| Purpose:Immune thrombocytopenia(ITP)is a common hemorrhagic disease,which is an acquired autoimmune disease.After the standard application of conventional treatment,there are still some refractory cases,which will seriously affect the life safety and quality of life.Therefore,in-depth exploration of the pathogenesis of ITP and active search for new therapeutic targets are very important for improving the quality of life of children with ITP.Current studies on the pathogenesis of ITP mainly focus on excessive platelet destruction due to humoral immunity or cellular immunity abnormalities.Studies have shown that autophagy affects platelet generation,but there are few studies on the dysfunction of megakaryocyte generation in children with ITP caused by autophagy.Therefore,this study preliminarily explored the expression level of autophagy related proteins in megakaryocytes in ITP,and explored the correlation between the level of autophagy in megakaryocytes in ITP and clinical factors,providing a new perspective for the treatment of ITP.Method:1.A total of 25 children diagnosed with ITP and other diseases were collected,and 10 children diagnosed with other diseases except iron deficiency anemia and with normal platelet count were collected in the control group.The age and gender of the control group matched the children with ITP(Megakaryocyte lines developed normally in children with iron deficiency anemia with normal platelets),bone marrow fluid was collected and smears were performed.2.Bone marrow smears were stained by immunohistochemical staining,and the positive expression rates of autophagy related proteins LC3Ⅱ and p62 in the whole film were calculated by two doctors in the tumor blood laboratory,and the average was taken.The patients with counting differences of more than 3% were re-examined by microscopy and photographed at the same time.3.SPSS26.0 and Graph Pad were used to analyze the differences in the expression of autophagy related proteins in megakaryocytes of ITP children and control group,and clinical data of ITP children were collected.The expression of autophagy related proteins and clinically relevant factors(age,disease stage,bleeding degree,platelet count,CD4+,CD8+,CD4+/CD8+,CD3+,CD19+,CD56+NK,total lymphocyte count,erythrocyte sedimentation,complement C3,complement C4,CRP,Ig G,Ig A,Ig M,etc.)were analyzed At the same time,children with ITP were divided into platelet membrane antibody GP Ⅱ b/ Ⅲ a positive group and platelet antibody negative group according to whether the platelet antibodies were positive,and the difference of the positive expression rate of autophagy related proteins in megakaryocytes was tested.Results:1.In order to clarify the expression levels of autophagy related proteins in the bone marrow of children with ITP,the expressions of LC3Ⅱ and p62 in the bone marrow smears of ITP patients were detected by immunohistochemical staining.The results show that: Compared with the control group,the positive rates of the autophagy related proteins LC3Ⅱ and p62 in the bone marrow smears of children with ITP were significantly higher than those of the control group,and the difference was significant(LC3Ⅱ 31.0±13.1% in the ITP group and LC3Ⅱ 12.0±3.9% in the control group).p62 in ITP group was 34.0±16.7%,and p62 in control group was13.0±2.0%.P <0.001).2.Pearson bivariate correlation analysis was used for clinical data with continuous normal distribution,and Spearman bivariate correlation analysis was used for clinical data with ordered variables and other non-normal distribution.The correlation between the positive rate of autophagy related protein expression and clinical data in ITP children was statistically analyzed.The results showed as follows:(1)There was a strong positive correlation between the positive expression rate of autophagy related protein LC3Ⅱ in the bone marrow smears of ITP children and the clinical stage of ITP children(r=0.742,P <0.001);There was a moderate positive correlation with Ig A(r =0.520,P<0.05).CD4+(r =-0.679,P<0.001),CD4+/CD8+(r=-0.735,P<0.001),CD3+ cells(r =-0.629,P<0.01)and total lymphocyte count(r=-0.596,P<0.01)was negatively correlated with CD8+,CD19+B CD56+NK,complement C3,C4,age,CRP,erythrocyte sedimentation,bleeding degree,and platelet count.(2)The positive expression rate of p62 was positively correlated with the clinical stage of ITP children(r=0.522,P<0.05);There was a moderate positive correlation with Ig A(r =0.433,P<0.05).CD4+(r =-0.614,P<0.01),CD4+/CD8+(r=-0.560,P<0.01),CD3+ cells(r =-0.543,P<0.05)and total lymphocyte count(r=-0.510,P<0.05)was negatively correlated with CD8+,CD19+,CD56+NK,complement C3,C4,age,CRP,erythrocyte sedimentation,bleeding degree,and platelet count.3.Independent sample t test was used to detect the difference in the positive expression rates of autophagy related proteins between the platelet antibody GPⅡb/Ⅲ a positive group and the platelet antibody negative group in ITP children.The results showed that there was no statistical significance in the positive expression rates of autophagy related proteins LC3Ⅱ and p62 between the positive and negative groups in ITP children(P>0.05).Conclusion:1.Autophagy occurs in megakaryocytes in children with ITP and in children with iron deficiency anemia.2.Compared with the normally developed megakaryocytes,the autophagy expression level of megakaryocytes in the bone marrow smears of children with ITP was abnormal,and the initiation of autophagy was abnormally activated and the process was abnormally blocked.3.In children with newly diagnosed,persistent and chronic ITP,the abnormal level of megakaryocyte autophagy,namely the degree of abnormal activation and process block,gradually increases.4.Autophagy may be involved in the pathogenesis of cellular and humoral immunity disorders in ITP children,and the degree of abnormal autophagy may be related to cellular and humoral immunity disorders.5.Autophagy levels of megakaryocytes in platelet membrane antibody GPⅡb/Ⅲa positive ITP children were not significantly different from those in antibo-negative ITP children. |
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