ObjectiveTo observe whether lipopolysaccharide (LPS) derived fromEscherichia coli (E.coli) can induce apoptosis of murine platelets in vitro.Methods Washed platelet suspension was prepared and adjusted to the final concentration of 3×108/mL. According to the difference in stimulants, samples were divided into control group (non-calcium Tyrode buffer), thrombin-treated group (1 U/mL final concentration and non-calcium TB) and LPS in different concentrations treated groups (1, 10 and 100μg/mL final concentration respectively and non-calcium TB). To each specimental group corresponding stimulus was added and incubated 30 minutes at room temperature. Chemiluminescence was adopted to determine the concentration of adenosine triphosphate (ATP) and the activity of cysteinyl aspartate specific proteinase-3 (caspase-3). The percentage of Annexin V positive platelets was determined by flow cytometry to reflect the level of phosphatidylserine (PS) exposure. Mean channel fluorescence (MCF) of platelets was determined by flow cytometry for reflecting the level of mitochondrial inner transmembrane potential (ΔΨm) depolarization.Results Compared with control group, the ATP concentration in thrombin-treated group was decreased obviously [relative light unit (RLU): (5.46±0.14)×105 vs. (6.25±0.26)×105,P< 0.05], Annexin V positive ratio [(50.43±2.45)% vs. (1.58±0.25)%,P< 0.05] and caspase-3 activity [RLU: (26.92±1.60)×103 vs. (1.30±0.10) ×103,P< 0.05] were increased obviously, and platelets MCF was lowered significantly [(8.32±0.58)×104 vs. (13.05±1.10)×104,P< 0.05], suggesting an increase inΔΨm depolarization. After being treated with different concentrations of LPS, ATP concentration, Annexin V positive ratio and caspase-3 activity were increased obviously, platelet MCF was decreased obviously, suggestingΔΨm depolarization was increased in a concentration-dependent manner. Compared with control group, 1μg/mL LPS could increase Annexin V positive ratio [(10.45±1.08)% vs. (1.58±0.25)%,P< 0.05], elevate caspase-3 activity [RLU: (14.06±0.61)×103 vs. (1.30±0.10)×103,P< 0.05], and decrease MCF significantly [(9.48±0.50)×104 vs. (13.05±1.10)×104,P< 0.05]. The ATP concentration, Annexin V positive ratio and caspase-3 activity reached maximum levels after the treatment with 100μg/mL LPS, and they were higher obviously than those of the control group [ATP (RLU): (7.00±0.03)×105 vs. (6.25±0.26)×105, Annexin V positive ratio: (55.35±2.42)% vs. (1.58±0.25)%, casepase-3 (RLU): (32.00±3.75)×103 vs. (1.30± 0.10)×103, allP< 0.05], and platelets MCF reached trough levels, and they were obviously lower than those of the control group [(4.69±0.55)×104 vs. (13.05±1.10)×104,P< 0.05].ConclusionE.coli LPS can induce an increase in ATP, PS exposure,ΔΨm depolarization and activity increase of caspase-3 on mouse platelet in vitro, which indicate that LPS can induce apoptosis of platelets in a concentration-dependent manner.

