| The incidence of malignant lymphoma has increased in recent years,and it has become one of the major tumours that seriously threaten human health and life.Natural killer/T-cell lymphoma?NKTCL?is a highly invasive subtype of non-Hodgkin lymphoma?NHL?associated with rapid progression,short patient survival time and poor prognosis.Currently,although NKTCL treatment has advanced,for example,with the use of L-asparaginase-and gemcitabine-based regimens,many patients are resistant to the drugs and have poor responses due to multidrug resistance?MDR?.Moreover,the molecular pathogenesis of NKTCL is unclear.Studies have shown that abnormal expression and activation of some key signalling pathway factors results in the pathological development of a variety of lymphomas.Therefore,further in-depth study of the key NKTCL molecules and signalling pathways is a potential direction for improving the efficacy of chemotherapy and the prognosis of patients.Platelet-derived growth factor receptor?PDGFR?is the founding member of a family of receptor tyrosine kinases that includes PDGFR?,PDGFR?,stem cell factor receptor?c-KIT?,fms-like tyrosine kinase?Flt-3?and colony stimulating factor 1receptor?CSF1R?.The PDGFR family ligands,platelet-derived growth factors?PDGFs?,are major mitogens for connective tissue cells,glial cells,and certain other cell types,primarily cells of mesenchymal origin.Structurally,PDGFs consist of four homodimers,namely,AA,BB,CC,and DD,and an AB heterodimer.Signal transduction is activated by combining these ligands with PDGFRs.Normal PDGFR signalling is important in processes such as gut villus formation,formation of alveolar smooth muscle,hair follicle development,vasculars mooth muscle development,development of kidney mesangial cells,among others.PDGFs are involved in cell proliferation,survival and migration.Dysfunction of PDGF signalling has been observed in a wide array of pathological conditions,such as cancer,fibrosis,neurological conditions and atherosclerosis.High expression levels of PDGFRs and their ligands?PDGFs?have been observed in many cancers,suggesting an association with poor prognosis in various tumours.Thus far,several studies have shown PDGFR?expression in lymphoma,but little evidence has demonstrated its significance in NKTCL.The objective of the present study was to detect PDGFR?expression and determine whether it is correlated with NKTCL clinical parameters and prognosis.Furthermore,we investigated the biological roles of PDGFR?in NKTCL cell proliferation,cell cycle,apoptosis,and tumour cell growth and the preliminary mechanism of action in NKTCL.The results will likely provide important evidence to delineate the functional roles of PDGFR?in NKTCL and identify a potential strategy for targeted therapy against NKTCL.Chapter ?:The expression and significance of PDGFR?in NK/T cell lymphomaObjective:Immunohistochemistry?IHC?,RT-PCR and western blotting were used to detect the expression of PDGFR?and p-PDGFR?in NK/T cell lymphoma tissues and cell lines.The correlation between the expression of PDGFR?and the clinical parameters and prognosis of NK/T cell lymphoma were analyzed.Materials and methods:1.We collected paraffin blocks of patients with NK/T cell lymphoma,and analyzed the expression of PDGFR?in NK/T cell lymphoma and reactive lymphoid hyperplasia of the nasopharynx by immunohistochemical method.2.Pearson?2 test was used to detect the correlation between PDGFR?expression and clinical parameters.3.The Kaplan-Meier survival curve was used to analyze the correlation between expression of PDGFR?and survival.The Cox regression was used to detect the prognostic factor of OS.4.The expression of PDGFR?in NK/T cell lymphoma cell line?YTS and NKL?was detected by RT-PCR and Western blot.Result:1.PDGFR?and p-PDGFR?were highly expressed in NK/T cell lymphoma and cytoplasm stained.The percentage of PDGFR?positive expression was 89.7%and73.5%in NK/T cell lymphoma