| BackgroundCoronary atherosclerotic heart disease, a kind of disease caused by myocardial ischemia and hypoxia, is lead by vascular stenosis because of coronary atherosclerosis. It is the most common type of organ injury lead by atherosclerosis. The morbidity of this disease has been increasing year by year, and it’s a common disease which is seriously harmful to human health. It’s a kind of disease caused by a variety of factors, such as age, gender, blood pressure, blood lipids, blood glucose, genetic, etc, are all considered relevant to this disease, so the pathogenesis of coronary heart disease has being explored for the recent decades in the medical field. Early studies showed that the most important reason of coronary atherosclerotic heart disease was atheromatous plaque coming into arterial intima, blood lipid deposition in the arterial intima was the the main lesion characteristics. For this reason, researchers believed the deposition of blood lipid in the arterial lead to atherosclerosis, which was the traditional theory of "lipid deposition". Hyperglycemia was considered to be an important risk factor of coronary heart disease, but only about50%of the coronary heart disease patients showed increased blood lipids. Further studies showed that the atherosclerosis was not only a pure fat deposition disease, chronic inflammation mechanism of organism played an important role in the formation and development of atherosclerotic plaque. In the90th of the last century, Dr.Russell Ross proposed the "injury" theory for the first time, he believed that atherosclerosis was a chronic inflammation in the vascular intima, this theory improved people’s understanding of the mechanism of atherosclerosis greatly. The early stage of atherosclerosis plaque formation begins from functional or morphological endothelial injury by a series of mechanical, chemical stimulation, which increased expression of cell adhesion molecules, synthesis of cytokines such as interleukin-8(IL-8), monocyte chemotactic factor-1(MCP-1),etc. Monocytes and macrophages were then activated and accumulated in the arterial wall, the next, fat accumulated, growth factor (GF) and cytokine released and smooth muscle cell and lymphocyte activation. The plaques gradually grew and matured with the process continuing. Platelets may aggregate in the place endothelial cells damaged. When the fatty cell died, the core part of plaque formed, and the plaque remodel and became fibrosis. Inflammatory mechanism mediated by macrophages, endothelial cells, smooth muscle cells, platelets and cells factors such as cytokines, growth factors, tumor necrosis factor and oxygen free radical released by these cells involved in the pathogenesis of atherosclerosis, promotes the development and deterioration of atherosclerosis. Inflammatory reaction plays a key role in the course of atherosclerosis, and be relevant to the prognosis of coronary atherosclerotic heart disease.Research showed that, inflammation is very active in atherosclerotic plaque, there are lots of inflammatory cells such as monocytes, macrophages, dendritic cells and T lymphocytes infiltrate. Active inflammatory reaction can make the stable atheromatous plaque change into unstable plaque, this is the cause of acute thrombosis in coronary artery. So that, blocking the inflammatory pathway became the hotspot and difficulty of coronary heart disease and immune therapy.Programmed death ligand1(PD-L1、B7-H1or CD274) is a kind of type I transmembrane protein composed of290amino acids, originally found high expressed in tumor cells, so it was assumed to relate to the tumor cell invasion. But with the deepening of the study,people found that in addition to tumor cells outside, PD-L1is expressed in various tissues and inflammatory cells such as vascular endothelial cells, kupffer cells, stellate cells, dendritic cells, bone marrow-derived mast cells, placental trophoblast cells, lymphocytes, macrophages, etc. In addition to tumor cells invasion, PD-L1have a close relationship with many pathological processes, such as microbial graft rejection, autoimmune diseases, infection (viral infection), etc. Recent studies suggested that as a member of the B7family molecules, the receptor for PD-L1is programmed death1(PD-1或CD279), there are two tyrosine residues