Objective To set up a method to determine cytoplasmic free calcium [Ca~(2+)]_(i) in human platelets with flow cytometry.Methods As a calcium indicator,Fluo 3-AM was used to determine the changes,induced by thrombin,of the level of platelet plasma free calcium [Ca~(2+)]_(i) in presence or absence of outside calcium [Ca~(2+)]_(o),in order to elucidate where the platelet plasma free calcium [Ca~(2+)]_(i) mainly come from and it's role in the activity of platelets.Results The geometric mean of fluorescence intensity of the platelet,labeled with Fluo 3-AM,was increased obviously.The concentration of platelet plasma free calcium [Ca~(2+)]_(i) was increased significantly by thrombin in a dose-and [Ca~(2+)]_(o)-dependent manner.Conclusion The increased level of platelet plasma free calcium [Ca~(2+)]_(o),induced by thrombin,mainly comes from [Ca~(2+)]_(o) and partly from the released [Ca~(2+)]_o of intra-platelet probably.

Objective To analyze the effects of fetal anoxia, respiratory and metabolic acidosis on the activity of antioxidation in fetal distress Methods Blood samples were taken from umbilical artery in 386 neonates for blood gas analysis and detection of the concentration of superoxide dismutase (SOD) Normal situation, anoxia, acidosis, respiratory acidosis, metabolic acidosis and mixed acidosis were diagnosed in all neonates according to the results of blood gas values, and the neonate asphyxia was diagnosed according to the Apgar scores (one minute) The effect of anoxia and acidosis to SOD were analyzed with multiple factor analysis of variation Results (1) Among the all 386 cases, 317 were normal, 31 with anoxia, 17 with acidosis, and 21 with both anoxia and acidosis Among the total cases of acidosis, 8 respiratory, 21 metabolic, and 9 mixed acidosis (2) The plasma levels of SOD of umbilical artery blood in anoxia, acidosis, both anoxia and acidosis, and normal sitution were (118 5?7 1) mmol/L, (122 0?11 4) mmol/L,(140 0?7 0)mmol/L, and (98 5? 2 6) mmol/L,respectively The results of unvariate analysis of variance showed that anoxia: F =4 999 ( P 0 05) (3) The plasma levels of SOD with respiratory acidosis, metabolic acidosis and mixed acidosis were (127 3?18 4) mmol/L, (126 0?8 1) mmol/L, (150 0?10 4) mmol/L The results of univariate analysis of variance showed that respiratory acidosis: F =4 404 ( P 0 05) Conclusion The superoxidation and antioxidation can be effected by factors like anoxia and acidosis, respiratory acidosis and metabolic acidosis However, the mechanisms of these effects are different. There is additive, but not synergistic effects among them

The paper illustrates the role of humanistic multi-element administrate model in the clinical laboratory operation on various aspects such as the construction of the institution,the education of professional personnel and the enhancing of the personnel's enthusiasm.

Objective To detect the significance of the SHP-1,p15 and SOCS-1 genes methylation status in the genesis and development of diffuse large B-cell lymphoma (DLBCL).Methods The methylation state of SHP-1,p15 and SOCS-1 genes CpG islands were measured by methylation-specific polymerase chain (MSP) reaction.Results The positive rates of aberrant methylation for SHP-1,p15 and SOCS-1 genes in 50 specimens of DLBCL were 96 ％ (48/50),52 ％ (26/50) and 56 ％ (28/50) respectively.In control group,however,15 specimens of benign lymph node proliferation showed no methylation in CpG island of SHP-1,p15 and SOCS-1 genes promoter.Conclusion Aberrant methylations of the SHP-1,p15 and SOCS-1 genes exist in the patients with DLBCL.The methylations of SHP-1,p15 and SOCS-1 genes may be associated with the occurrence and development of DLBCL.This study provides a theoretical basis of treatment methylation for DLBCL.

