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The people SR-PSOX/CXCL16 gene transfection and silence , and its impact on atherosclerosis -related functions

Author: QuanZhiHua
Tutor: YangYongZong
School: Nanhua University
Course: Pathology and Pathophysiology
Keywords: atherosclerosis SR-PSOX lentiviral expression vector RNA interference
CLC: R543
Type: PhD thesis
Year: 2007
Downloads: 313
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Abstract


BackgroundAtherosclerosis(AS) is a multi-factor-induced chronic disease. Recent studies demonstrated that chemokine and its receptor are involved in the pathogenesis and development of many cardiovascular diseaes,such as AS, myocarditis and cardiomyopathy. It is encouraging to find that inhibition of the immunity of chemokine and its receptor can inhibit the development of AS. SR-PSOX(scavenger receptor that binds phosphatidylserine and oxidized lipoprotein),which is the same molecule as CXCL16(CXC chemokine ligand 16), initially cloned in the cDNA bank of PMA-induced macrocyte, is a new member of chemokine-αfamily, existing as member-binding and two-protein-secreting forms. SR-PSOX is up to today the second member-binding chemokine with extracellular chemokine domain binding musin-like stem, structurally similar to irregular chemokine(Fractalkine).Consisting of Clig-like transmembrane domain and cytoplasma-tail like structure.SR-PSOX/CXCL16 is mostly expressed in antigen-presenting cells such as dendritic cells and macrocyte. Immunohistochemistry and quantitative reverse transcription PCR showed that SR-PSOX/CXCL16 are expressed in macrocyte, arterial SMC and vascular endothelial cells in AS, rheumatic valvular disease and infective endocarditis, but not in normal arterial tissues. And its receptors CXCR6 is expressed in CD8+ T cells, NK cells and CD4+ T cells. SR-PSOX/CXCL16 can strongly chemoattracts activated CD8+ T cells amounting to 40%, but less strongly chemoattracts activated CD4+ T cells. SR-PSOX/CXCL16 binds its receptor superselectively, and induces intracellular Ca2+ flow. Like other scavenger receptor family member, SR-PSOX/CXCL16 ,with the help of its chemokines participates in the macrophage and antigen capture of bacteria and virus by T cells, thus initiating immune response. Therefore, SR-PSOX/CXCL16 is a multi-functional molecule, acting as a link between scarenger receptor and chemokine family. At present, domestic research about its functions is inadequate, and controversies still exists about its role in the pathogenesis of AS. In this paper,consisting of three parts,is to perform functional analysis of SR-PSOX/CXCL16.Part 1 The expression of SR-PSOX/CXCL16 in the peripheral blood in ACS patientsObjectives: To observe the expression of chemokines in the peripheral blood in ACS patients, and investigate its role in the mechanism of coronary heart disease.Method: We choose 40 ACS patients,20 AMI,20 UAP and 20 normal controls. Monocytes in the peripheral blood are extracted,and we compare the change of expression of CXCL16/ SR- PSOX mRNA using RT-PCR, withβ-actin as internal control. We compare the expression of CXCL16/ SR- PSOX in the ACS subgroups, using western-blot to analyze protein expression levels, and then do statistical analyze protein expression levels, and then do statistical analysis. Wax slides are made from infective endocarditis, cirrhosis, lung cancer patients and normal controls. And the expression of CXCL16/ SR- PSOX is analyzed with co-focal microscopy.Results: The expression of CXCL16/ SR- PSOX mRNA and protein in the monocytes of peripheral blood in ACS patients is significantly higher than normal control group(P<0.05), however, there is no significant difference of CXCL16/ SR- PSOX expression between UAP group and AMI group.(P>0.05). Immunofluorescence showed that there are mild expression of SR-PSOX in normal vascular endothelial cells, and enhanced expression in every layer of the infected vessels, and spreading from endothelial cells to surrounding tissues as infection worsens. Co-focal microscopy showed that the expression of SR- PSOX is enhanced and also existent in the infiltrated lymphocytes in the cirrhosis, the expression level is proportionate to the degree of inflammation in hilum and lobuli.Conclusions: The expression of CXCL16/ SR- PSOX in the monocytes of the peripherial blood in ACS patients is significantly higher than in the control group.(P<0.05), CXCL16/ SR- PSOX-mediated inflammation may contribute to the pathogenesis of ACS and CXCL16 may play an important role in the pathogenesis and development of As in humans. Part 2 Construction and functional analysis of a lentiviral expression vector containing a scavenger receptor(SR-PSOX) that binds uniquely phosphatidylserine and oxidized lipoproteinObjectives: To construct a lentiviral expression vector containning a scavenger receptor (SR-PSOX) that binds uniquely phosphatidylsedne and oxidized lipoprotein with the six histidines tag in order to investigate the function of SR-PSOX in atherosclerosis.Method: To enable directional cloning, the forward primer of SR-PSOX contains the sequence, CACC, at the 5’end of the primer. And the reverse PCR primer was removed the native stop codon. The interest blunt-end sequence was amplified using RT-PCR strategy and was performed directional TOPO Cloning reaction following the manufacturer’s recommendations. Use a combination of the CMV forward primer and the reverse primer of SR-PSOX, the correct clones were determined by PCR and sequencing. The ViraPowerTM Packaging Mix and SR-PSOX-pLenti6 /V5 TOPO expression plasmid were cotransfected into the 293FT cell line with help of LipofectamineTM 2000 Reagent, 48 hours after cotrans- fection, viral supernatant was collected、concentrated and determined the titer. Using SDS-PAGE,Western blotting and indirect immunofluorescence assay, then we tested the expression of endogenic and recombinational SR-PSOX and TNF-αprotein respectively in the multiplicate foam cell models at the different times by incubating with 50 mg/ L oxLDL and viral supernatant. Results: It was confirmed by DNA sequencing that RT-PCR product of SR-PSOX is according with the data in GeneBank, there is not incorrect and base mutation in the orientation of SR-PSOX-pLenti6/V5 TOPO recombinant. The expressions of SR-PSOX and TNF-αproteins were upregulated in the foam cell models by Western blotting and indirect immunofluorescence analysis.Conclusions: Our data suggested that over expression of recombinational human SR-PSOX protein can promote the forming of foam cells and upregulate the expression of inflammation factor (TNF–α) protein .Part 3 The construction of clip SiRNA lentiviral vector and its effect on the expression of SR- PSOX in humansObjectives: To construct the clip SiRNA lentiviral vector to clarify its functions.Method: The study is to construct clip SiRNA lentiviral vector by gene cloning and then to construct the shRNA lentiviral vector using LR recombinant technique after sequencing.We obtained proper recon with PCR identification and package it into viral host 293FT cells using liposome-mediated transfection.And collect supernatant to infect HepG2 cell in vitro.We detect the expression of SR- PSOX using RT-PCR,indirect immunofluorescence and western-blotting.Results: shRNA lentiviral vector can reduce significantly the expression of SR- PSOX mRNA and protein by 80%,and effectively silence SR- PSOX gene in cultured cells.Conclusions: The construction of SR- PSOX-siRNA lays foundation for the study of the function of the gene.

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CLC: > Medicine, health > Internal Medicine > Heart, blood vessels ( circulatory ) disease > Vascular disease
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