ObjectiveTo explore the relationship between thrombocytopenia (TCP) induced by lipopolysaccharide (LPS) and coagulation or inflammatory response in mouse.Methods Forty-eight C57BL/6 mice were divided into control group, low-dose, and high-dose LPS treatment groups by random number table method, and each group was subdivided into 4-hour and 24-hour subgroups randomly, with 8 mice in each subgroup. 0.5 mg/kg or 50 mg/kg LPS was injected intraperitoneally in low-dose or high-does group respectively, and equal amount of normal saline was injected in control group. Blood was collected from endocanthal vein at the specified time point, platelet count (PLT) was counted, and the levels of thrombin antithrombin complex (TAT), D-dimer, fibrinogen degradation product (FDP), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were determined by enzyme linked immunosorbent assay (ELISA).Results Compared with control group, PLT (×109/L) at 4 hours and 24 hours in low-dose and high-dose LPS groups was significantly decreased (4 hours: 660.65±180.48, 568.55±117.99 vs. 1 199.13±110.54; 24 hours:505.63±218.92, 256.33±72.86 vs. 1 229.13±1 189.37, allP< 0.05), and the changes were more obvious in high-dose LPS group compared with those of the low-dose LPS group (allP< 0.05). Factorial analysis showed that the changes in PLT were related with LPS dosage and time (F1 = 135.660,P1 = 0.000;F2 = 12.120,P2 = 0.001). It was also found that there was an interactive effect of the dose of LPS and time on PLT (F = 5.580,P = 0.007). Compared with control group, TAT, TNF-α, and IL-6 at 4 hours and 24 hours in low-dose and high-dose LPS groups were significantly decreased [TAT (ng/L) at 4 hours: 1.10±0.59, 0.22±0.13 vs. 3.47±1.73; 24 hours: 1.18±0.68, 0.39±0.29 vs. 3.19±1.27;TNF-α (nmol/L) at 4 hours: 87.35±12.29, 93.70±5.25 vs. 101.59±10.96, 24 hours: 81.94±8.26, 93.23±4.71 vs. 102.84±10.56; IL-6 (ng/L) at 4 hours: 81.78±7.82, 78.59±9.06 vs. 110.88±9.66, 24 hours: 76.03±9.85, 71.34±3.69 vs. 110.88±10.35, allP< 0.05]. TAT at 4 hours and 24 hours in high-dose LPS group was further decreased, and TNF-αat 24 hours was increased as compared with those of low-dose LPS group (allP< 0.05). TAT, TNF-α and IL-6 were influenced only by different dosage of LPS (TAT:F = 42.350,P = 0.000; TNF-α:F = 14.810,P = 0.000; IL-6:F =81.910,P = 0.000), not time (TAT:F = 0.002,P = 0.967; TNF-α:F = 0.342,P = 0.562; IL-6:F = 2.973,P = 0.092). Changes in TAT was not found to be related with the dose of LPS and its time of action, or levels of TNF-α and IL-6 (TAT:F = 0.236,P = 0.791; TNF-α:F = 0.572,P = 0.569; IL-6:F = 0.774,P = 0.468). The dosage of LPS and time of admission showed no influence on D-dimer (F1 = 2.448,P1 = 0.099;F2 = 0.024,P2 = 0.877). The effect of different doses of LPS and time of administration showed no influence on FDP (F1 = 0.106,P1 = 0.900;F2 = 0.013,P2 = 0.908), and no interactive effects were found (D- dimer:F = 0.002,P = 0.998; FDP:F = 0.582,P = 0.563).Conclusion LPS can induce TCP in mouse, but this effect may not related to the activation of coagulation system and excessive inflammatory response.

Objective To explore the relationship between the fibroblast growth factor 2 (FGFR2) gene polymorphism (rs 2981582 ,rs 1219648 ,rs 2420946) and the breast cancer risk in Tibetan population ,Qinghai province .Methods This is a case con‐trol study .Peripheral blood samples from 210 breast cancer patients and 230 healthy women in Qinghai area were collected .DNA samples were extracted from peripheral blood cells .FGFR2 gene polymorphism (rs 2981582 ,rs 1219648 ,rs 2420946) were typed by Taqman‐MGB probe based on PCR and DNA sequencing ,then analyzed its correlation with breast cancer in Tibetan population , Qinghai province .Results The genotype frequencies of rs 2981582 CC ,CT and TT were 40 .48% ,39 .05% and 20 .47% among the breast cancer patients while 36 .09% ,48 .69% and 15 .22% among the controls .The genotype frequencies of rs 1219648 GG ,AG and AA were 24 .76% ,26 .19 % and 49 .05% among the patients while 23 .91% ,47 .39% and 28 .70% among the controls .The genotype frequencies of rs 2420946 CC ,CT and TT were 29 .05% ,45 .24% and 25 .71% among the patients while 30 .87% , 51 .74% and 17 .39% among the controls .The genotype frequencies of all genetic loci had no significant difference between rs 2981582 and rs 2420946 (P>0 .05) .But the genotype frequencies of rs 1219648 AA have statistical sense (P< 0 .05) ,compared with GG ,the incidence of breast cancer was remarkably increased with AA [OR=1 .65 ,95% CI= (1 .01 ,2 .69)] .Conclusion This study shows that FGFR2 rs1219648 AA is related to breast cancer risk among Tibetan population .