Objective To explore the significance of the suppressor of cytokine signaling-1 (SOCS-1)gene methylation in the genesis, development and prognosis of diffuse large B-cell lymphoma (DLBCL).Methods The methylation state of CpG island in SOCS-1 gene were detected by methylation-specific polymerase chain reaction (PCR) and the level of SOCS-1 gene was measured by the real-time PCR. The clinical data of 30 patients with DLBCL were collected, and according to the international prognosis index (IPI), they were divided into low risk group and high risk group. Results Aberrant methylation of SOCS-1 in 17 DLBCL patients (56.7 %) were positive, however, in control group aberrant methylation was negative(P ＜0.01).The methylation level of DLBCL patients with positive SOCS-1 methylation was higher than that of patients with negative (P ＜0.05). Combined with the clinical data, the positive rate of methylation in patients with high level of serum LDH or the numbers of extra-nodal lesions＞l were significantly higher than that in patients with normal LDH level or the numbers of extra-nodal lesions ≤ 1, respectively. Hence, the positive rate of methylation in the high risk group of DLBCL was higher than that in the low risk group (P ＜0.05). Conclusion There were aberrant methylations of the SOCS-1 gene in the patients with DLBCL. The methylations of SOCS-1 can silence the gene expression, which indicates that SOCS-1 and its methylations play some role on genesis and development of DLBCL and can evaluate the prognosis of the patients with DLBCL.

Objective To investigate the suppressive effect of exogenous carbon monoxide (CO) on abnormal platelet exocytosis and its possible molecular mechanism. Methods Venous blood was collected from healthy volunteers. Platelet-rich plasma (PRP) was isolated from the blood by differential centrifugation. The PRP was randomly divided into five groups by random number table, namely normal control group, lipopolysaccharide (LPS) group (challenged with 10 mg/L LPS), inactively exogenous carbon monoxide releasing molecule 2 (iCORM-2) group (given 10 mg/L LPS + 50 μmol/L iCORM-2 for intervention), exogenous carbon monoxide releasing molecule 2 (CORM-2) 10 μmol/L and 50 μmol/L groups (given 10 mg/L LPS + CORM-2 10 μmol/L or 50 μmol/L for intervention). After 30 minutes, enzyme linked immunosorbent assay (ELISA) was used to determine the platelet-derived growth factor BB (PDGF-BB) and matrix metalloproteinase 2 (MMP-2). Chemical fluorescein method was used to determine the platelet adenosine triphosphate (ATP). Flow cytometer was used to determine the expression of P-selectin. The expressions of Toll-like receptor 4 (TLR4), phosphorylation of protein kinase Cθ (PKCθ) and syntaxin binding protein 1 (STXBP-1) were determined by Western Bolt. The soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (SNAREs) complex formation [syntaxin 2-synaptosomal-associated protein 23-vesicle associated membrane protein 8 (STX2-SNAP23-VAMP8)] mediated by STXBP-1 was determined by immunoprecipitation. Results ① Compared with normal control group, the platelet release of PDGF-BB, MMP-2 and ATP was significantly increased after LPS challenge, and the P-selectin expression of platelet was also obviously up-regulated [PDGF-BB (μg/L): 127.53±1.78 vs. 94.35±5.84, MMP-2 (ng/L): 51.87±9.20 vs. 35.83±3.17, ATP (μmol/L): 1.288±0.056 vs. 0.975±0.010, P-selectin: (3.93±0.19)% vs. (0.44±0.10)%, all P < 0.05]. The increases in platelet release of PDGF-BB, MMP-2 and ATP were suppressed by 10 μmol/L or 50 μmol/L CORM-2 administration, as well as high-expression of P-selectin in a dose-dependent manner [PDGF-BB (μg/L): 114.68±1.35, 97.08±6.14 vs. 127.53±1.78, MMP-2 (ng/L): 32.67±8.00, 24.63±1.63 vs. 51.87±9.20, ATP (μmol/L): 0.999±0.015, 0.965±0.008 vs. 1.288±0.056, P-selectin: (1.95±0.27)%, (0.94±0.11)% vs. (3.93±0.19)%, all P < 0.05]. ② Compared with normal control group, LPS challenge resulted in a significant increase in the expression of TLR4 and the phosphorylation of PKCθ and STXBP-1 [TLR4 (gray value): 1.21±0.38 vs. 0.67±0.06, p-PKCθ (gray value): 1.36±0.20 vs. 0.44±0.03, p-STXBP-1 (gray value): 1.13±0.06 vs. 0.59±0.04, all P < 0.05]. The increases in above parameters were suppressed by 10 μmol/L or 50 μmol/L CORM-2 administration in a dose-dependent manner [TLR4 (gray value): 0.76±0.05, 0.65±0.04 vs. 1.21±0.38; p-PKCθ (gray value): 0.71±0.07, 0.47±0.10 vs. 1.36±0.20; p-STXBP-1 (gray value): 0.56±0.02, 0.48±0.01 vs. 1.13±0.06, all P < 0.05]. ③ Compared with normal control group, the SNAREs proteins in platelet that combined with STXBP-1, including STX2, SNAP23 and VAMP8, were obviously increased after LPS challenge [STX2 (gray value): 1.35±0.06 vs. 0.57±0.04, SNAP23 (gray value): 0.97±0.04 vs. 0.30±0.12, VAMP8 (gray value): 1.37±0.12 vs. 0.77±0.10, all P < 0.05]. The increases in SNAREs complex formation were suppressed by 10 μmol/L or 50 μmol/L CORM-2 administration in a dose-dependent manner [STX2 (gray value): 0.77±0.02, 0.39±0.03 vs. 1.35±0.06, SNAP23 (gray value): 0.41±0.03, 0.22±0.08 vs. 0.97±0.04, VAMP8 (gray value): 0.85±0.07, 0.66±0.07 vs. 1.37±0.12, all P < 0.05]. There was no significant difference in the above mentioned parameters between iCORM-2 group and LPS group. Conclusions LPS-induced abnormal secretion of platelet was suppressed by CORM-2 administration. The mechanism may involve the TLR4/PKCθ/STXBP-1 signaling pathway activation and the SNAREs complex formation.

