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Objective To investigate the mechanism of STAT5-ROS pathway to mediate IM resistance of K562 cells.K562 cells were cultured with imatinib at gradually increased concentrations to generate resistance cell line.Methods CCK-8 assay was used to clarify the resistance ratio.The STAT5A and STAT5B mRNA levels were detected by RT-PCR.Flow cytometry assay was used to detect the level of ROS and cell apoptosis.The expression of STAT5 protein was detected by Western blot.Results Imatinib resistance cell line K562/G was successfully induced by gradually increasing concentrations of IM.The IC50 of K562/G was eighty times higher than K562 by CCK-8.Cell growth curve showed that K562/G was not inhibited in 20 μmol/L imatinib, whereas the K562 cell was significantly inhibited by up to 0.1 μmol/L imatinib.Intracellular level of ROS in K562/G was obviously higher than that of K562 cells(P=0.000 1).The apoptosis ratio of K562 was lower than that of K562/G when the same concentration of IM react on the two cell lines for the same time(P<0.05).The expression of STAT5A and STAT5B mRNA in K562/G was higher than in K562(P=0.000 1,P=0.017 0).Then,the level of STAT5 proteins in K562/G cells was significantly increased (P=0.009 0).Conclusion The STAT5 is highly expressed in CML.STAT5-ROS is closely related to the formation of imatinib resistance of chronic myeloid leukemia.
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Objective To discuss the clinical application of minimal residual disease in acute myelogenous leukemia(AML) by wt1 mRNA quantitative combined with multi-parameter flow cytometry (FCM).Methods Real time quantitative polymerase chain reaction (qRT-PCR) method was established for detecting wt1 gene expression level in 35 AML patients.The indexes were detected by different subtypes;And 9 cases of ease and 4 cases of recurrence in patients was followed-up and detected the wt1 level.The multiparameter flow cytometry was used to analyze the minimal residual disease in AML.Results The expression of wt1 gene was significantly higher than that of the control group.Significant difference was found(P<0.05).In the newly diagnosed AMLs, wt1 was the highest in M2 and the lowest in M6.Follow-up of 4 AML patients showed that wt1 gene expression level was markedly decreased after CR, but obviously increased after relapse.The proportion of abnormal myeloid cells in different phases significantly changed by FCM.There was no difference of minimal residual disease in AML by qRT-PCR and multiparameter flow cytometry.The ROC curve was used to analyze the recurrent cases to get the threshold value(3.33%).Conclusion The quantitative analysis of wt1 combined with multi-parameter flow cytometry can be used to monitor minimal residual disease in leukemia patients, assess the treatment efficacy and prognosis, and predict the risk of recurrence.
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Purpose To explore the accuracy of ALK fused gene expression by immunohistochemistry ( IHC) in non-small cell lung cancer ( NSCLC) patients, and to investigate the clinical and pathological features of ALK-positive NSCLC patients. Methods By u-sing rabbit monoclonal D5F3 antibody, ALK IHC was performed on 234 NSCLC patients. ALK positive cases were confirmed by reverse transcription-polymerase chain reaction ( RT-PCR) . Results The positive incidence of ALK by IHC in 234 NSCLC specimens was 8. 97% (21/234), the positive rate of ALK fused gene verificated by RT-PCR was 5. 98% (14/234). There was significant difference with histological type, age, stage (P120, the consistency rate was 100%. Conclusion Although immunohistochemical expres-sion of ALK fused gene may have a certain false positive, IHC or immunohistochemical score> 120 show very high value for ALK fused gene RT-PCR followed by ALK immunohistochemistry in lung cancer is a economical and feasible method for the valuation of ALK fused gene.
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Objective To study the significance of morphologic, immunophenotype, cytogenetic features, molecular biology (MICM) and prognosis of t (8;21) acute myeloid leukemia (AML) patients.Methods Morphological, FAB subtypes, flow cytometric immunophenotyping, G-binding technique and RTPCR were performed in 70 AML patients with t (8;21) and AML1-ETO fusion transcripts as compared with normal karyotype 70 AML patients. Results In 70 AML patients with t(8;21), 1 case of M1, 64 cases of M2, 3cases of M4 and 2 cases of ambiguity AL according to FAB classification. The CD13, CD33, CD34 and CD117expression were higher, meanwhile CD19 was positive in 40 %, CD15 was 11%, CD11b was 10 % and CD7 was 7 %. Cytogenetically, 50 % cases had additional chromosomal abnormalities, and main associated recurrent additional abnormalities were loss of a sex chromosome, 9q- and hyperdiploid. AML1/ETO fusion gene transcripts were detected in all 70 AML patients with t(8;21) by RT-PCR. CR rate of t(8;21) AML with CD19were 72 %, t(8;21) AML with CD19 and CD7 were 0; in normal karyotype AML were 31%. Conclusion The t(8;21) is the characteristic chromosome abnormality of M2. In the t(8;21), CD19, CD34 and CD117 expression are high, while CD7 are low. These antigen expression in t(8;21) AML closely correlated with karyotype. CD19 is a marker of good prognosis, but CD7 is a marker of low CR.
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Objective To analyze the fusion genes derived from 29 types of chromosome structural aberrations in leukemia patients,and the significances on the MICM typing,risk grouping,and minimal residual disease(MRD)monitoring of leukemia.Methods The bone ulan-ow or blood samples from 141 leukemia patients were analyzed with a novel multiplex nested RT-PCR.In addition.chromosomal karyotypes were investigated in some patients.Results Of the 141 leukemic samples,66(46.8%)carried 13 types of MLL/AF6,MLL/AF9,dupMLL MLI/ENL,CBFβ/MYH11 and TLS,ERG.Fusion genes were positive in 27 of 57 ALL patients(47.4 q%),and 33 of 78 AML patients(42.3%),respectively.In these ALL or AML patients,7 or 6 chromosome structural aberrations were found. Conclusion This multiplex nested RT-PCR reaction could screen 29 types of chromosome structural aberrations at the same time. It may be helpful for the diagnosis, risk grouping,prognosis evaluation and the detection of minimal residual diseases after chemotherapy and bone marrow transplantation in these leukemia patients.