Objective:To analyze the clinical and laboratory features of patients with Coronavirus Disease 2019(COVID-19), for providing reference for the diagnosis and treatment of the disease.Methods:The clinical data of 72 patients confirmed as COVID-19 in Qishan hospital during January 23, 2020 to February 13, 2020 were collected. The data included the general information, clinical manifestations, laboratory tests and CT findings of the lungs. According to the severity of the disease, the patients were divided into the non-severe group and the severe group, and the laboratory tests characteristics of the two groups were compared. SPSS 22.0 statistical software was used for data analysis.Results:In the 72 patients, 40 were males and 32 females, the average age was (46.0±14.8) years. The main clinical manifestations were fever (84.7%) and cough (77.8%). Among them, 45 cases (62.5%) had expectoration with a small amount of white sticky sputum. The main laboratory results showed that lymphocyte count decreased in 26 cases (36.1%), the absolute number of T lymphocytes decreased in 18 cases (25.0%), CD4 + T cell count decreased in 8 cases (11.1%), CD8 + T cell count decreased in 14 cases (19.4%), ESR increased in 42 cases (58.3%), and CRP increased in 31 cases (43.1%). Peripheral blood lymphocyte (L), albumin, CD4 + T cell, CD8 + T cell, T-lymphocyte in severe group (n=61) were lower than those in non-severe group (n=11), while C-reactive protein (CRP), serum amyloid A (SAA), erythrocyte sedimentation rate (ESR), fibrin (FIB), aspartate aminotransferase (AST), creatine kinase (CK), lactate dehydrogenase (LDH) in severe group were higher than those in non-severe group ( P<0.05). In 72 patients, the negative time of the twice nucleic acid detection from nasopharyngeal swabs and sputum specimens were (21.48±7.9) days and (27.79±7.4) days, there were significant differences between the two groups of specimens for two consecutive negative times ( t=-4.532, P=0.000). The main pulmonary imaging manifestations of COVID-19 patients: two lungs in 58 cases, patchy opacities in 51 cases and ground glass shadows in 61 cases. Conclusions:Most of the COVID-19 patients in Yantai and Weihai were common type. The abnormal T lymphocyte, subcohorte and other laboratory indicators may indicate that the patients are more serious.

