| Objective:Part ?:Whole blood cell reduction is a very common blood disease among which pathogenesis of IRP is that due to irritation of some unknown pathogen,major antigen presenting dendritic cell subsets become abnormal,which causes regulatory unbalance of the downstream T lymphocytes resulting in B lymphocyte number,subgroup,functional abnormality.This further produces autoantibodies only to bone marrow hematopoietic cells.By mediated macrophage phagocytosis,activated complement in situ hemolysis or functional proteins on the closed membrane of hematopoietic cells,the autoantibodies prevent hematopoietic cell proliferation and differentiation leading to bone marrow failure or ineffective hematopoiesis.It is vital to have correct diagnosis and to distinguish hematological diseases characterized by it and other panhematopenia.Recently,a lot of experimental studies are focused on the relevance of genetic material in the nucleus and hematologic diseases.D-loop region in mt DNA detected from the experiment engage in whether or not the extracellular DNA mutation is relevant to immune hematological system disease and it commits to provide new ideas for the pathogenesis and clinical diagnosis of the disease.Mitochondrion,except for nucleus,it is the only organelle containing DNA in the nucleus of higher animal cells.It does not rely on genome for duplicating,transcription and translation.However.As mt DNA is lack of histone protection,it is exposed to oxidative phosphorylation environment prong to oxygen free radical damage.Since it lacks necessary repair mechanism,its mutation rate is more than 10 times higher than that of the nuclear DNA.Mitochondrial DNA oxidative damage is relevant to human aging,diabetes,cancer risk and disease prognosis.The position of D-loop region is 16028bp-577 bp,between t RNApro and t RNAphe,around 1122 bp.It accounts for 6% of the all mt DNA.As the biggest region of the whole mitochondrial genome sequence,length,genetic variation,it is rich in A-T base group.Mt DNA replication origin of heavy and light chains are located in this region mainly for mt DNA transcriptional regulation and duplication.Nearly all regulatory sequence relevant to mt DNA duplication,transcription and translation are in this region.Thus,it becomes the research hotspots of mitochondrion DNA in recent years.Single nucleotide polymorphism(SNP),It is mainly considered that the polymorphism of the DNA sequence is due to the single nucleotide mutation in the genomic level,the frequency in humans are often more than 1%,is one of the most common genome sequence changes,and the proportion of it is more than 90% in all of the known polymorphisms.So SNP are considered to relate with the species diversity,individual differences in drug treatment,and susceptibility to certain diseases.Because the human genome is linkage disequilibrium,so while most of the SNP will not directly affect the expression of the gene product,but it will still be seen as a kind of genetic markers,which is used to locate some disease-causing changes in the structure or function of the adjacent genes.If the SNP is in promoter of some certain gene,and plays a role in the gene encoding and regulation,the SNP are likely to have an impact on the gene.This time will be study the D-loop region of mt DNA replication control region,the starting and ending point of light chain and heavy chain of mt DNA and the important area bear the regulation of mt DNA transcription and replication,the highly polymorphic genetic of them will have a certain impact on the mitochondria.As a result,we conduct studies on the significance of polymorphism of the peripheral blood lymphocyte mitochondria DNA(mt DNA)D-LOOP region of the immune associated thrombocytopenia,correlation of immune index,and morphological correlation studies of patient bone marrow and peripheral blood cells.Part ?:Prolymphocytic leukemia(PLL)Is a special type of lymphocytic Leukemia,is generally considered as rare Variant of chronic lymphocytic leukemia,but some others believe it is acute lymphoblastic leukemia(type II).Auer corpuscle is a diagnosis morphologic characteristics of acute lymphoblastic leukemia,it has he clinical significance of diagnosis in the detection of acute myeloid leukemia,distribution of acute myeloid leukemia and relationship with the clinical diagnosis.This time what we discussed is that Auer body like inclusions whose morphology is Auer like in the cytoplasm of prolymphocytic leukemia cells.Method: Part ?: 1.Extract the peripheral blood lymphocyte mitochondria DNA of 43 newly diagnosed IRP patients and 41 normal controls and conduct PCR proliferation for the D-LOOP region.Conduct detection of PCR amplified gene sequence of its D-LOOP region by direct and reversing sequencing method.Compare the detected result with Cambridge sequence of human mitochondrial DNA library record and relevant data of Polymorphic Sites sub database of mt DB database.2.Use FACS(flow cytometry)to detect bone marrow hematopoietic cells CD15+?Gly Co A+ and positive rate of cell membrane antibody(Ig G and Ig M)of 43 newly diagnosed IRP patients 3.Use FACS(flow cytometry)to detect the positive rate of peripheral blood B lymphocyte and T lymphocyte subsets of 43 newly diagnosed IRP patients 4.Use Wright's staining method to smear 43 newly diagnosed IRP patients' marrow,count 200 marrow cells and respectively record bone granule,oil drop,proliferative degree,erythroid proportion,morbid hematopoiesis,lymphocyte ratio,megakaryocyte quantity and platelet and so on.5.Use Wright's staining method to smear 43 newly diagnosed IRP patients' peripheral blood,count 100 peripheral blood cells and respectively record its granulocyte proportion,mature erythrocyte and lymphocyte ratio,monocyte ratio and platelet and so on.Part ?:We observe bone marrow cell morphology and cell staining characteristics by microscope,detect bone marrow cells by flow cytometry,and analysis of its components by electron microscope scanning.Result: 1.In the peripheral blood lymphocyte mt DNA D LOOP region of 43 IPR patients,there are totally 110 mutation sites,among which 62 are polymorphic loci,48 are mutation sites in which 14 site are the new mutation sites which is not mentioned in the database.2.In 110 mutation sites,we found 516 base alteration of which the main change is 410 base substitution.The common base substitutions are T/C(184/410)and A/G(113/410)3.In 110 mutation sites,the high frequency mutation sites are 73 and 263.The mutation rate of site 311 is 32/43,of site 310 and 16224 is 27/43,of 16519 is 25/43,of 489 and 16362 is 24/43.4.In relevance statistics between polymorphism of the peripheral blood lymphocyte mitochondria DNA(mt DNA)D-LOOP region and bone marrow mononuclear cells found that in 35 adult group divided by age 18,the lymphocyte cell mt DNA polymorphism of D-LOOP region is significantly relevant to CD15 Ig M?Gly Co A+cell Ig M,CD34+cell Ig G,and CD34+cell Ig M bone of marrow mononuclear cells.5.Bone marrow cell morphology tips for prolymphocytic leukemia,some Auer body like inclusions found in the cytoplasm.It analyses as B lymphocytes by cytochemical staining and flow cytometry analysis,and it is the degeneration of mitochondria with the matrix density increased and the internal structure disappeared by electron microscope scan.Conclusion: 1.The peripheral blood lymphocyte mitochondria DNA(mt DNA)D-LOOP region of IRP patient exists high frequency mutation 2.The main base change type are T/C and A/G 3.The high frequency mutation sites respectively are 73?263?311?310?16224?16519?489 and 16362 4.In adult group(aged over 18),it is obviously relevant to bone marrow mononuclear cell combining autoantibody and it may be an important link of immune associated cell reduction pathogenesis.5.Some Auer body like inclusions found in the lymphatic system in cytoplasm of tumor cell after the degeneration of mitochondria by Wright's stain.Auer is a valuable discovery of body morphology,but not only by morphological,also in relation to other relevant checks further to category type of leukemia cells. |
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