in the cytoplasmic tail of PD-l,the tyrosine residues at N side takes part in an immunoreceptor tyrosine based inhibitory motifs(ITIM), the tyrosine residues at C side takes part in an immunoreceptor tyrosine-based switch motif(ITSM), the ITSM is highly conserved, prompt its important function. When PD-1combinate with its ligand, the occurrence of ITSM phosphorylation, make the downstream effectors dephosphorylation, play a negative regulatory role. PD-L1is the ligand of PD-1, play immunomodulatory effect when combined with PD-1. Research showed that the role of PD-1/PD-L1signal system on immune function, also associated with chronic inflammation of atherosclerosis. Gotsman detected the expression of PD-L1in atherosclerotic plaques by immunofluorescence technique in study of coronary artery plaque. The APC extracted from PD-L1, PD-L2, LDL receptor three gene knockout (triple knockout, TKO) mice shows a strong stimulus to the inflammatory reaction of T cells on ox-LDL, compared with the LDL receptor knockout mice, the damage at aortic arch area, descending aorta increases3times in TKO mice. Lee and his colleagues found that in patients with coronary heart disease, T lymphocyte phenotype of PD-1and PD-L1phenotypes of peripheral blood dendritic cells significantly decreased than healthy people. Our research group previous studies have shown that in patients with stable angina and acute coronary syndrome the expression of PD-L1protein in peripheral blood T lymphocytes is higher than that of normal control group, for this reason we conjecture that PD-L1plays an important role in the formation process of atherosclerotic.The integrity mechanism of expression of PD-L1protein in cells and PD-L1proteins play a role in immune regulation is still unclear. The different research objects or different stimuli, lead to different result. To sum up, the signal pathway that may influence on the expression of PD-L1are JAK/STAT signal pathway, PI3K/Akt signal pathway, MEK/Erk signal pathway, NPM/ALK signal pathway, MAPK signal pathway,etc. and transcription factor such as IRF-1, STAT-3may also take part in this physiological process. p38MAPK signal pathway is an important branch of MAPK pathway, It changes in gene expression through phosphorylation of transcription factors, participate in a variety of intracellular information transfer process, can widely react to extracellular signal, mediate the process of cell growth, development, differentiation and death. Our research group previous studies have shown that, p38MAPK pathway is involved in the regulation of expression of PD-L1protein in dendritic cell differentiation. But there is not relevant research on vascular endothelial cells which play a key role in the initiation and progression of atherosclerosis by now. For this reason, we plan to take human umbilical vein endothelial cells were cultured in vitro as the object of this study, simulate the inflammatory process by stimulating with inflammatory cytokines (TNF-α), observe the change of PD-L1expression on the process of human umbilical vein endothelial cells Influenced by TNF-α, make clear the effect of TNF-α on the equression of PD-L1protein firstly, then block the p38MAPK patlway with P38specific inhibitor SB203580. further inquiry the relationship between expression of PD-L1protein and p38MAPK signal pathway, probe into the molecular mechanism of TNF-α induced PD-L1protein expression in human umbilical vein endothelial cells, improve our understanding of negative costimulatory signals (PD-1/PD-L) mechanism to control chronic inflammatory process, to provide theoretical foundation for the study of immune mechanism of atherosclerosis.Part1The ideal concentration of TNF-a inducing human umbilical vein endothelial cells by CCK-8experimentObjective:To study the effect of TNF-α on the activity of human umbilical vein endothelial cells in different concentrations, select concentration gradient of TNF-α which is effective and significantly different.Object and methods:1. Object Healthy human umbilical vein endothelial cells cultured in vitro(HUVECs)2. Methods2.1Isolation and culture of human umbilical vein endothelial cellsTake neonatal umbilical cord about25centimeters long in aseptic condition (Provided by the First Affiliated Hospital of Zhongshan