and reactive lymphoid hyperplasia of the nasopharynx.The percentage of patients with p-PDGFR?positive expression was 98.7%and11.8%in NK/T cell lymphoma and reactive lymphoid hyperplasia of the nasopharynx.There were significant differences between PDGFR?and p-PDGFR?expression in NK/T cell lymphoma and reactive lymphoid hyperplasia of the nasopharynx tissues?P=0.028,P=0.000?.2.There was no significant difference between PDGFR?expression with gender,age,Ki67,?2-MG,EBV-DNA copy and treatment efficiency.There were significant differences between PDGFR?expression with LDH level and clinical staging?P=0.028,P=0.013?.3.Compared with PDGFR?low expression group of NK/T cell lymphoma patients,the PFS and OS of PDGFR?high expression group was significantly worse?P=0.005,P=0.011?.COX multivariate analysis showed that the clinical stage?P=0.028?and PDGFR??P=0.048?were independent prognostic factors for NK/T cell lymphoma patients.4.Compared with normal NK cells,the mRNA expression of PDGFR?was higher in YTS?P=0.0002?and NKL?P=0.0004?.the mRNA expression of PDGFR?in YTS cells was higher than that in NKL?P=0.0192?.Compared with normal NK cells,the protein expression of PDGFR?was higher in YTS?P=0.0004?and NKL?P=0.0022?.the protein expression of PDGFR?in YTS cells was higher than that in NKL?P=0.0322?.Conclusion1.PDGFR?was highly expressed and activated in the tumor of NK/T cell lymphoma.2.The expression of PDGFR?has no significantly relation with gender,age,Ki67,?2-MG,EBV-DNA copy number and treatment efficiency,and was significantly related to LDH level and clinical staging.3.The expression PDGFR?was one of the independent prognostic factors for NK/T cell lymphoma.4.The expression of PDGFR?was higher in YTS and NKL.It was higher in YTS cells than NKL.Chapter ?:The function and mechanism of PDGFR?in NK/T cell lymphoma cell lineObjective:The PDGFR?signaling pathway was activated and blocked by the specific PDGFR?agonist PDGF-AB and inhibitor Imatinib.We explored the effect of PDGFR?on NK/T cell lymphoma YTS and NKL cell proliferation,cell cycle and apoptosis and its mechanism.Materials and methods:1.The proliferation rate changes of YTS and NKL cells with PDGF-AB or Imatinib treatment were detected by CCK-8.2.The apoptosis rate changes of YTS and NKL cells with PDGF-AB or Imatinib treatment were detected by flow cytometry.3.Flow cytometry was used to detect the changes of YTS and NKL cell cycle after PDGF-AB or Imatinib treatment.4.Western blotting was used to detect the expression key proteins?p-AKT,AKT,Erk and p-Erk?changes of the downstream pathways in YTS and NKL cellsResult:1.Compared with control group,the OD value in 5 ug/L?25 ug/L?50 ug/L groups of YTS cells were increased after PDGF-AB treatment?P=0.0166,P=0.0002,P=0.000?.Compared with control group,the OD value in 5 ug/L?25 ug/L?50 ug/L groups of NKL cells were increased after PDGF-AB treatment?P=0.0165,0.0014,P=0.0045?.The growth rate of YTS and NKL cells were getting slowly following by Imatinib concentration.The growth rate of YTS and NKL cells was decreased with time dependence.2.After the treatment of PDGF-AB,there was no significant change in the apoptosis rate of YTS and NKL cells.After the treatment of Imatinib,the apoptosis rate of YTS and NKL cells increased?P=0.0002,P=0.0006?.3.After PDGF-AB treatment,the proportion of S phase cells in YTS and NKL cells increased significantly?P=0.0007,P=0.0027?,and the proportion of G1/G0phase decreased?P=0.0022,P=0.0063?.After Imatinib treatment,the proportion of G1/G0 phase cells in YTS and NKL cells increased significantly?P=0.0000,P=0.0000?,and the proportion of G2/M phase decreased?P=0.0004,P=0.0001?.4.Western blotting results showed the expression of p-PDGFR and p-Erk was reduced with higher Imatinib concentration in YTS cells,while no significant changes with PDGFR,AKT,Erk and p-AKT expression.Conclusion:1.PDGF-AB promoted the proliferation of NK/T cell lymphoma cell lines with