OBJECTIVETo explore the effects of exogenous carbon monoxide-releasing molecule 2 (CORM-2) on formation of human neutrophil extracellular traps (NETs) stimulated by endotoxin/lipopolysaccharide (LPS) and its relevant mechanism.

METHODSVenous blood samples were collected from a healthy adult volunteer to isolate neutrophils. The neutrophils were divided into normal control (NC) group, LPS group, LPS+ 10 μmol/L CORM-2 group, LPS+ 50 μmol/L CORM-2 group, and LPS+ inactive CORM-2 (iCORM-2) group according to the random number table. No treatment was given to the neutrophils in NC group. The neutrophils in LPS group underwent LPS stimulation (1 μL, 1 μg/mL). The neutrophils in LPS+ 10 μmol/L CORM-2 group, LPS+ 50 μmol/L CORM-2 group, and LPS+ iCORM-2 group underwent the same LPS stimulation as that in LPS group and treatment of 10 μmol/L CORM-2, 50 μmol/L CORM-2, and 50 μmol/L iCORM-2, respectively, with the volune of 1 μL. After conventional culture for 1 h, the number of NETs was determined with propidium iodide staining method; the early cell apoptosis rate was determined with flow cytometer; the generation level of reactive oxygen species (ROS) was assessed with dihydrogenrhodamine 123 fluorescent probe staining method (denoted as mean fluorescence intensity); the expression level of phosphorylated extracellular regulated kinase 1/2 (p-ERK1/2) was determined by Western blotting. The sample numbers of each group in the 4 experiments were all 5. Data were processed with one-way analysis of variance and SNK test.

RESULTS(1) The numbers of NETs per 400-time visual field in cells of LPS and LPS+ iCORM-2 groups were close to the number in NC group (with P values above 0.05). The number of NETs per 400-time visual field was significantly larger in cells of LPS+ 10 μmol/L CORM-2 and LPS+ 50 μmol/L CORM-2 groups than in NC and LPS groups (with P values below 0.05). The number of NETs per 400-time visual field in cells of LPS+ iCORM-2 group was close to that of LPS group (P>0.05). (2) The early cell apoptosis rate was significantly increased in LPS, LPS+ 10 μmol/L CORM-2, LPS+ 50 μmol/L CORM-2, and LPS+ iCORM-2 groups than in NC group (with P values below 0.05). The early cell apoptosis rates in LPS+ 10 μmol/L CORM-2, LPS+ 50 μmol/L CORM-2, and LPS+ iCORM-2 groups were close to the rate in LPS group (with P values above 0.05). (3) The generation level of ROS was significantly higher in cells of LPS, LPS+ 10 μmol/L CORM-2, and LPS+ iCORM-2 groups than in NC group (with P values below 0.05). The generation level of ROS in cells of LPS+ 50 μmol/L CORM-2 group was close to that of NC group (P>0.05). The generation level of ROS was lower in cells of LPS+ 10 μmol/L CORM-2 and LPS+ 50 μmol/L CORM-2 groups than in LPS group (with P values below 0.05), while the generation level of ROS in cells of LPS+ iCORM-2 group was close to that of LPS group (P>0.05). (4) The expression levels of p-ERK1/2 in cells of LPS and LPS+ iCORM-2 groups (respectively 0.0311±0.001 and 0.0309±0.0018) were close to the level in NC group (0.0304±0.0046, with P values above 0.05). The expression level of p-ERK1/2 was significantly higher in cells of LPS+ 10 μmol/L CORM-2 and LPS+ 50 μmol/L CORM-2 groups (respectively 0.7891±0.0201 and 1.2970±0.0056) than in NC group (with P values below 0.05). The expression level of p-ERK1/2 was significantly higher in cells of LPS+ 10 μmol/L CORM-2 and LPS+ 50 μmol/L CORM-2 groups than in LPS group (with P values below 0.05). The expression level of p-ERK1/2 in cells of LPS+ iCORM-2 group was close to that of LPS group (P>0.05).

