.631,P=0.014].There was a negative correlation between the level of IL-17 and apoptosis of B cells in patients with RA (r=0.36,P＜0.01).Conclusion The elevated bcl-2 and reduced caspase-3 of B cells in patients with RA further proves there is abnormal apoptosis of B cells in RA patients.There is negative correlation between the expression of IL-17 and apoptosis of B cells in patients with RA and IL-17 can inhibit B cell apoptosis.
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Objective To study the potential use of the urinary beta-trace protein ( βTP) for diagnosis of type 2 diabetic renal injury.Methods 174 patients with type 2 diabetic mellitus (T2DM) were classified into 2 groups according to the ratio of urinary albumin to creatinine (Alb/Cr):diabetes without renal injury group (group A) and diabetes with renal injury group (group B).70 healthy subjects served as normal control group ( group C).The level of urinary βTP and αl microglobulin (α1MG) was measured by latex particle enhanced immunoturbidimetry assay.The urinary Alb and Cr were determined by nephelometry and Jaffe method respectively.The level of uriuary βTP among all groups was compared and ROC curve analysis was performed.The relevant analysis on urinary βTP,urinary α1MG and other related indexes was made.Results The median level of urinary βTP/Cr in group B was 9.1mg/g Cr,significantly higher than 3.1mg/g Cr of group A and 2.0mg/g Cr of group C.The difference had statistical significance ( H =45.5,P ＜ 0.01).The other indexes ( Alb/Cr,α1MG/Cr,SCr) were all higher in group B than in the other 2 groups ( H =110.9,38.3,11.4 respectively,P ＜0.01).The relevant analysis showed that urinary βTP/Cr was positively correlated with urinary α1MG/Cr (r =0.894,P ＜ 0.01),SCr (r =0.367,P ＜ 0.05 ),HbA(J) C ( r =0.242,P ＜ 0.05 ),systolic pressure ( r =0.162,P ＜0.05 ),and the course of the disease ( r =0.251,P ＜ 0.05 ).No correlation was found between urinary βTP/Cr and diastolic pressure,fasting blood glucose(FBG) or BMI.ROC curve analysis showed the area under the curve (AUC) was 0.86 (95％CI,0.78-0.93)for urinary βTP/Cr and 0.76 (95％ CI,0.67-0.85) for urinary α1MG/Cr.The best cut-off value of urinary βTP/Cr and α1MG/Cr was 4.1mg/g Cr vs 10.9mg/g Cr,the sensitivity was 68.5％ vs 59.7％,and the specificity was 89.8％ vs 80.3％.The difference had statistical significance (P ＜ 0.05).Conclusions Urinary βTP has better diagnostic value for type 2 diabetic patients with renal injury than urinary α1MG.It can sensitively reflect renal tubular injury and can be used as a novel available biomarker to evaluate the renal tubular injury in clinic.
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Objective To investigate the clinical significance of apoptosis of B cells in the peripheral blood of patients with rheumatoid arthritis (RA).Methods The proportions of B cells in the peripheral blood (PB) of 51 active and 30 remission patients with RA and 80 healthy controls were detected by flow cytometry.B cells in the PB of 10 active,10 remission and 10 healthy individuals were isolated by MACS.The apoptosis of cultured B cells,which were collected at 24,48,72,96 h respectively,were assessed by flow cytometry.ANOVA,t test and,Spearman correlation analysis were used for statistical analysis.Results The proportions of B cells marked as CD19 and CD22 in the PB of active and remission patients with RA and healthy controls were (26±11)％,(12±8)％,(10±7)％,(26±10)％,(12±8)％,(11±5)％ respectively.The proportions of B cells in the PB of active patients was significantly higher than that of remission patients and healthy controls (P＜0.01).There was positive correlation between B cell proportion in the PB of active patients and DAS 28 and IgG level.The proportion of apoptosis of B cells in the PB with active patients was less than healthy controls.Conclusion The pathway of apoptosis of B cells in the PB of active patients is inhibited,which could increase B cell proportion.Moreover,the high proportion of B cells in the PB of active patients is closely related to disease activity.
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Objective Analysis of the expression of CD38,CD133 antigen and their clinical significance in myelodysplastic syndrome (MDS).Methods CD38 and CD133 antigen were analyzed by flow cytometry in 31 cases of MDS patients.Results CD38 was expressed in 18 cases (58.1% ),among them,12 cases were found to be myelodysplastic syndrome refractoryanermia ( MDS-RA ),accounting for 57.1%,6 cases were found to be MDS-RAEB,accounting for 66.7%.CD133 was expressed in 20 cases(64.5% ) ,among them,11 cases were found to be MDS-RA ( 52.4% ),1 case MDS-RAS,and 8 cases of MDS-RAEB,accounting for 88.9% .CD38 expressed significantly higher in MDS than anemia and relatively normal group ( P ＜ 0.05 ).CD133 expression in anemia groups was different from MDS-RA without statistical significance ( P ＞ 0.05 ),but was significantly different from relatively normal group (P ＜0.05).CD133 expression was significantly higher in these with MDS-RAEB than those in anemia and normal group ( P ＜ 0.05 ).Conclusions Combining with conventional antibodies,flow cytometry used in detection of CD38 ,CD133 ,could improve the diagnostic rate of MDS.
