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Role of Advanced Glycation End Products and Their Receptors System on Adventitial Fibroblast Function

Author: LiuYaYang
Tutor: WuZongGui
School: Second Military Medical University
Course: Internal Medicine
Keywords: Advanced glycation endproducts Advanced glycation endproducts receptor Vascular adventitial fibroblasts Candesartan Galectin -3 Small RNA interference
CLC: R587.2
Type: PhD thesis
Year: 2009
Downloads: 233
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Abstract


Adventitia through direct or indirect effects on vascular structure and function of regulation play an important role in regulating. Vascular adventitial fibroblasts (AF) is the major cellular components of vascular adventitia in the development of atherosclerosis, poor vascular remodeling and vascular restenosis play an important role. Advanced glycation end products (AGEs) and AF in diabetes-related complications, tissue AGEs concentration and atherosclerosis plaque severity, AGEs direct effect as well as its effect upon binding to the receptor, can cause cause arterial atherosclerosis and a variety of factors related to cell activation. Diabetic patients and elderly population adventitia of AGEs gradually accumulate on the outer membrane of cells such as AF impact, but its internal mechanism is poorly understood, the study of cell migration from the AF AGEs, oxidative stress and inflammation related aspects are discussed in . Main content divided into five parts: The first part of advanced glycation end products on vascular adventitial fibroblasts glycation endproducts receptor expression Objective: To investigate the AF AGEs advanced glycation endproducts receptor (Receptor for Advanced Glycation Endproducts, RAGE) expression. Methods: Organization paste block culture of SD rats AF. Fluorescence immunocytochemistry cells. Cell synchronization process, different concentrations (50,100,150,200,300 μg / ml) for advanced glycation of human serum albumin (Advanced Glycation Endproducts-Human Serum Albumin, AGE-HSA) intervention for 24 hours to 200μg / ml human serum albumin (Human Serum Albumin, HSA) as a control. By RT-PCR and Western-blot detection of RAGE gene and protein expression. Results: 50,100,150,200,300 μg / ml respectively of AGE-HSA increased RAGE mRNA and protein expression, and the expression of RAGE 150μg/ml, 200μg/ml, 300μg/ml and control groups significantly different (P lt ; 0.05), 200μg/ml reached the peak (P lt; 0.05). This effect can be p38 antagonist, ERK1 / 2 antagonist, JNK antagonist, candesartan suppressed. Conclusion: AGEs can increase AFs RAGE expression candesartan can inhibit this effect. The second part of advanced glycation endproducts promote vascular adventitial fibroblast migration mechanism and the intervention effect of candesartan Objective: To observe the migration of AGEs on AF, its mechanism and the concept of Chakan candesartan rats. Methods: Organization paste block culture of SD rats AF, with the transwell chamber detection AGE-HSA for AF migration. Western blot technique to observe AGE-HSA for AF mitogen-activated protein kinase (MAPK) pathway phosphorylation affected. Results: AGE-HSA can promote p38, ERK1 / 2, JNK phosphorylation, JNK in 20min, p38, ERK1 / 2 peaked at 30min (P lt; 0.05). p38 antagonist, ERK1 / 2 antagonist, JNK antagonists were inhibited p38, ERK1 / 2, JNK phosphorylation, RAGE neutralizing antibody, candesartan inhibits p38, ERK1 / 2, JNK phosphorylation. Different concentrations of AGE-HSA (50,100,150,200,300 μg / ml) may promote vascular adventitial fibroblast migration, 200μg/ml reached the peak (P lt; 0.05). RAGE neutralizing antibody, p38 antagonist, ERK1 / 2 antagonist, candesartan can inhibit AGE induced fibroblast migration (P lt; 0.01). Conclusion: AGEs via RAGE affect MAPK pathway promote vascular adventitial fibroblast migration, candesartan by blocking this pathway inhibits vascular adventitial fibroblast migration, which may be a new mechanism of vascular protective effects. The third part of advanced glycation end