CONCLUSIONSCORM-2 can obviously increase the production of NETs in LPS-induced neutrophils, and it might be attributable to the promotion of inhibition of ROS generation and phosphorylation of ERK1/2.

Apoptosis ; Carbon Monoxide ; metabolism ; Extracellular Traps ; Humans ; Lipopolysaccharides ; pharmacology ; Organometallic Compounds ; pharmacology ; Phosphorylation ; drug effects

OBJECTIVE: To analyze the action of miRNA-326 on its target gene BCL-XL and the molecular mechanism of platelet apoptosis regulated by miRNAs. METHODS: Dual-luciferase vectors containing respectively the wild-type and mutant 3'-untranslated region (3'UTR) fragments of the BCL-XL gene were constructed with firefly and renilla luciferases and transfected into 293T cells. Relative fluorescence intensities of the transfected cells were measured. RESULTS: Dual-luciferase reporter gene vectors for PsiCHECK- BCL-XL -3'UTR-WT (wild-type) and PsiCHECK- BCL-XL -3' UTR-MT (variant) were respectively constructed. Relative fluorescence intensities of the 293T cells co-transfected by miRNA-326 and PsiCHECK- BCL-XL -3'UTR-WT plasmid were significantly lower compared with the control group (co-transfected by a miRNA-326 negative sequence and PsiCHECK- BCL-XL -3' UTR-WT plasmid) ( P = 0.034). The relative fluorescence intensity was also significantly reduced in cells co-transfected by miRNA-326 and PsiCHECK- BCL-XL -3' UTR-WT plasmid compared with the mutant control group co-transfected by miRNA-326 and PsiCHECK- BCL-XL -3'UTR-MT plasmid (P = 0.022). CONCLUSION miRNA-326 may participate in the regulation of platelet apoptosis by acting on the 3'-UTR of the BCL-XL gene.

Objective: To explore the improvement effect of hypnotherapy on dysmenorrhea symptoms of female college students with primary dysmenorrhea, in order to provide reference for the intervention and treatment of female college students with primary dysmenorrhea. Methods: From Septerber to December 2021, 90 female college students diagnosed with primary dysmenorrhea in Qinzhou First People s Hospital were randomly divided into hypnotic suggestion group( n =30), hypnotic relaxation group ( n =30) and control group( n =30). The 10 session hypnotic suggestion and 10 session hypnotic relaxation interventions were carried out while the control group received no intervention. Participants in the three groups were assessed by using the Visual Analogue Scale(VAS), Beck Depression Inventory Ⅱ(BDI Ⅱ), State Anxiety Inventory(SAI), Questionnaire of Quality of Life of College Students(QOLCS), Cox Dysmenorrhea Symptom Scale (CMSS) before and after intervention. Results: After intervention, the VAS, BDI Ⅱ and SAI scores of the hypnotic suggestion group and the hypnotic relaxation group significantly decreased compared to those before the intervention( t =7.04, 13.32, 3.58, 2.15, 2.52, 2.01, P <0.05). There were statistically significant differences in VAS, BDI Ⅱ and SAI scores among the three groups( F =24.71, 29.57, 6.60, P <0.01). After intervention, the QOLCS total score, physical, psychological and behavioral dimension scores in the hypnotic suggestion group and the hypnotic relaxation group significantly improved( t =-4.61, -3.36, -3.12, -2.81, -2.71, -2.19, -2.69, -2.28, P <0.05). There were statistically significant differences in QOLCS total score, physical, psychological, behavioral, environmental, and social support dimension scores among the three groups( F =10.36, 4.14, 5.14，4.81, 7.07, 5.53, P <0.05). After the intervention, the CMSS dysmenorrhea severity and dysmenorrhea duration scores in the hypnotic suggestion group and the hypnotic relaxation group were significantly lower than those before the intervention( t =5.66, 4.70, 3.09, 2.21, P <0.05). There were significant differences in CMSS dysmenorrhea severity and dysmenorrhea duration scores among the three groups( F=15.33, 12.33, P <0.05). Conclusion Hypnotherapy can help relieve pain of female college students with primary dysmenorrhea，improve depression and anxiety.

