| Objective 1. Analyse the expression of Btk and NFκB in leukemia cells from acute leukemia patients, to evaluate the role of Btk and NFκB in the development, progress, prognosis and the therapeutic outcome of acute leukemia(including ALL and AML).2. To investigate the role of PCI-32765(Btk inhibitor) and Bortezomib(NFKB inhibitor) on the proliferation and apoptosis of Raji, Ramos cells, and to explore the possible mechanisms.Methods 1. Bone marrow mononuclear cells:Bone marrow samples from 35 ALL and 14 AML patients were collected. and the mononuclear cells were separated by Ficoll density gradient centrifugation methods. Btk and NFκB expression are detected at RNA and protein level by RT-PCR and Western blot methods.2. The cell lines of B cell malignance:Raji and Ramos cells were treated with PCI-32765 and Bortezomib, the experiments were divided into four groups:control group, Bortezomib group, PCI-32765 group and the PCI-32765 and Bortezomib combination group. The cell proliferation and apoptosis were detected by CCK-8 and Flow cytometry methods, and the expression of Btk, NFκB, c-IAP1, BCL-XL and Caspase-3 were detected by Western blot.Results 1. The results of AML:1.1 Btk and NFκB are expressed in all the samples at RNA and protein level.1.2 At protein level, Btk and NFκB expression is higher in the cells from newly diagnosed AML patients than in the cells from complete remission(CR) stage patients (p< 0.05).2. The results of ALL:2.1 Btk and NFκB are expressed in all the samples at RNA and protein level, the expression level is different. 2.2 At protein level, Btk and NFκB are expressed in all newly diagnosed 35 ALL patients, and 27 patients have higher expression level of Btk,23 patients have high NFκB expression level. Btk and NFκB expression is higher in the cells from newly diagnosed ALL patients than in the cells from CR stage patients (p< 0.05), and the Btk and NFκB expression is higher in relapsed ALL patients.2.3 Btk, NFκB expression and the ALL patients follow-up:2.3.1 among 27 patients who have high expression of Btk,20 patients achieved CR, the CR rate is 74.1%(20/27), among the 20 CR patients,8 patients achieved CR after first cycle of therapy, one cycle CR rate is 29.6%(8/27). About the 20 CR patients,12 patients were relapsed in short time(less than six months), the early relapse rate is 60%(12/20).2.3.28 newly diagnosed patients have low expression level of Btk, among these patients,6 patients achieved CR, the CR rate is 75%(6/8), among the 6 CR patients,5 patients achieved CR after first cycle of therapy, one cycle CR rate is 62.5%(5/8). About the 6 CR patients,1 patient was relapsed in short time(less than six months), the early relapse rate is 16.7%(1/6).2.3.323 newly diagnosed patients have high expression level of NFκB, among these patients,16 patients achieved CR, the CR rate is 69.6%(16/23), among the 16 CR patients,6 patients achieved CR after first cycle of therapy, one cycle CR rate is 26.1%(6/23). Among the 16 CR patients,9 patients were relapsed in short time(less than six months), the relapse rate is 56.3%(9/16).2.3.412 newly diagnosed patients have low expression level of NFκB, among these patients,10 patients achieved CR, the CR rate is 83.3%(10/12), among the 10 CR patients,7 patients achieved CR after first cycle of therapy, one cycle CR rate is 58.3%(7/12). About the 10 CR patients,4 patients were relapsed in short time(less than six months), the early relapse rate is 40%(4/10).2.3.522 newly diagnosed patients have high expression level of Btk and NFκB, among these patients,14 patients achieved complete CR, the CR rate is 63.6%(14/22), among the 14 CR patients,4 patients achieved CR after first cycle of therapy, one cycle CR rate is 18.2%(4/22). About the 14 CR patients,9 patients were relapsed in short time(less than six months), the early relapse rate is 64.3%(9/14).2.3.66 newly diagnosed patients have low expression level of Btk and NFκB, among these patients,4 patients achieved CR, the CR rate is 66.7%(4/6), among the 14 CR patients,3 patients achieved CR after first cycle of therapy, one cycle CR rate is 50%(3/6). About the 4 CR patients,1 patient was relapsed in short time(less than six months), the early relapse rate is 25%(1/4).3. The results of cell lines:3.1 Both PCI-32765 and Bortezomib can inhibit the proliferation of Raji and Ramos cells, the effects shows dosage and time dependent manner, and PCI-32765 and Bortezomib have the synergy effects.3.2 The Raji and Ramos cell lines were treated with PCI-32765 (2μmol/l) and Bortezomib(20nmol/L) for 48 hours.3.2.1 In Raji cell experiment, the cell apoptosis rate in the control group, PCI-32765 group, Bortezomib group and PCI-32765 and Bortezomib combination group are 10.34±0.53%,24.26±0.91%,43.66±1.08% and74.06±0.72% respectively, and the differences was significant among the groups(p< 0.05). Cpmpared with the control group, The expression level of Btk, NFκB, c-IAPl and BCL-XL are lower in the single drug and combination groups, But the expression of Caspase-3 is in the different way.3.2.2 In Ramos cell experiment, the cell apoptosis rate in the control group, PCI-32765 group, Bortezomib group and PCI-32765 and Bortezomib combination group are 15.16±1.49%,71.36±0.82%,75.32±2.36% and 84.30±0.91% respectively, the differences was significant among the groups(p< 0.05). Cpmpared with the control group, The expression level of Btk, NFκB, c-IAPl and BCL-XL are lower in the single drug and combination groups, But the expression of Caspase-3 is in the different way.Conclusion 1. Btk and NFκB may play an important role in the development and progress of acute leukemia.2. PCI-32765 and Bortezomib has synergistic effect in the proliferation and apoptosis of Raji and Ramos cell lines, the mechanism of the effects were down-regulation the expression level of NFκB, c-IAP1 and BCL-XL and up-regulation the expression of Caspase-3.3. Btk and NFκB may be used as a potential therapeutic targets in ALL, they may be used as prognosis factor in ALL and AML. |
