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Objective To investigate the frequency of JAK2 V617F mutation and JAK2 V617F mutation allele burden in patients with essential thrombocythemia (ET), and explore the relationship between mutation and hematological parameters and coagulation function. Methods The clinical and laboratory parameters of 90 ET patients were analyzed. JAK2 V617F mutation was detected by AS-PCR and the mutation allele burden of JAK2 V617F was detected by qPCR. The correlation between mutation frequency and mutation burden of JAK2 V617F and blood laboratory parameters were investigated in ET. Results JAK2 V617F mutation was found in 50 patients (55.6 %). RBC [(4.67±0.89)×109/L vs (4.04±0.99)×109/L, P =0.003], WBC (11.64±5.20)×109/L vs (9.11±4.11)×109/L, P = 0.014], HCT (0.41±0.07) vs (0.36±0.07), P =0.005) in the JAK2 V617F mutated group were higher than those in the wild-type group. PT in mutated patients was longer than that in wild-type group [(13.18±1.63) s vs (12.02±1.24) s, P = 0.000]. The JAK2 V617F mutation allele burden was (29.91 ±18.63) %. No significant correlation was found between JAK2 V617F mutation allele burden and hematological parameters such as WBC, RBC and Plt (all P>0.05), but the JAK2 V617F mutation allele burden had a significant correlation with FDP (r = 0.456, P = 0.001). Conclusions JAK2 V617F mutation occurs in significant percentage patients with ET. Detection of JAK2 V617F mutation allele burden at diagnosis may play an important role in the early prevention of vascular events.
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Objective To explore clinical characteristics and diagnosis of aggressive natural killercell leukemia (ANKL),and evaluate the value of flow cytometry (FCM) in diagnosing it.Methods A case of ANKL was reported and literatures were reviewed.Results The patient presented with persistent high fever,progressive pancytopenia and hepatosplenomegaly.The untypical cells could be seen by morphology.By FCM,NK cells consisted 83.3 ％ of total lymphocytes in bone marrow and immunophenotypes were CD34-,CD2+,CD7+,CD3-,CyCD3+,CD5-,CD16+,CD56+,CD30-,CD4-,CD8-,CD117-,CD11c,CD19-,CD45++,SSC+-++.T-cell receptor (TCR) and IgH gene rearrangement were negative and chromosome was normal.The patient was diagonised ANKL eventually.Conclusions ANKL is a quite rare disease with highly aggressive,poor prognosis and could be misdiagnosed easily.FCM combined with morphology is a convenient,handy,practicable and less invasive device for the diagnosis,and can be a preferred detection technique in some cases.
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Objective To investigate the expression features and clinical significance of CD4+ CD25+ regulatory T cells in elderly patients with newly diagnosed acute myelocytic leukemia.Methods Totally 65 patients newly diagnosed as acute myeloid leukemia (AML group) and 72 healthy volunteers (control group) were divided into the elderly group (aged over 60 years) and the young group aged (44.6±2.9) years.The expression of CD4+ CD25+ regulatory T cells was detected by CD4,CD25 and CD127 three-color fluorescein-labeled and multiparameter flow cytometry.The expression features and clinical significance of CD4+ CD25+ regulatory T cells in each group were analyzed.Results After being gated on CD4+ lymphocytes,the expression level of CD4+ CD25+regulatory T cells was significantly increased in AML group as compared to control group [(7.06±2.60) ％ vs.(5.61 ± 1.06) ％,t =4.19,P=0.000].The expression level of CD4 + CD25 + regulatory T cells was higher in elderly AML patients than in elderly controls [(7.55 ± 2.78)％ vs.(5.98 ±1.08) ％,t=3.42,P=0.001].The expression of CD4+ CD25+ regulatory T cells was higher in young AML patients than in young controls [(6.09±1.91)％ vs.(5.14±0.82)％,t=2.21,P=0.036].The expression level of CD4+ CD25+ regulatory T cells was higher in elderly AML patients than in young AML patients [(7.55±2.78)％ vs.(6.09±1.91)％,t=2.19,P=0.032].Conclusions The dual roles of immunosenescence and tumor may cause the excessive accumulation of CD4+ CD25+regulatory T cells in elderly patients with newly diagnosed acute myeloid leukemia.
