| Background and ObjectiveAcute T-cell lymphoblastic leukemia (T-ALL) is an aggressive malignant disease.Despite the significant progress in combination chemotherapy and hemopoietic stem cell transplantation, the patients continue to have a particularly poor prognosis. Furthermore, many patients get medicine resistance during the process, even experience significant toxicity. Therefore, it is important to explore new targeted treatment strategies that improve survival in high risk patients and decrease toxicity in standard risk patients.Notch signaling is an evolutionarily conserved signaling pathway that plays an essential role in regulating cell fate determination, differentiation, proliferation, and apoptosis. As a very important receptor in Notch family, Notch1 plays an essential role in the generation of T leukemia cells. 50% to 60% of human T-ALL cell lines and primary patient samples were shown to harbor activating mutations in the Notch1 gene that result in aberrant Notch1 signaling. By using the small molecule inhibitors ofγ-secretase inhibitors which can reduce the Notch1 signal pathway transduction to induce apoptosis in T-ALL has become one of the targeted therapy agent.Forkhead box protein 3(Foxp3), which is the specific marker of CD4+CD25+ regulator T cells (Tregs), plays an important role in the regulation ,activation and differentiation of T cell. Foxp3 + Tregs, not only play an important role in maintaining peripheral tolerance, but also occupy an important position in the process of tumor development and progression. The expression of Foxp3 is closely related to many mice and human malignant tumors, which are ovarian carcinoma and pancreatic, breast adenocarcinoma, as a result, Foxp3 is likely to become a clinical indicator of assessment of prognosis of malignant, furthermore, it will be a therapeutic target of some tumors. In this study, the Jurkat cell line is used to research. On the basis of confirming the high expression of Foxp3 in Jurkat cell ,this study detected the effect of DAPT, which is the inhibitor of Notch1 signaling pathway, on the expression of Foxp3,and determined the effect of the proliferation, apoptosis and differentiation of Jurkat cell,explored whether Notch1 signaling pathway was involved in the regulation of Foxp3 and how to control it.The lab techniques included cell culture, Real-time PCR method, Western-blot, flow cytometry and other cell molecular biology techniques.As a result,the dissertation would provide valuable experimental evidence for the role of inhibitors of Notch1 signaling pathway in T cell leukemia, and explore a new method for the targeted therapy of T cell leukemia .MethodsPart one The expression of Notch1 and foxp3 in Jurkat cell line1. The mRNA of Notch1 of Jurkat cell line was determined by RT-PCR,which was compared with normal Human peripheral blood mononuclear cells.2. The expression of Notch1 of Jurkat cell was determined by Western-blot,which was compared with normal Human peripheral blood mononuclear cell.3. The expression of Foxp3 of Jurkat cell was determined by Western-blot,which is compared with normal Human peripheral blood mononuclear cell.Part two Impact of DAPT on the Biological characteristics of Jurkat Cell line1. Jurkat cells in culture medium were treated with various concentration of DAPT(1,2.5,5,10,20umol /L) for 4,8,12,24,48 ,72 hours in vitro.Cell proliferation was evaluated by cell counting kit -8(CCK-8) assay.The morphology of cell was observed under inverted microscope.2. The cell apoptotic rate was detected by flow cytometry using Annexin V/PI double staining after the cells were treated with various concentration of DAPT(1,5,10,20umol /L) for 48 hours.3. The morphological variations of apoptotic cells were observed after the cells were treated for 48 hours with DAPT through Wright-Giemsa staining. 