OBJECTIVETo investigate the levels and influencing factors of phthalate internal exposure in pregnant women (gestation age ≤ 16 weeks).

METHODSDuring April to June in 2013, 1 020 pregnant women (gestation age ≤ 16 weeks) who had established the maternal care manual were recruited in maternal and child health hospital of Siming District, Xiamen city. Participators were asked to complete a questionnaire to obtain information on socio-demographic characteristics, lifestyle behaviors, and antenatal examination and to provide a urine sample. Finally, 998 pregnant women who provided a urine sample and completed the questionnaire were enrolled. Adopting systematic sampling method, 100 ones were selected randomly among 998 pregnant women. High performance liquid chromatography-electrospray ionization-tandern mass was used to determine the concentration of five phthalate monoesters in each urine, including mono-n-methyl phthalate (MMP), mono-ethyl phthalate (MEP), mono-butyl phthalate (MBP), mono-benzyl phthalate (MBzP), mono-ethylhexyl phthalate (MEHP). Based on the measurements and questionnaire data, multivariate logistic regression was used to analyze the association between the phthalate monoester levels and potential influential factors.

RESULTSThe detection rates of MMP, MEP, MBP, MBzP and MEHP in 100 pregnant urine samples were 94%, 93%, 87%, 83%, 99%, respectively. And the urinary median uncorrected concentrations of MMP, MEP, MBP, MBzP and MEHP in 100 urine samples were 20.56, 17.62, 10.15, 2.03, and 5.12 ng/ml, respectively. Specific gravity-corrected concentration were 20.81, 20.36, 12.88, 2.58, 5.00 ng/ml, respectively. The results of multivariate logistic regression analysis indicated that: education degree was negatively associated with urinary concentration of MMP, MEP, MBP, MBzP and MEHP, OR (95% CI) were 0.495 (0.253-0.966), 0.380 (0.191-0.755), 0.379 (0.186-0.774), 0.401 (0.196-0.819), 0.373(0.183-0.762), respectively. Participants who had hair permed and dyed during pregnancy had higher urinary level of MBP and MBzP, OR (95% CI) were 12.867 (1.240-133.525), 15.982 (1.367-186.911), respectively; Participants who use cosmetics during pregnancy had higher urinary level of MEP and MBP, OR (95% CI) were 2.977 (1.012-8.757), 4.440 (1.485-13.272), respectively; plastic bottled water consumption was positively associated with urinary concentrations of MEP and MEHP, OR (95% CI) were 3.780 (1.417-10.083), 2.699 (1.039-7.010), respectively; annual household income was negatively associated with urinary concentration of MMP, OR (95% CI) was 0.597 (0.372-0.959); individuals who took medications during pregnancy had higher urinary level of MEHP than non-takers, OR (95% CI) was 4.853 (1.084-21.732).

CONCLUSIONPregnant women whose gestation age was less than 16 weeks are generally exposed to phthalate. Phthalate internal exposure levels are significantly associated with most measured factors and the influencing factors with different phthalates internal exposure levels are different.

Chromatography, High Pressure Liquid ; Dibutyl Phthalate ; urine ; Female ; Humans ; Life Style ; Maternal Exposure ; Phthalic Acids ; urine ; Pregnancy ; Surveys and Questionnaires ; Tandem Mass Spectrometry