products on vascular adventitial fibroblast activation mechanism of oxidative stress Objective: To observe the AF AGEs oxidative stress, and to explore its mechanism. Methods: Organization paste block culture of SD rats AF, with DCFH-DA detection AGE-HSA for AF ROS expression. By RT-PCR, Western-blot detection of NADPH oxidase subunit p22phox, p47phox expression. Results: AGE-HSA, respectively from the gene and protein levels increased p22phox, p47phox expression. Oxidase inhibitor DPI (diphenyliodonium), APO (apocynin), anti-RAGE neutralizing antibodies can be suppressed candesartan stimulated by AGE-HSA p22phox, p47phox upregulated (P lt; 0.05). AGE-HSA can promote the upregulation of ROS. Oxidase inhibitor DPI, APO, anti-RAGE neutralizing antibodies can be suppressed candesartan stimulated by AGE-HSA upregulation of ROS. CONCLUSION: AGE-HSA via RAGE affect ROS and p22phox, p47phox upregulation, ROS upregulation and p22phox, p47phox expression. NADPH oxidase inhibitors and candesartan can be reduced p22phox, p47phox AF decreased expression of ROS generation. The fourth part of the advanced glycation endproducts promote vascular adventitial fibroblasts and inflammatory response mechanisms candesartan Objective: To observe the effects of AGEs AF inflammatory mechanisms and candesartan rats. Methods: Organization paste block culture of SD rats AF, RT-PCR detection of MCP-1, IL-6, VCAM-1 mRNA expression, ELISA detection of supernatant MCP-1, IL-6, VCAM-1. Western-blot and EMSA detected NF-κB and I-κB-α. Results: AGE-HSA 200μg/ml role of AF after 0.5h, 1h, 2h nuclear NF-κB transcriptional activity was enhanced role after 0.5h, 1h I-κB-α phosphorylation. AGE-HSA 200μg/ml deal to promote NF-κB nuclear transfer, plus candesartan inhibits NF-κB nuclear shift, I-κB-α phosphorylation also be candesartan suppressed. Different concentrations of AGE-HSA (50,100,200,300 μg / ml) can make the AF IL-6, VCAM-1, MCP-1 mRNA upregulation, 50,100 μg / ml compared with the control group mRNA levels were significantly different (P lt; 0.05), 200,300 μg / ml mRNA expression compared with the control group, with significant difference (P lt; 0.01). With RAGE neutralizing antibodies, NF-κB inhibitor MG-132, candesartan pretreatment reduced the AGE-HSA induced IL-6, VCAM-1, MCP-1 mRNA expression and protein concentration in the supernatant ( P lt; 0.05). CONCLUSION: AGE-HSA through RAGE, NF-κB pathway promote inflammation and upregulated. Candesartan can effectively inhibit I-κB-α phosphorylation, NF-κB nuclear transfer, expression of inflammatory cytokines, suggesting that it has a role in the treatment of vascular complications of diabetes. The fifth part adventitia fibroblasts galectin-3 expression and galectin-3 RNA interference Objective: To observe the AF galectin -3 (galectin-3) expression of galectin-3 RNA interference construct lentiviral vector observation AGEs galectin-3 receptor on vascular function of fibroblasts. Methods: RT-PCR detection of galectin-3 expression, RNA interference target sequence design, synthesis short hairpin RNA galectin-3 was cloned into vector, restriction enzyme analysis and sequencing recombinant vector. Work carrier and packaging plasmid were transfected 293T cells 72 hours after the collection of lentiviruses, adding cultured vascular fibroblasts. Western blot analysis of galectin-3 expression after. Results: 50,100,150,200,300 μg / ml of AGE-HSA, respectively from the gene and protein level increased expression of galectin-3. Galectin-3 screening effective target for RNAi, galectin-3 was successfully constructed a small RNA interference lentiviral vector, which can effectively transfected AFs, protein galectin-3 to achieve effective knockdown. Conclusion: The constructed lentiviral vector can effectively inhibit adventitial fibroblasts galectin-3 expression.

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CLC: > Medicine, health > Internal Medicine > Endocrine diseases and metabolic diseases > Islet disease > Diabetic coma and other complications
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