a concentration dependence.Imatinib inhibited the proliferation of NK/T cell lymphoma cell lines,and the inhibitory effect is concentration and time dependent.2.PDGF-AB didn't influence the apoptosis of NK/T cell lymphoma cell lines.Imatinib promoted the apoptosis of NK/T cell lymphoma cell lines.3.PDGF-AB promoted NK/T cell lymphoma cells into S phase.Imatinib blocked the progression of cell cycle in NK/T cell lymphoma cells at G1/G0 phase.4.Imatinib inhibited the phosphorylation of Erk,and the inhibitory effection was concentration dependent.Chapter ?:The function and mechanism of PDGFR?in NK/T cell lymphoma in vivoObjective:We construct the animal model of NK/T cell lymphoma,explored the effect of PDGFR?on the growth of tumor cells in NK/T cell lymphoma mouse model through the specific inhibitor Imatinib blocking the PDGFR?signaling pathway,and investigated its mechanism by western blot.Materials and methods:1.YTS and NKL cells were used to construct the animal model of NK/T cell lymphoma.2.We divided the YTS and NKL animal models into 2 groups,control group and imatinib group.3.Daily observation of the growth of the tumor mice in each group and the changes in tumor volumes.4.On the 24th day,the size of the tumor and weight changes of the mice in each group was measured.5.Histopathology HE staining and immunohistochemistry on the tumor tissues.6.Western blot was used to detect the expression key proteins?p-AKT,AKT,Erk and p-Erk?changes of the downstream pathways in YTS cells.Result:1.YTS and NKL lymphoma animal models were conducted successfully.2.Imatinib can inhibit the growth of NK/T cell lymphoma animal model.Compared with the blank control group,the volume of tumor tissue decreased significantly in imatinib groups of YTS and NKL lymphoma animal model?P=0.0002,P=0.0026?,the average tumor weight of these imatinib groups decreased?P=0.0001,P=0.0162?.The tumor suppressor rates of imatinib groups of YTS and NKL lymphoma animal model were 31.4%,21.4%.3.Immunohistochemical staining showed that NK cells labeled CD56,T cells labeled CD3,Proferin andGrB were all expressed in varying degrees.There was no significant difference between the drug groups about the expression of PDGFR??P=0.505,P=0.906?.Compared with the blank control group,the expression of p-PDGFR?in imatinib groups of YTS lymphoma animal model decreased?P=0.021?,the expression of p-PDGFR?in imatinib groups of NKL lymphoma animal model decreased slightly without significant difference?P=0.053?.Compared with the blank control group,the expression of p-Erk in imatinib groups of YTS and NKL lymphoma animal model decreased slightly without significant difference?P=0.053?.4.Western blotting results showed compared with the blank control group,the expression of p-PDGFR?and p-Erk in imatinib groups of YTS?P=0.0052,P=0.0076?and NKL?P=0.0067,P=0.0310?lymphoma animal model was reduced,while no significant changes with PDGFR?,AKT,Erk and p-AKT expression.Conclusion:1.YTS and NKL lymphoma animal models were conducted successfully.2.Imatinib inhibited the tumor growth in mouse model of NK/T cell lymphoma by blocking PDGFR?.3.Imatinib downregulated the expression of p-Erk and may play an anti-tumor role by acting on the MAPK/Erk signaling pathways.Summary1.PDGFR?was highly expressed in the tumor tissue and cell lines of NK/T cell lymphoma.High PDGFR?expression was an independent factor for the adverse prognosis of NK/T cell lymphoma patients.2.PDGF-AB induced cells into S phase to improve the proliferation of NK/T cell lymphoma cell lines.3.Imatinib inhibited the proliferation of NK/T cell lymphoma cell lines,promoted cell apoptosis and blocked the progress of cell cycle through inhibiting the phosphorylation of Erk.4.Imatinib inhibited can inhibit the tumor growth in mouse models of NK/T cell lymphoma and might play an anti-tumor effect through MAPK/Erk signaling pathway. |
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