University, puerpera informed consent, all the operation complied with the ethical standards in the Helsinki declaration), cut off the clamps at both ends, extruded the residual blood, repeated washing with PBS until the fluid is colorless. Pinch the extremitas anterior of umbilical veins,0.1%inject collagenase I at0.1%and trypsin (within EDTA) at concentration of0.125%, digest the vein to obtain umbilical vein endothelial cells, inoculate them in25cm2cell culture bottle with special culture medium for human umbilical vein endothelial cells, observe the cell morphology and adherent condition with optical microscope every12hours, exchange the medium totally every48hours, about72hours later, cells bespread the bottom of the plate, present a typical cobblestone-like pattern, subculture to next generation, The3th-5th generation cells are chosen for our experiment, adopt the factor VIII related antigen staining to identify the cells. The composition of special medium for Human Umbilical Vein Endothelial Cells (Every100ml medium Contain:FBS20ml (20%) from Gibicol; Beta-ECGF5ng/ml from sigma; L-glutamine,1mM; Penicillin-Streptomycin; heparin2500U/100ml; M-19980ml from Gibicol)2.2Frozen and reserve the human umbilical vein endothelial cellsAt the logarithm growth period of the third generation cells, discarded the upper medium, washing the cells gently two times with no Ca2+, Mg2+PBS, to remove the medium composition, digested the cells by adding0.125%trypsin(containing EDTA) in each flask, added in serum into cells medium to terminate the digestion when the spindle retracts into the sphere, washed two times with PBS again, added3ml M199medium composed of20%FBS,10ng/mL β-ECGF,25U/mL heparin, repeated pipetting until most of the human umbilical vein endothelial cells fall off the the bottom of the bottle, concentration of cells was adjusted to105/ml, draw3ml for cell identification test and Cell Counting Kit-8(CCK-8) Test for the detection of cell activity. The remaining cells are centrifugated under1000r/min,5min, discard the upper medium, added frozen stock solution and kept in liquid nitrogen ryopreservation. The Component of frozen stock solution:85%M199medium,10%fetal bovine serum,5%DMSO.2.3Grouping and detection of cell activity by CCK-8experimentCultivate the human umbilical vein endothelial cells(3th generation, concentration of105/ml) into25cm2cell culture bottle, subculture to4th generation according to1:3, when the cells grow to the logarithmic growth phase discarded the medium, flushed, digested, collected the cells, suspended the cells with culture medium special for human umbilical vein endothelial cells, adjust the concentration of cells to105/ml. Cultivate the HUVECs into the96holes culture plate on the basis by eight groups,5holes each group,100μl each hole, numbered to be1,2,3,4,5,6,7,8groups. Set another "background group",numbered to be0group, add100μl culture medium into the five holes, without human umbilical vein endothelial cells, for the background value of detection of CCK-8experiment.9groups,45holes in all. In order to reduce the error caused by evaporation reagent, edge culture holes in the96holes culture plate were abandoned and occupied with PBS solution. After that, place the96holes culture plate in constant temperature incubator to culture the cell for24hours preincubation, add in the HUVECs special culture medium, culture medium containing TNF-α at the concentration of2μg/ml after preincubation, adjust the total capacity to200μL each hole. The concentration of TNF-α in group1to8was Ong/ml,5ng/ml,10ng/ml,20ng/ml,40ng/ml,60ng/ml,80ng/ml,100ng/ml respectively, add the special culture medium to make the capacity to be200μL in every hole of0group. Continue to culture the cells for48hours, added Cell Counting Kit-8(CCK-8) assay for cell viability test,10μl every hole, detect the absorbance value (OD) at the wavelength of450nm.3. The statistical method All the data were processed using SPSS13.0software. Measurement data were representated in the form of the mean±standard deviation (x±S), statistical analysis using single factor analysis of variance(one-way ANOVA), analysis between two data using LSD-t method, P value<0.05was considered significant.Results1.Cell morphology of Human umbilical vein endothelial cellsPrimary generation