4. The cell cycle state was analyzed by flow cytometry using GENMED after the cells were treated with various concentration of DAPT(1, 5,10,20umol /L) for 48 hours.5. The expression of T cell differentiation antigens were detected after being treated with DAPT for 48 hours by flow cytometry.6. The RNA of Jurkat cells were extracted after being treated with DAPT in different concentration (1, 5, 10,20umol /L) for 24, 48, 72hours, respectively.The Real-time PCR was used to detect the mRNA of Notch1-Cleaved, Hes1 and Foxp3, respectively.7. The protein of Jurkat cells were extracted after being treated with DAPT in different concentration (1, 5, 10,20umol /L) for 24, 48, 72hours, respectively.The Western-blot was used to detect the protein of Notch1-Cleaved, Hes1 and Foxp3, respectively.8. The expression of Foxp3 was detected after being treated with DAPT in different concentration (1, 5, 10,20umol /L) for 48, 72 hours by flow cytometry.9. The protein of Jurkat cells were extracted after being treated with DAPT in the concentration of 10umol /L for 48, hours, respectively.The Western-blot was used to detect the protein of NF-κB, p-ERK1/2 and STAT1, respectively.ResultsPart one1. The expression of mRNA and protein of Notch1 were determined in Jurkat cells.Meanwhile,the normal human peripheral blood mononuclear cells did not express.There was statistical significance between them(P﹤0.05).2. The expression of protein of Foxp3 were (88±2%)and (5±3.5%) in Jurkat cells and normal human peripheral blood mononuclear cells respectively.There was statistical significance between them(P﹤0.05).Part two1. CCK-8 analysis showed there were no apparente inhibition in cell growth with different concentration of DAPT treated after 4,8,12 hours(P﹥0.05).The inhibitory rate caused by 20umol/L DAPT treated after 48 hours was 33±2.3%(P﹤0.05),which was highest at all.However, the inhibitory capability was decreased gradually after 72 hours.2. DAPT could induce apoptosis in Jurkat cells after treated for 48 hours in dose-dependent.3. The obvious morphological changes of Jurkat cells were detected by Wright-Giemsa staining.Membrane integrity and chromatin distribution of normal Jurkat cells were observed in the control group.Some apoptotic cells were identified by the presence of cell shrinkage, nucleolus breakage,or cytoplasm outflow in the cells treated with with DAPT in the concentration of 10umol /L after 48 hours.4. DAPT could arrest the Jurkat cell cycle at the G0/G1 phase in dose-dependent,and then,the cells in S and G2/M phases decreased.5. The expression of CD7 after being treated with DAPT in the concentration of 10 umol/L for 48 hours were detected by flow cytometry,which were decreased compared with the control group(P﹤0.05). Meanwhile,the expression of CD2,CD5 had no changed with DAPT treated.6. The expression of mRNA of Notch1 after being treated with DAPT in different concentration (1,5,10,20umol /L) for 48 hours were detected by RT-PCR, which were decreased in a dose-dependent manner compared with the control group.The expression of mRNA of Hes1 after being treated with DAPT in different concentration (1,5,10,20umol /L) for 48 hours were detected by Real-Time PCR, which are 90.12±1.4%,57.3±2.2%,42.1±3.3% and 41.8±6% compared with the control group(P﹤0.05), respectively. The expression of mRNA of Hes1 after being treated with DAPT in the concentration of 10umol /L for 24,48,72 hours were 53.59±12.7%,28.95±4.2% and 27.35±1.4% compared with the control group, respectively.(P﹤0.05).7. The semiquantitative ratio of protein of Notch1-Cleaved ,Hes1, STAT1, p-ERK1/2 ,NF-κB after being treated with DAPT in the concentration of 10umol/L for 48 hours were detected by Western-blot,which were 72.5±3.8%,32.1±2.9%,compared with the control group(P﹤0.05), respectively.8. The expression of protein of Foxp3 after being treated with DAPT in different concentration (1,5,10,20umol /L) for 48 hours were detected by flow cytometry, which were 65.5±3.5%,60.9±2.4%,58.8±2.8% and 50.7±1.9% compared with the control group(P﹤0.05), respectively.9. The semiquantitative ratio of protein of NF-κB , p-ERK1/2 and STAT1 after being treated with DAPT in the concentration of 10umol /L for 48 hours were detected by Western-blot,which were 48.7±1.4%,50.1±2.9%,68.8±3.8%,compare with the control group(P﹤0.05), respectively.Conclusion1. DAPT, the inhibitor of Notch1 signaling pathway, plays a role in the growth inhibition of Jurkat cell line with time and concentration dependent,moreover, affects the apoptosis and differentiation of Jurkat cells.2. DAPT reduces the expression of Foxp3 in Jurkat cells dose dependently, which may be followed by the regulation of NF-κB, p-ERK1/2, STAT1. Foxp3 may be an important downstream target molecule of Notch1 signaling pathway.3. DAPT has some potential in the anti-leukemia therapy. Foxp3 may be a good target in the treatment of T cell leukemia. |
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