Objective:To investigate the effect of myogenic differentiation protein 1(Myod1)on the proliferation inhibition and apoptosis of the SH-SY5Y cells induced by oxygen-glucose deprivation(OGD),and to elucidate its mechanism.Methods:Real-time quantitative fluorescence PCR(RT-qPCR)method was used to detect the mRNA levels of Myod1 and long non-coding RNA(lncRNA)small nucleolar RNA host gene 15(SNHG15)in peripheral blood of the subjects in normal group and the patients in ischemic cerebral infarction group as well as the normal cultured SH-SY5Y cells(control group)and the cells in OGD model(OGD group).After transfecting SH-SY5Y cells with si-Myod1,pcDNA3.0-Myod1,si-SNHG15,pcDNA3.0-SNHG15、si-NC,Vector,miR-NC,and miR-24-3p mimics,the cells were treated with OGD,and then the SH-SY5Y cells were divided into control group,OGD group,OGD+Vector group,OGD+Myod1 group,OGD+si-NC group,OGD+si-Myod1 group,OGD+si-SNHG15 group,OGD+si-SNHG15+Vector group,OGD+si-SNHG15+Myod1 group,OGD+miR-NC group,OGD+miR-mimics group,OGD+miR-mimics+Vector group,and OGD+miR-mimics+SNHG15 group.CCK-8 method was used to detect the cell activities in various groups;5-ethynyl-2'-deoxyuridine(EdU)staining was used to detect the rates of EDU positive cells in various groups;the rates of TdT-mediated dUTP nick end labeling(TUNEL)positive cells in various groups were detected by TUNEL staining;Western blotting method was used to detect the expression levels of cleaved caspase-3,cleaved caspase-9,B-cell lymphoma 2(Bcl-2)and Bcl-2 associated X protein(Bax)proteins in the cells in various groups;the association between Myod1 and SNHG15 was evaluated by chromatin immunoprecipitate(CHIP);dual luciferase reporter gene experiment was used to evaluate the targeting relationships between Myod1 and SNHG15 as well as SNHG15 and miR-24-3p.Results:Compared with normal control group,the expression levels of Myod1 and SNHG15 mRNA in peripheral blood of the patients in ischemic cerebral infarction group were significantly increased(P＜0.05).Compared with control group,the expression levels of Myod1 and SNHG15 mRNA in the SH-SY5Y cells in OGD group were significantly increased(P＜0.05).Compared with OGD group,the cell activities and rates of EdU positive cells in OGD+Myod1 group at 48 and 72 h were decreased(P＜0.01),and the rates of TUNEL positive cells were increased(P＜0.05);the cell activities and rates of EdU positive cells in OGD+si-Myod1 group were increased(P＜0.05),while the rates of TUNEL positive cells were decreased(P＜0.01).Myod1 binded to the promoter sequence of SNHG15.SNHG15 could absorb miR-24-3p,and there were target relatronships between Myod1 and SNHG15 as well as SNHG15 and miR-24-3p.After SNHG15 knockdown,compared with OGD group,the cell activities and rates of EdU positive cells in OGD+si-SNHG15 group at 48 and 72 h were increased(P＜0.01),and the rates of TUNEL positive cells were decreased(P＜0.01),the expression levels of Bax,cleaved caspase-3 and cleaved caspase-9 proteins were decreased(P＜0.01),and the expression levels of Bcl-2 protein were increased(P＜0.01).Compared with OGD+si-SNHG15 group,the cell activities and rates of EdU positive cells in OGD+si-SNHG15+Myod1 group at 48 and 72 h were decreased(P＜0.05),the rates of TUNEL positive cells were(P＜0.05),the expression levels of Bax,cleaved caspase-3,and cleaved caspase-9 proteins were increased(P＜0.05),and the expression levels of Bcl-2 were decreased(P＜0.05).After over-expression of miR-24-3p and SNHG15,compared with OGD group,the cell activities and rates of EdU positive cells in OGD+miR-mimics group at 48 and 72 h were increased(P＜0.01),the rates of TUNEL positive cells were significantly decreased(P＜0.01),the protein expression levels of Bax,cleaved caspase-3 and cleaved caspase-9 were decreased(P＜0.05),and the expression levels of Bcl-2 were increased(P＜0.01).Compared with OGD+miR-mimics group,the cell activities and rates of EdU positive cells in OGD+miR-mimics+SNHG15 group at 48 and 72 h were decreased(P＜0.05),and the rates of TUNEL positive cells were increased(P＜0.05),the expression levels of Bax,cleaved caspase-3 and cleaved caspase-9 proteins were increased(P＜0.05),and the expression levels of Bcl-2 protein were decreased(P＜0.05).Conclusion:Myod1 can promote the proliferation inhibition and apoptosis of OGD-induced SH-SY5Y cells by binding to the SNHG15 promoter region and then absorbing miRNA-24.