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Objective To investigate the relationship between CD4+CD25+ regulatory T(T-Reg)ceils and the higher prevalence of tumor in elderly people. Methods The frequencies of T-Reg in peripheral blood from 64 health adults.52 healthy elderly people and 64 elderly cancer patients were determined by multi-color detection and multi-parameter flow cytometry.and the relationships among the 3 groups were analyzed and the correlation among CD4+CD25high,CD4+CD25+FoxP3+and CD4+CD25+CD127low were studied. Results Be gated on CD4+ lymphocytes,the reference ranges of CD4+CD25high T cells in the groups of health adults.healthy elderly people and elderly cancer patients were(1.390±0.412)%,(1.729±0.247)%and(2.150±O.769)%respectively,while CD4+CD25+FoxP3+ T cells were(1.1 80±0.343)%,(2.477±0.400)%and (4.980±2.177)%respectively,and CD4+CD25+CD127low T cells were(5.213±0.942)%,(6.186±1.196)%and(7.194±1.538)%respectively.There was the same change rule in CD4+CD25high,CD4+CD25+FoxP3+and CD4+CD25+CD127low T cells of the 3 groups.elderly cancer patients was at the highest.then was healthy elderly people,and the last was health adults.The 3 markers were positively correlated among 3 groups(all P＜0.05).There were gender difference in 3 groups of CD4+CD25high,CD4+CD25+FoxP3+ and CD4+CD25+CD127low T cells. Conclusions The percentages of CD4+CD25high are increased with age and its excessive accumulation is possibly correlated with immunosenescence and the tumor development.
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Objective To analyze the clinical and biological features of mixed acute leukemia(MAL).Methods Bone marrow specimens of 38 MAL patients were evaluated to prove the diagnosis and the classification by morphoiogic,immunologic examinations.These patients were treated with protocols suitable for both acute myeloid leukemia(AML)and acute lymphoblastic leukemia(ALL).Results All MAL patients had a leukemia syndrome.Morphologically,the subtypes of M1,M2 and M5 were predominant in AML,as L2 Was in ALL.Immunologically,coexpression of myeloid and B lineage associated antigens was predominant,about 68.4%;cytogenetically,Ph chromosome was observed in 33.3%(5/15)of MAL patients,and immunophenotype was B-M;1 Ph chromosome(+)MAL patient,fusion gene bcr-abl 190(+)and immunophenotype was B-M.In 38 cases,32 patients received chemotherapy.The complete remission rate was 28.1%(9/32).CR of.normal karyotype was significantly higher than that of abnormal ones.Conclusion Patients with MAL have unique biological features and the complete remission rate was low and the prognosis was poor.
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Objective To evaluate the proportion and clinical significance of CD4+CD25+ regulatory T cells in childhood acute lymphocyte leukemia(AEL)during different therapeutic stages.Methods 55 peripheral blood samples from 40 children patients with ALL were detected by muhiparameter flow cytometry with fluoresce-hbeled monoclonal antibody.Results Treg cells phenotypically express not only CD62L but also FoxP3 protein.In patients with ALL standard-risk the proportion of CD4+CD25Hi was(1.04±0.33)% in the first course of induction treatment, (1.60±0.44)% in maintenance treatment groups, and(1.29±0.30)% in complete remission groups respectively,while in patients with ALL the intermediate and high risk during maintenance therapy was(2.24±0.75)%.Conclusion Compared with healthy children,the proportion of Treg ceHs in ALL is significantly higher,and may be related to the effect of chemical treatment and severity of ALL.The elevated proportion of Treg may contribute to disease relapse.
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Objective To evaluate the diagnostic significance of detecting serum-DNA concentration and clonal gene rearrangement in lymphogenous malignant patients.Methods Serum-DNA in 72 diagnosed patients were measured and analyzed by SPSS11.0.Serum and mono-nuclear cells samples were collected. IgH CDRⅢ, TCR?V9 region were amplified by PCR.Results The serum-DNA concentration of disease groups were (418?172)?g/ml,(426?192)?g/ml, (388?170)?g/ml and(400?110)?g/ml, each was higher than those of control group(77?47)?g/ml(P0.05).Conclusion The concentration of serum-DNAwere higher in lymphogenous malignant patients and tumor associated DNA could be easily detected, so they have important diagnostic value in lymphogenous malignant patients.
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<p><b>OBJECTIVE</b>To assess the platelet and plasma concentrations of fibronectin (Fn) and fibrinogen (Fg) in congenital fibrinogenopenic (FgP) patients and explore their role in inducing platelet adhesion and aggregation.</p><p><b>METHODS</b>A FgP family was selected as study group and the platelets isolated and purified to assess concentrations of Fn and Fg in platelets, alpha-granules and plasma with Western blotting, immuofluoresence staining and flow cytometry (FACS), respectively, the expression of platelets GP II b/III a by FACS.</p><p><b>RESULTS</b>The concentration of platelets Fn in FgP patients is higher than that in controls, and is higher in homozygote than in heterozygote. In contrast, plasma Fn levels were identical in all samples. The amount of platelet Fg from FgP patients is lower than that from the controls and positively correlated with the concentration of their plasma Fg. No difference in the expression of platelet GP II b/III a had been found.</p><p><b>CONCLUSION</b>It suggested that increased platelet Fn could partially compensate the lack of Fg and lead the platelet adhesion and aggregation.</p>