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Objective To investigate the clinical significance of flow cytometry (FCM) assay in following up of the minimal residual disease (MRD) used for predicting relapse and guiding chemotherapy. Methods The clinical data of 43 acute leukemia patients diagnosed by MIC were collected in our hospital from 2005 July to 2008 June.Bone marrow aspirates were collected from 43 patients with newly diagnosed acute leukemia after induction therapy and during constimulation therapy. The cells with leukemia associated with immunophenotype were investigated using FCM, as immunologic target of MRD. Results MRD were detected earlier in predicting the relapse than those of the traditional bone marrow cells morphology assay by an average of 4-6 months. The results of the MRD following up: MRD was negative at CR in 26 cases, 6 cases relapse, 20 cases of them were kept negative during following up. MRD was positive in 17 cases at CR, 9 cases of them were relapse. 4 cases after intensified chemotherapy the MRD became negative and kept egative for more than one year. The MRD of the 43 cases at CR were divided into 3 groups, MRD less than 1×10-4 group (A group) MRD between 5×10-3 and 1×10-4 group (B group) and MRD above 5×10-3 group(C group). By chi square test. There was no statistical significance between A group and B group, but there was tatistical significance between B group and C group (P=0.02). Conclusion The application of FCM in detecting MRD has important clinical significance in predicting relapse and guiding chemotherapy.
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Objective To establish a method for detecting urinary vanillylmandelic acid (VMA), homovanillic acid (HVA) and creatinine (Cr) simultaneously by high performance capillary electrophoresis (HPCE). Methods The separations were carried out using a 120 mmol/L phosphate buffer (pH 6.80) in a fused-silica capillary tube of 47 cm×75 μm I.D. by capillary zone electrophoresis (CZE). Injections were made by using the pressure mode for 4 s at 1 p. s. i. after samples were centrifuged and diluted. The detections were monitored by a diode-array detector (DAD) at 200 nm after samples were separated at a voltage of 20 kV. The method developed was validated systematically and applied to urine samples from healthy adults (n = 100) and children (n = 100) for establishing the reference ranges of VMA/Cr and HVA/Cr, respectively. Results Under these conditions, the separations of VMA, HVA and Cr could be completed within 13 min. The linearity ranges of VMA, HVA and Cr were 0-500, 0-500 and 0-4 000 μmol/L, respectively, with the correlation coefficients (r) between 0.997 2 and 0. 999 1 (P < 0.01). The detection limits (S/N= 3) were 1.0 μmol/L for VMA, 1.0 μmol/L for HVA and 50.0 μmol/L for Cr. The mean within-run (n = 10) CVs of migration time for VMA, HVA and Cr in urine were 0.58%, 0.56% and 0.25% respectively, while the mean between-run (n = 10) CVs of migration time were 0.95%, 1.00% and 0.48% respectively. The mean within-run (n = 10) CVs of peak area for VMA, HVA and Cr were 3.78%, 3.97% and 2.76% respectively, while the mean between-rim (n = 10) CVs of peak area were 4.60%, 4.08% and 4.42% respectively. The average recoveries were 98.36% for VMA, 93.56% for HVA and 98.85% for Cr. Other compounds in human urine such as catecholamines, 5-hydroxytryptamine and albumen didn't interfere with the assay. The correlation between CE method and HPLC method was good. And the correlation coefficients (r) of VMA and HVA were 0.954 9(P <0.01) and 0.945 1 (P < 0.01), respectively. Skewness distributions were presented for VMA/Cr and HVA/Cr in random urine from both adults and children, and the 95% reference ranges were established by the percentile method. For adults, the reference ranges of VMA/Cr and HVA/Cr were 0-4. 26 and 0-1.69 (μmol/mmol), respectively. For children, the reference ranges of VMA/Cr and HVA/Cr were 0-10.39 and 0-4.31 (μmol/mmol), respectively. Conclusions The CE method devised here for direct measurement of urinary VMA, HVA and Cr is simple, fast,precise and automatic with good repeatability. It is an ideal method for routine detection and mass screening of pheochromocytoma and neuroblastoms.
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Objective To investigate the expression of serum sHLA-G in systemic lupus erythematosus (SLE) patients and the association with the disease activity.Methods The serum concentration of sHLA-G in SLE patients and healthy controls was measured with enzyme-linked immunosorbent assay.Results Significant higher sHLA-G levels were detected in patients of SLE than control group (P＜0.01),The serum concentrations of sHLA-G in active SLE patients were markedly higher than stable SLE patients (P＜0.01).The expression level of sHLA-G showed positive correlations with SLE activity index (SLEDAI)(r=0.30,P=0.01).There was no correlation between sHLA-G levels and serum concentration of Anti-dsDNA,C3,C4 and Anti-ANA in SLE patients (P＞0.05).Conclusion The level of serum sHLA-G is significantly increased in SLE patients.Positive correlations are observed between sHLA-G levels and SLEDAI.These data indicated that sHLA-G may play certain roles in the pathogenesis and progress in SLE.
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Objective To investigate the relationship between levels of serum matrix metalloproteinases(MMPs)and brain edema and neurologic impairment in patients with intracerebral hemorrhage(ICH).Methods The levels of serum MMP-9 and MMP-2 in 31 patients with ICH were tested with ELISA for at 1 d,3 d,7 d and 2 weeks after onset.The volumes of hematoma and its peripheral edema were evaluated by CT,the neurologic impairment was evaluated by NIHSS at 1 d and 14 d after onset.Results Levels of serum MMP-9 and MMP-2 were significant higher in ICH group at each time point after onset than those in normal control group(allP