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Study of the Molecular Mechanism of Integrin-mediated Signaling during Neutrophil Directed Migration

Author: WangGang
Tutor: ZhangYanMing
School: Northwest University of Science and Technology
Course: Preventive Veterinary Medicine
Keywords: Cell Migration integrin β1 andβ2 subunits gene cloning eukaryotic expression prokaryotic expression protein purification protein interaction
CLC: S852.33
Type: Master's thesis
Year: 2009
Downloads: 36
Quote: 0
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Abstract


As the first line of host defense that prevents host body from being infected, neutrophils are one of important parts of innate immune system. As the key cell surface adhesion factors and cell signal transduction receptors, integrins express on all kinds of leucocytes and play an important role in cell adhesion and bi-directional cellular signal transduction. Before stimulation, neutrophils are in an inactive stage and their integrins have low affinity to extracellular matrix. When stimulated, neutrophils became active immediately, followed by upregulated integrins affinity to their lagand. This promotes neutrophils adhering to and transmigrating through vascular endothelium and reaching the areas of inflammation, where they mediate phagocytic and immune functions. However, excessive neutrophil accumulation and phagocytisis due to misregulated integrin activities can cause profound tissue damage such as chronic inflammation, allergy and autoimmune diseases. Thus, it is important to study of fundamental role of integrins during neutrophil adhesion and migration.In the study, using molecular biology and cell biology approaches, we study the molecular mechanism of integrin-mediated signal during neutrophil migration and get the following results.（1）Integrinβ1 andβ2 subunits have different locations during neutrophil migration by immunofluence,β1 locate to front of neutrophils, whileβ2 moves to the back of cells.（2）To make sure the location of integrinβ1 andβ2, we construct pEYFP-N1-β1 and pEYFP-N1-β2 eukaryotic expressive plamids, then construct pWPXLd-β1 and pWPXLd-β2 lentivirus plasmids and transfect HL-60 cells successfully. We got the same results as immunofluorescence data.（3）To find the moleculars which integrins interact with during neutrophil migration, intracellular region of integrinβ1 andβ2 subunits were amplified by PCR and subcloned into pGEX-4T2 vector to constuct GST-β1 and GST-β2 fusion protein express plasmids, then transfered to E.coli BL21（DE3）and induced with IPTG. Finally, we got high purity and soluble GST-β1 and GST-β2 fusion protein purfied by Glutathione SepharoseTM 4B. （4）In the following GST Pull-down assay, MLC and RhoA, which are the critical molecular for cell migration, bind to integrinβ1 andβ2 subunits intracellular region.

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