human umbilical vein endothelial cells grew as a monolayer, oval shape, boundary clear, abundant cytoplasm, the nucleus was oval. About2-3days later, cells overspread the bottom of cell culture flask, fusiform shape, boundary clear, abundant cytoplasm, the nucleus was oval, grew as a monolayer, adherently grow, a typical cobblestone-like, uniform distribution.2. Factor Ⅷ related antigen stainingWatch the cells by fluorescence microscopy, brown-yellow granules were seen iun the cytoplasm, blue nucleus, this proved the presence of factor Ⅷ related antigen of endothelial cell specific, the control group was negative reaction.3. Results of cells activity detection by CCK-8From group1to group7, as the the concentration of TNF-α rise from Ong/ml to100ng/ml,the absorbance value of cells in each groups reduce by and by, the deviation among groups was significant Statistically (P<0.05), the absorbance value reduced from (0.721±0.0129) to (0.548±0.0196). but the data between group7and group8has no statistical significance, we believe that may be related to the concentration gradient is too small between the two groups.Conclusion1. Tumor necrosis factor alpha (TNF-α) have a significant effect on the activity of human umbilical vein cells, the differences among0ng/ml,5ng/ml,10ng/ml,20ng/ml,40ng/ml,60ng/ml,80ng/ml groups were statistically significant. The difference between80ng/ml, and100ng/ml groups was not statistically significant.2. We selected the four concentrations of0ng/ml,5ng/ml,20ng/ml,80ng/ml for the next experiments. Part2TNF-α effect the expression of PD-L1protein in human umbilical vein endothelial cellsObjective:Stimulate the human umbilical vein endothelial cells with TNF-α in certain concentration for about48hours, detect the expression of PD-L1protein in each group by Western Blot method, to investigate the effects of TNF-α on the expression of PD-L1protein in human umbilical vein endothelial cells.Objects and methods1. Objects Healthy human umbilical vein endothelial cells cultured in vitro(HUVECs)2. Methods2.1Thawed and cultured the Human umbilical vein endothelial cellsTook the frozen umbilical vein endothelial cells out of the liquid nitrogen cabinet, put it into the37℃water thermostatic until it melt completely about2minutes later, collected the cells by centrifugation, added the culture medium special for HUVECs, repeated uniform mixing, cultivated the cells into the25cm2cell culture bottle, watch the cell morphology every12hours, the culture medium was changed totally every48hours, subcultured the cells to4th generation at logarithmic growth phase according to1:4.2.2Group setting and PD-L1protein detection by Western Blot methodIn logarithmic growth phase, change the culture medium into the culture medium containing tumor necrosis factor alpha, adjusted the concentration to be group A Ong/ml、group B5ng/ml、group C20ng/ml、group D80ng/ml, incubated the cells in constant temperature incubator for48hours, discarded the upper medium, wash2times repeatedly with PBS, trypsin digest, added the PBS solution and rinsed repeatedly, until the cells fall off the bottom of the bottle, harvested the cells by centrifugation. Washed the cells for2times again, added the lysate and extract the protein, quantified the total protein by BCA method, adjusted the volume of sample was25μg total protein/sample, make the sample, electrophoresis, blotting, closed for the night, PBST washing the membrane5times after closing, added first-Antibody, react for1.5hours at37℃, washed for5times with PBS, add the second-Antibody badged with Horseradish peroxidase, react for1hour at37℃, washed for5times with PBS again, dyeing, exposure, developed and fixed successively with BIORAD GELDOC XR Gel imaging system, analysis the protein bands of film with QuantityOne Basic Software. The test was repeated3times.3. The statistical methodAll the data were processed using SPSS13.0software. Measurement data were representated in the form of the mean±standard deviation (x±S), statistical analysis using single factor analysis of variance(one-way ANOVA), analysis between two data using LSD-t method, P value<0.05was considered significant.Results1. The unfrozen cells grew normally, overspread the bottom of cell culture flask adherent, fusiform shape, grew as a monolayer, a