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The Mechanism Of Lipid Rafts And Receptors Involved In Trans Fatty Acids Induced Apoptosis Of Human Umbilical Vein Endothelial Cells

Posted on:2015-05-22Degree:MasterType:Thesis
Country:ChinaCandidate:H RaoFull Text:PDF
GTID:2284330422977334Subject:Nutrition and Food Hygiene
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Cardiovascular disease is the high mortality and morbidity disease in the world.In China, there was a population of260million suffered obesity and high bloodpressure and there were300million people die from cardiovascular disease for oneyear, especially atherosclerosis. Chinese residents have eaten too many fat that farmore than the Nutrition Society recommended levels. Hydrogenated oils as thecommon oil used in food processing had a high content of trans fatty acids. It hasbeen shown that a high intake of TFAs could cause endothelial injury by elevatedserum LDL and TG in vascular, also by increased the soluble intercellular adhesionmolecule-1(SICAM-1), soluble vascular cell adhesion molecule-1(SVCAM-1) andE-selectin (E-selectin) expression. Although many studies have reported therelationship between TFAs and cardiovascular disease, but the mechanisms of TFAsimpact on vascular remain unclear. Some scholars have found that elaidic acid(9t18:1) and linolelaidic acid (9t12t18:2) which were widespread in the Chinese dailydiet would induce vascular endothelial cell apoptosis and promote endometrialinflammatory cytokine release by Caspase pathways. Epidemics evidence has beenconfirmed that the mechanisms of inducing AS by these two TFAs respectively hadsignificant differences. Recent studies indicated that the lipid rafts were rich in kindsof lipids and some components which related to signal transduction and might be aseffective signal transduction and provide a platform for "cross talk" of various signaltransduction pathways. It has been reported that polyunsaturated fatty acids such aseicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) might change thestructure of lipid rafts. DHA could also regulate interleukin-2-signal pathway bychanging the lipids component of lipid rafts. So we supposed that the impact of transfatty acids on the cells may related to the structure and function of lipid rafts. On theone hand, trans fatty acid induce cell apoptosis by altering the fatty acid andphospholipid composition of lipid rafts, on the other hand trans fatty acid activeddeath receptors on the lipid rafts. In our study, we will compare the effect of TFAs oncell signaling before and after when lipid rafts were disrupted by agents and to confirm the lipid rafts and their receptors as the key role in TFAs inducing apoptosis.This research would provide a scientific basis for the prevention and treatment ofcardiovascular disease.First, the reasonable time of lipid raft disrupting agents must be determined toestablish lipid rafts damage model. It would not cause morphological andphysiological changes of cells under this reasonable time. We observed the changes ofcell viability by MTT assay and tested the apoptosis and cell cycle by flow cytometryafter9t18:1or9t12t18:2supplement to cells and non-rafts cells. Furthermore, usingWestern-Blot method to test the expression levels of apoptosis-related proteins(caspase-3,-8,-9, Bax, Bcl-2, Bid, p53, etc.) and cell-cycle-related proteins (p21,p27, CDK2, CDK4, CyclinD1, etc.) to investigate the effect of lipid rafts on TFAsinduced apoptosis and cell cycle changes. According to the preliminary results, weanalyzed the expressions of death receptors on lipid rafts by Western-Blot method,and then using the fluorescence microscope to observe the death receptors on lipidrafts, and we also analyzed the changes of fatty acid composition by efficient gaschromatography after TFAs treated to cells and non-rafts cells. Taken together, it canbe concluded that apoptosis receptor pathway might involve in TFA inducedapoptosis of HUVEC. Meanwhile, the role of lipid rafts involved in different kinds ofTFAs induced physiological changes have been studied by using same method asabove. The main research results obtained are as follows:1. Lipid rafts damage models have been successfully established. Lipid raftdisrupting agent (MβCD) would not cause significant changes of cell viability whenMβCD treated on HUVECs at a concentration of10mM for less than60min.Otherwise it would cause changes in cell morphology even cell death.The membranecholesterol have significantly reduced after treated for40min. The expression ofmarker proteins (caveolin) on lipid rafts decreased significantly when MβCD treatedon HUVECs for50min. Taken together, the reasonable time (40-60min) of MβCDtreated on lipid rafts can be obtained. Under this reasonable time, MβCD can disruptthe function of lipid rafts but not influence other physiological functions.2. Lipid rafts involved in TFAs induced cell apoptosis. TFAs would decrease thecell viability and induce apoptosis. However, compared with treated on normal cells, the cell viability increased and the number of apoptotic cells decreased after TFAtreated on non-rafts cells. The expression of apoptosis-related proteins (caspase-3,-8,-9, Bax, Bcl-2, Bid, p53etc.) decreased, especially linolelaidic acid treated whenthese two TFAs at same concentration (50μmol/L). It is indicated that lipid raftsinvolved in TFAs induced apoptosis, and lipid rafts had stronger influence onlinolelaidic acid induced apoptosis of HUVECs.3. Lipid rafts involved in TFAs induced cell cycle change.9t18:1or9t12t18:2could accelerate non-rafts cells from the G0/G1phase to S phase, so that thepercentage of proliferating cells increased significantly. Lipid rafts also had effect oncycle-related proteins (p21, p27, CDK2, CDK4, CyclinD1etc.) expression levels.4. Both elaidic acid and linolelaidic acid could increase the expression ofinflammatory cytokines, such as ICAM-1, VCAM-1and E-selectin. Linolelaidic acidshowed a stronger pro-inflammatory effect on HUVECs under the sameconcentrations. After removal of lipid rafts, inflammation factor expression wassignificantly reduced, which indicating that disruption of lipid rafts could inhibitinflammation.5. Fas apoptosis receptor clustered on the lipid rafts after elaidic acid stimulatedendothelial cells. After removal of lipid rafts, the expression of Fas apoptosis receptorand its ligand decreased and its downstream apoptotic protein expression was alsoreduced. So we supposed that elaidic acid stimulated lipid rafts to make Fas receptorbinding with its ligand FasL and then activate the downstream apoptotic pathwayinduced apoptosis.
Keywords/Search Tags:lipid raft, elaidic acid, linolelaidic acid, HUVEC, receptor, signalpathway
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