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The Study of Expression of PINCH-1 in Leukemia Stromal Cells and the Influence on Leukemia Bone Marrow Hematopoietic Microenvironment

Author: XuZuo
Tutor: KongPeiYan
School: Third Military Medical University
Course: Internal Medicine
Keywords: Acute leukemia PINCH-1 ILK BIM Cell Adhesion Cell proliferation
CLC: R733.7
Type: Master's thesis
Year: 2010
Downloads: 104
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Abstract

The purpose and significance of bone marrow hematopoietic microenvironment of bone marrow stromal cells, extracellular matrix (of extracellular matrix ECM) as well as a variety of hematopoietic growth factors, extracellular matrix (ECM) and its related cell adhesion molecules (cell adhesion molecules , CAMs) is formed between hematopoietic cells and the basis for interaction between hematopoietic cells and stromal cells. In pathological cases, a variety of factors led to abnormal secretion of extracellular matrix proteins and adhesion molecules, and the formation of abnormal signal transduction, abnormal bone marrow microenvironment and leukemia. Bone marrow stromal cells by secreting a variety of cytokines, and in contact with hematopoietic cells, adhesion to regulate the proliferation and differentiation of hematopoietic cells in abnormal conditions, abnormal secretion of cytokines and abnormal regulation of bone marrow stromal cells, can cause abnormal proliferation and differentiation of hematopoietic cells obstacles. Leukemia is a clonal disease of hematopoietic stem cells, with the deepening of the study, the therapeutic effect of leukemia has been considerable progress, but there are still some cases of refractory and relapsed still incurable; leukemia drug in the treatment process, the bone marrow stromal cells and the adhesion of leukemic cells protect leukemia cells to escape the killing effect of the drug interaction is one of the important reasons that cause residual leukemia cells, drug resistance and relapse. PINCH-1 (Particulary Interesting New Cysteine-Histicline rich protein) protein is a recently discovered protein is closely related with the tumor-associated matrix and adhesion molecules, by 5 LIM (containing Lin-11, ISL-1 and MEC-3 domain) of the protein structure, as well as a C-terminal is constituted. Now been found that there PINCH-1 and PINCH-2 two subtypes, wherein PINCH-1 is involved in cell adhesion to achieve PINCH, the main function of the signal transduction subtypes. PINCH-1 mRNA is widely present in a variety of tissues and cells, PINCH-1 protein is mainly confined to the stromal cell cytoplasm and cell - matrix adhesion connection site. PINCH-1 protein LIM1 domain with the integrin-linked kinase ILK (Integrin-linked kinase) relative to the role, to participate on cell proliferation, differentiation, migration, and other activities through ILK regulation. HT-1080 cells, PINCH-1 is able to inhibit the expression of pro-apoptotic protein Bim cell apoptosis tolerated, Bim cells currently known of BCL-2 inhibition and antagonist in many cell plays a pro-apoptotic role. Detection intervention PINCH-1 BIM whether increased expression of bone marrow stromal cells, then bring increased apoptosis of bone marrow stromal cells, is the way of a possible transformation of the bone marrow microenvironment. PINCH-1 expression and its role in the bone marrow microenvironment in leukemia research at home and abroad has not been reported. PINCH-1 expression after antagonizing Bim, ILK protein expression was observed, this project intends From this point start PINCH-1 protein expression levels in leukemia bone marrow stromal distribution and PINCH-1 expression by specific blockade changes in the situation, and at the same time observe the changes in the proliferation of bone marrow stromal cells. This experiment through the comprehensive study to explore the PINCH-1 leukemia bone marrow stromal cells and their interaction with leukemia cells may be changed, and to provide a theoretical basis for exploring a new perspective to the treatment of leukemia. Materials and Methods 1. PINCH-1 expression and distribution of leukemia bone marrow stromal cells collected 15 cases of acute myeloid leukemia patients, 17 patients with acute lymphoblastic leukemia (both onset leukemia patients), and 30 normal controls bone marrow stromal cells. (1) bone marrow stromal cells cultured normal and acute leukemia patients; (2) RT-PCR method to detect PINCH-1 mRNA expression in normal and acute leukemia bone marrow stromal cells; PINCH-1 protein (3) Western blot assay expression in normal and acute leukemia bone marrow stromal cells, immunofluorescence and immunohistochemistry of PINCH-1 in the bone marrow stromal cells distributed. 2. RNAi by inhibiting the expression of bone marrow stromal cells PINCH-1 collection of patients with primary acute leukemia (acute lymphoblastic four cases, two cases of ANLL) bone marrow samples of six cases: (1) develop acute leukemia bone marrow stromal cells; (2) build for PINCH-1 lentiviral RNA interference vector fragment; (3) a lentivirus vector RNAi silence PINCH-1 expression; (4) RT-PCR was used to detect the RNAi bone marrow stromal cells before and after the intervention of acute leukemia bone marrow stromal cells PINCH -1 mRNA expression changes; (5) Western blot assay RNAi silencing PINCH-1 before and after acute leukemia bone marrow stromal cells PINCH-1, Bim, ILK protein expression changes; (6) MTT assay RNAi silencing PINCH-1 before and after acute leukemia bone marrow stromal cell proliferation changes. Results 1. Acute leukemia bone marrow stromal cells PINCH-1 expression was significantly higher than that of normal bone marrow stromal cells: in acute lymphoblastic leukemia and acute non-lymphocytic leukemia in patients with bone marrow stromal cells, PINCH-1 mRNA and protein levels than normal bone marrow stromal cells increased (P lt; 0.01); acute lymphoblastic leukemia PINCH-1 protein expression was significantly higher than that of acute non-lymphocytic leukemia (P lt; 0.01), but mRNA expression was no significant difference; 2. immunocytochemistry and immunofluorescence PINCH-1 protein expression in the cytoplasm, the nucleus showed no expression; 3. successfully constructed lentiviral vector fragment of RNA interference; 4. lentiviral vector RNAi silencing of bone marrow stromal cells in the bone marrow stromal cells PINCH-1 expression, silence, then: (1) of PINCH-1 expression at the RNA and protein levels were significantly reduce; (2) BIM protein expression increased; (3) ILK protein expression reduced; (4) Inhibition leukemia bone marrow stromal cell proliferation activity was significantly decreased. Conclusion 1. Leukemia bone marrow stromal cells exist PINCH-1 expression; 2.PINCH-1 may by ILK formation of metastable structures involved in the regulation of leukemia bone marrow stromal cell adhesion function 3.PINCH-1 may promote leukemia proliferation of bone marrow stromal cells and thus participate in the formation of the abnormal bone marrow microenvironment leukemia, this effect may be related to PINCH-1 inhibition Bim proapoptotic role. leukemia bone marrow stromal PINCH-1 expression may stromal cells by inhibiting withered apoptosis and promote proliferation of the change matrix support and protect the role of the leukemia cells, thereby contributing to the malignant proliferation of leukemia cells and extensive infiltration

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