typical cobblestone-like, boundary clear, abundant cytoplasm, the nucleus was oval.2, As the the concentration of TNF-α rise from Ong/ml to80ng/ml,the expression of PD-L1protein gradually declined, the gray value of Western blot were respectively groupA (1.797±0.1550), groupB (1.512±0.1022), groupC (1.108±0.0799), groupD (0.845±0.0542), the differences between groups was statistically significant.ConclusionTumor necrosis factor alpha can significantly inhibit the expression of PD-Ll protein in concentration-dependented manner in Human umbilical vein endothelial cells.Part3TNF-α effect the expression of PD-Ll protein through p38MAPK Signal pathway in HUVECsObjective:Detect the expression of PD-L1protein in human umbilical vein endothelial cells stimulated by TNF-α after blocking the p38MAPK signaling pathway with special protein inhibitor SB203580, study the relationship between the expression of PD-L1protein and P38MAPK signal pathway.Objects and methods1. Objects Healthy human umbilical vein endothelial cells cultured in vitro(HUVECs)2. Methods2.1Thawed and cultured the Human umbilical vein endothelial cells.Took the frozen umbilical vein endothelial cells out of the liquid nitrogen cabinet, put it into the37℃water thermostatic until it melt completely about2minutes later, collected the cells by centrifugation, added the culture medium special for HUVECs, repeated uniform mixing, cultivated the cells into the25cm2cell culture bottle, watched the cell morphology every12hours, the culture medium was changed totally every48hours, cultured the cells to4th generation at logarithmic growth phase according to1:3.2.2Group settingsPlant the HUVECs(the fourth generation) into the6hole culture plate, set3groups according to the use of TNF-α and p38MAPK signaling pathway inhibitor SB203580,2holes each group. The concentration of TNF-α was80ng/ml, and SB203580was dissolved with two dimethyl sulfoxide (DMSO).Group1was normal culture group:the HUVECs were cultured with special culture medium in constant temperature incubator as control group.Group2was intervented by TNF-α:the HUVECs were cultured with special culture medium containing the TNF-α at the concentration of80ng/ml, cultured in the constant temperature incubator for48hours.Group3was intervented by TNF-α and SB203580together. Add the TNF-α and P38MAPK blocker SB203580into the special culture medium, cultured the cells in the constant temperature incubator for48hours. The concentration of TNF-α was80ng/ml, the SB203580was dissolved with two dimethyl sulfoxide (DMSO).2.3PD-L1protein detection by Western Blot methodHarvested the cells, added the lysate and extract the protein, quantified the total protein by BCA method, adjusted the volume of sample to be25μg total protein/ sample, make the sample, electrophoresis, blotting, closed for the night, PBST washed the membrane5times after closing, added first-antibody, react for1.5hours at37℃, washed for5times with PBS, add the second-antibody and badged by horseradish peroxidase, react for1hour at37℃, washed for5times with PBS again, dyeing, exposure, developed and fixed successively with BIORAD GELDOC XR Gel imaging system, analysed the protein bands of film with QuantityOne Basic Software. The test was repeated3times.3. The statistical methodAll the data were processed using SPSS13.0software. Measurement data are representated in the form of the mean±standard deviation (x±S), statistical analysis using single factor analysis of variance(one-way ANOVA), analysis between two data using LSD-t method, P value<0.05was considered significant.ResultsThe expression of PD-L1protein in the group intervented by TNF-α was lower evidently compared with co-stimulated group and normal group, the gray value ratio were (0.486±0.0571)、(1.069±0.0401)、(1.215±0.0506) respectively, P<0.05, the differences between groups was statistically significant, and the expression of PD-L1protein in normal group was lower than co-stimulated group.Conclusion1. The p38MAPK pathway is involved in the process that TNF-α affect the Expression of PD-L1protein.2. The Expression of PD-L1protein in normal group is lower than co-stimulated group, we speculate that there may be endogenous TNF-α or other cytokines affect the expression of PD-L1. |
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