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The Screening of Tumor Specific PK-M2and Study on Its Basic Characterisitcs in Acute Leukemia

Author: CuiYaJuan
Tutor: WangGuangPing
School: Central South University
Course: Clinical
Keywords: Acute leukemia Proteomics Pyruvate kinase M2 Expression Basic characteristics
CLC: R733.71
Type: Master's thesis
Year: 2013
Downloads: 4
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Abstract


Background and Purpose:Acute leukemia (AL) is a hematopoietic malignant disease caused by stem cell clones proliferation, but its diagnosis and treatment are still not specific since the pathogenesis of leukemia is unkonwn so far in detail. Therefore, it is great important to strengthly study the pathogenesis mechanism and find out leukemia-specific and/or related molecular markers for the accurate diagnosis and targeted therapy of AL. In view of this reason, in this study, differentially expressed proteins in CD34-CD13+cells from acute myeloid leukemia M2-type (AML-M2) were firstly screened, and the expressions, and other basic characteristics of the tumor specific pyruvate kinase isozyme type M2(PK-M2) in AL, which was one of the differentially expressed proteins picked out from the CD34-CD13+cells, were further analyzed. We believe that this study might set a foundation for further study the role of PK-M2in the pathogenesis and diagnosis of ALMethods:Firstly, magnetic activated cell sorting (MACS) method was applied to sort the CD34-CD13+cells from AML-M2patients and normal subjects followed by detecting the purity of the sorted CD34-CD13+cells. Subsequenly, differentially expressed proteins in the CD34-CD13+cells between AML-M2patients and normal subjects were screened by proteomic technique. Finally, the expressions, distribution, plasma levels and activity of tumor specific PK-M2, one of the differentially expressed proteins picked out from the CD34-CD13+cells in AL were further studied by fluorescence real-time quantitative reverse transcrption-polymerase reaction chain (RT-PCR), Western blot, immunohistochemistry and ELISA.Results:After sorting, the average purity of CD34-CD13+cells was96%for AML-M2patients and90%for normal subjects.2-D DIGE proteomic results showed that21proteins dots were found to be different expression with1.1-fold change at least in the CD34-CD13+cells between the patients and normal subjects, and the mass fingerprints of15proteins dots were obtained, Among, PK-M2was one of the incresed expression protein in patient CD34-CD13+cells. Real-time quantitative RT-PCR results showed that, compared with the normal group, PK-M2mRNA expression in AML and ALL patients were higher with11.43-fold and7.72-fold increase, respectively. Statistical analysis showed that the expression of PK-M2mRNA between AML and ALL patients with normal subjects was significant different with P values of0.002and0.028, respectively. Similarly, the protein expression of PK-M2also increased in AML and ALL patients compared with the normal subjects. Immunohistochemistry analysis showed that PK-M2was mainly expressed in the cell nucleus, and there was less or no expression in nucleolus and cytoplasma, the positive rate of PK-M2expression in AML patients was87.5%,84.6%in ALL and no expreesion in normal subjects. Moreover, the plasma PK-M2level for AML patients was (10.54±15.58) IU/ml, but ALL patients and normal control group were unable to detect PK-M2in plasma. Furthermore, plasma PK activity was (1.0889±0.9335) mU/ml,(0.9736±0.4145) mU/ml and (1.8475±0.3597) mU/ml in AML, ALL and normal group, respectively. There was statistically significance between AML and ALL patients with the normal group (P=0.012, P=0.000), but no statistically significance between AML and ALL patients (P=0.703).Conclusions:Tumor specific PK-M2with increased expression in CD34-CD13+cells from the patients with AML-M2was successfully screened out by proteomics technique. The high expression, both at the level of mRNA and protein, and nuclear distribution and decreased plasma activity in AL may mean that the tumor specific PK-M2plays an important role in the the pathogenesis and the diagnosis of AL

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CLC: > Medicine, health > Oncology > Hematopoietic and lymphoid neoplasms > Leukemia > Acute leukemia
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