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Objective To explore the feasibility and significance of detecting Cyclin D1 and bcl-2 with flow cytometry in the diagnosis of B-cell lymphoma.Methods 45 patients patients with confirmed hematologic diseases in final diagnosis were collected and recorded 45 cases in detail included in the study.25 healthy persons showing normal results in routine physical examinations and laboratory tests were also selected and matched according to the age and gender.In addition,according to the age and gender matching principle,selected routine physical examination and all tests were within the normal range in 25 healthy persons as a healthy control group.Select the pathologically confirmed Cyclin D1 or bcl-2 positive cases were selected as the positive control group,select and the negative cases as the negative control group.Four-color monoclonal antibodies directly immunofluorescence monoclonal antibody labelinged with immunofluorescence were used to analyze the surface and cytoplasmic antigens,and multi-parameter flow cytometry was used to investigate the peripheral blood Cyclin D1 and bcl-2 subsets in lymphoma patients.Results The normal values of Cyclin D1 MFI and bcl-2 were obtained from the 25 normal volunteers by analyzing the (x)±s values and 95 ％ confidence interval,thus establishing the diagnostic criteria.Compared to the healthy control (0.356±0.159,1.938±0.324),all B-cell lymphoma patients had increased expression in Cyclin D1 (1.824±0.312)and bcl-2 MFI (4.259±0.541) in their peripheral blood,results showing statistical significance (P ＜ 0.01).Patients with different subtypes of lymphoma expressed differing Cyclin D1 MFI.In patients with Hodgkin' s lymphoma,Cyclin D1 MFI (0.386±0.112) was significantly lower than that in the non-Hodgkin' s lymphoma patients (1.623±1.987) (P ＜ 0.01).Further,in non-Hodgkin' s lymphoma patients,mantle cell lymphoma was 100 ％ positive for Cyclin D1,while the remaining subtypes were negative.Patients with different subtypes of lymphoma also showed differences in bcl-2 expression.In the Hodgkin' s lymphoma,bcl-2 (2.045±0.877) was significantly lower than the non-Hodgkin' s lymphoma (4.045±0.499) (P ＜ 0.01).In non-Hodgkin' s lymphoma,T cell lymphoma expressed the lowest,while MCL and FL showed 100 ％ positivity.In patients with positive Cyclin D1 or bcl-2,the mean fluorescence intensity level of Cyclin D1 (before treatment 3.099±0.349; after treatment 1.008±0.279) or bcl-2 (before treatment 7.814± 1.030,after treatment 3.131±0.522) was significantly lowered after treatment (P ＜ 0.01).Conclusion Using the method of flow cytometry for detecting Cyclin D1 and bcl-2 in lymphoma cells is feasible,and it can be applied clinically to evaluate the treatment.
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Objective:To study the molecular mechanism for momordin in inducing apoptosis of multidrug-resistant human chronic leukemia K562/A02 cells. Methods:The growth inhibition value of K562/A02 cells was detected by CCK-8 method. Cell apoptosis was analyzed by Annexin Ⅴ flow cytometry (FCM) and cell morphological examination. FCM was also used in determining expression of P-glycoprotein, p53 protein, bcl-2 protein and caspase activity. Results:Momordin inhibited the proliferation of K562/A02 cells in a dose-dependent manner. It also induced cell apoptosis, reduced the expression of P-glycoprotein, p53 protein and bcl-2 protein, and increased caspase-3 and caspase-8 activity.Conclusion:Momordin reversed the inhibition of apoptosis in multidrug-resistant K562/A02 cells. The molecular mechanism may be related with down-regulation of expression of p53 protein, P-glycoprotein, and bcl-2 protein and up-regulation of caspase-3 and caspase-8 activities.
