| Background:Idiopathic thrombocytopenic purpura(ITP)is an acquired autoimmune disease characterized by bleeding from the skin and mucous membrane,reduced number of platelets,shortened life expectancy,normal or increasing number of megakaryocytes and small spleen,and its pathogenesis is complex.With the advent of new drugs,the overall efficiency of ITP has been significantly improved,but there is still a low rate of long term remission or no effective treatment for patients with nearly 1/3,most of which are chronic or refractory ITP.ITP patients have serious bleeding risk,unpredictability of disease prognosis,fear of disease,long time treatment,and reduction of social activities,and other factors seriously affect their quality of life,or even lower than cancer patients.Therefore,it is an important way to improve the quality of life of ITP patients by digging out the pathogenesis of ITP and exploring new therapeutic targets.Objectives:At present,the research on the pathogenesis of ITP mainly focuses on two aspects: humoral immunity,excessive destruction of platelets mediated by cell immunization and the formation of thrombocytopenia in megakaryocytes.There are few studies on the formation of thrombocytopenia in megakaryocytes of ITP patients.Therefore,This study is divided into three parts to study the relationship between megakaryocyte apoptosis and autophagy in ITP patients,and further explore the relationship between the two mechanisms,providing a new perspective for the treatment of ITP.Method:This experiment is divided into three parts:1.14 cases of ITP were collected,and a control was set up.The bone marrow was collected and smeared.The plasma was collected after the peripheral blood centrifugation.The bone marrow smear was stained with the Wright Giemsa stain.Then the megakaryocyte was counted and photographed at the same time.2.The Meg-01 cells of adult megakaryocyte leukemia were selected to represent megakaryocyte,co-culture with serum free 1640 culture fluid,normal human plasma and ITP patients.The morphologic changes were evaluated by Wright Giemsa staining,Lyso-Tracker Red,MDC and flow cytometry were used to detect autophagy;Annexin V-FITC/PI double staining and flow cytometry were used to detect apoptosis,and autophagicinhibition was used.The effect of chloroquine on autophagy and apoptosis was tested by autophagy inhibition assay.3.The Meg-01 cells of adult megakaryocyte leukemia were selected to represent megakaryocyte,co-culture with serum free 1640 culture fluid,normal human plasma and ITP patients,and the protein expression of Bcl-2,Bax,Beclin-1,LC3 B and Cleaved caspase-3 were detected by Western blot,and autophagic inhibitor chloroquine was used to inhibit the expression of the above protein.Results:1.Compared with the normal control group,the total number of megakaryocytes in ITP patients was not statistically significant.Compared with the control group,the number of megakaryocytes in the early stage of ITP patients was significantly higher than that in the control group(P<0.01),while the number of megakaryocytes in the late ITP patients was significantly lower than that of the control group(P<0.05).The cytoplasm of granulosa megakaryocytes in ITP patients was less,the granules in the cytoplasm were small and sparse,but the cytoplasm of granulosa megakaryocytes in the normal control group was rich and the granules in the cytoplasm were thicker and denser.The chromatin of megakaryocytes in ITP patients was loose,but the chromatin of the normal control group was dense.2.The morphology of Meg-01 cells in the 2.ITP plasma group wasvacuolated and nuclear agglutination was obvious,but in the no plasma group.By fluorescence analysis,the apoptosis of ITP plasma group was significantly lower than that of normal plasma group,and the autophagy increased(P<0.05).FCM showed that compared with the control group,the early apoptosis rate in the ITP plasma group and the no plasma group decreased with statistical difference(P<0.05 or P<0.01),but there was no statistical significance in the advanced apoptosis rate in each group.The autophagy rate in ITP plasma group and non plasma group increased by flow cytometry.There was a statistically significant difference in the autophagy rate between the plasma group and the control group(P<0.05).Through autophagy inhibition test,it is proved that megakaryocytes in ITP patients inhibit normal apoptosis through autophagy.3.In the no plasma group and the ITP plasma group and the normal control group,the level of Bcl-2,Beclin-1 and LC3 B are increased(P<0.01 or P<0.05),Bax,Cleaved caspase-3 are decreased simultaneously(P<0.01 or P<0.05).After the addition of chloroquine(CQ),the level of Beclin-1and LC3 B are increased in the normal plasma+CQ group and the normal plasma group,the ITP plasma+CQ group and the ITP plasma group,the normal plasma group and the ITP plasma group(P<0.01 or P<0.05).The level of Bcl-2 are decreased in the normal plasma group and the ITP plasma group,the ITP plasma+CQ group and the ITP plasma group(P<0.01 or P<0.05).The level of Bax and Cleaved caspase-3 in the normal plasmagroup and the ITP plasma+CQ group are higher than that of ITP plasma group(P<0.01 or P<0.05),but the level of Bcl-2 and Bax in normal plasma group was not statistically significant compared with the normal plasma+CQ group(P>0.05).Conclusion:1.By means of the analysis of bone marrow smear,statistics of clinical data and the phenomenon of vacuolization and nuclear agglutination in the morphology of ITP plasma and Meg-01 cells,it provides strong evidence for the existence of autophagy in the megakaryocytes of ITP patients.2.through fluorescence and flow cytometry,the apoptosis rate and autophagy of Meg-01 cells in ITP plasma group were decreased.After adding the autophagic inhibitor chloroquine,the early apoptosis rate of Meg-01 cells increased and the autophagy decreased correspondingly,suggesting that the megakaryocytes of ITP patients inhibit their normal apoptosis through autophagy.3.Western blot test showed that compared with the normal control group,the Bcl-2,Beclin-1 and LC3 B in the non plasma group and the ITP plasma group increased,and the caspase-3 of Bax and Cleaved decreased.After adding the autophagic inhibitor chloroquine,the Bcl-2,Beclin-1 and LC3 B in the plasma free group and the ITP plasma group decreased,and Bax and Cleaved caspase-3 increased.It suggested that the mechanism ofthe megakaryocyte to inhibit its normal apoptosis by autophagy is Bcl-2through the down-regulation of Bax mediated mitochondrial apoptosis pathway to achieve the autophagy switching. |
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