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Objective To determine the expression of phosphatidyhnositol 3-kinase (PI3K), phosphorylated protein kinase B (P-AKT) and protein kinase B (AKT) in cervical cancer and explore the correlations with the genesis and development of cervical cancer. Methods Western blot was used to detect the expression of PI3K, P-AKT and AKT proteins in 66 cases of cervical tissues, including 33 cervical cancers, 23 cervical intraepithelial cancer, 10 normal cervical tissues, and analyzed the clinical significances according to clinical informations. Results In cervical cancers, cervical intraepithelial cancer and normal cervical tissues, the expression of PI3K protein were 1.3880±0.0435, 0.5330±0.0939, 0.2427±0.0888 and had significant differences among them, but there was no significant difference in the ratio of P-AKT and AKT, which were 5.8702±0.0543, 5.0755±0.0888, 3.8353±0.0056. The ratios of P-AKT and AKT had association with histological type, the ratios in squamous cancer of the cervix and in adenocancer and denosquamous cancer were 6.7823±0.7745 and 0.7621±0.0709, and the ratios were much higher in squamous cancer of the cervix than adenocancer and denosquamous cancer. Conclusions Overexpression of PI3K protein is involved in the occurrence of human cervical cancer. The ratio of P-AKT and AKT plays a more important role in squamous cancer of the cervix.
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Objective To investigate gene expressions of matrix metalloprotease (MMP-2, MMP-9) and tissue inhibitor of metalloproteinase-2 (TIMP-2) and RECK ( reversion inducing cysteine-rich protein with Kazal motifs) in the liver tissue of rats with Vibrio vulnificus sepsis. Methods Vibrio vulnificus sepsis model was induced by injection of 1 mL/100 g Vibrio vulnificus (9 × 106 cfu/mL) at hind limbs in 50 Sprague-Dawley male rats of clean grade, each time 10 animals were sacrificed at 2, 6, 9, 12 and 16 h after injection and liver samples were taken. Ten rats served as control group. The ethological changes were observed. The total RNA was extracted from liver tissue and the gene expressions of MMP-2, MMP-9, TIMP-2 and RECK were evaluated by semi-quantitative RT-PCR. Results The rats manifested shortness of breath, fever and swelling in hind limbs at 4 h after injection of Vibrio vulnificus. These symptoms gradually deteriorated, and the rats presented cyanosis and convulsion at 12 h. The gene expressions of MMP-2 and MMP-9 were markedly up-regulated, while those of TIMP-2 and RECK were down-regulated. Conclusion The study suggests that the elevation of MMP-2 and MMP-9 and down-regulation of TIMP-2 and RECK is associated with the development of Vibrio vulnificus sepsis.
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[Objective] To explore effect of momordin on reversion of multidrug resistance(MDR) in K562/A02 cells,and expression and function of P-glycoprotein(P-gp).[Methods] IC50 was detected by CCK-8,and expression of P-gp and retention of ADM in K562/A02 cells were detected by flow cytometry(FCM).[Results] Momordin can improve sensitivity of K562/A02 cells to many kinds of chemotherapeutical drugs,result in decrease of expression of P-gp,and raise concentration of ADM within cells.[Conclusion] Momordin can partly reverse drug resistance of K562/A02 cells,the mechanism of reversion is related with down regulation of expression of P-gp.
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AIM: To explore the change of the expression of cell adhesion molecule CD_ 54 and CD_ 44 in erythroleukemic K562 apoptosis cells induced by momordin from momordica charantia seeds and study the effects of cell adhesion molecule CD_ 54 and CD_ 44 on cell apoptosis induced by momordin. METHODS: After the treatment of K562 cells with appropriated concentration momordin, CCK-8 test was employed to determine K562 cells growth; flow cytometry FACScan (FITC-Annexin V staining) and electron microscopy were used to detect apoptosis; The expression of CD_ 54 and CD_ 44 were examined by flow cytometry FACScan (FITC-CD_ 54 and PE-CD_ 44 staining). RESULTS: CCK-8 test showed K562 cells growth was significant inhibited by momordin; the apoptosis was detected by cell morphology and flow cytometry FACScan (FITC-Annexin V) in K562 cells after treatment by appropriated concentration momordin. The expressions of CD_ 54 and CD_ 44 in momordin treated K562 cells were 18.62 % and 1.32 % respectively, and in negative momordin treated K562 cells were 0.25 % and 0.17 % respectively, and momordin could up-expresses the protein of CD_ 54 18.37 % and CD_ 44 1.15 %. CONCLUSION: Momordin can markedly induce the K562 cell to apoptosis. The up-expressions of CD_ 54 exist in the process of apoptosis induced by momordin. The change of cell adhesion molecule maybe one of the key factors in the mechanisms of apoptosis induced by momordin, and its mechanism maybe involve in adhesion-dependent apoptosis.