| BackgroundCoronary Atherosclerosis heart disease(CHD) is one of the diseases with thehighest incidence rate and most disservice at preasent. It becames one of theconsequence question for discussion nowdays how the pathogenetic to CHD goes onand how to prevent and cure it. Therefor there were several theories to interpret theetiopathogenisis of CHD from difference points. Howevre some reseachers found thatautoimmune was a key factor to the etiopathogenisis of CHD. That is to say CHD isan autoimmune disease for some reasons. Firstly some phlegmasia transmitters andimmune substance were found in the blood and affected tissues with CHD patients.Secondly some autologous antibodies were detected with CHD patients. Thirdly therewere some special immune cells such as T leukomomocyte cell which participateautoimmune in affected tissues.The initiation step to priming immune response is the immediate contactbetween leukomomocyte cell and antigen presenting cell(APC). APC is a kind ofspecial immune cell who can capture and process antigens, then present thoseantigens to antigenic special leukomomocyte cell. Acroding to whether MHC-Ⅱmolecules and its co-stimulus molecules were expressed in the surface ofcells or not, APC included two kinds of cells which was special and unspecial APC.Dendritic cell is the most functional APC in our bodys. T leukomomocyte cells couldbe intensively activited to priming special immune response with slight dendritic cells.And its capability to active T leukomomocyte cell was as much 100～10000 times asmacrophaga and B leukomomocyte cells. As we know the activationg of Tleukomomocyte cell was key to form plaques in CHD.More and more researchs focused on Oxidized low density lipoprotein(Ox-LDL)with more cytotoxicitys which could result in more foam cells developed. Howeverfollowing researches showed us that LDL had some immunogenicitys similar toforeign proteins caused, which LDL was descompounded and reassociated inoxidative process and some new epiposition formed. Some finished clinicalresearches had drawed conclusions that Ox-LDL could not only damage endothelialcells and smooth muscle cells but also show some chemotaxis to histomonocytessome else. The binding between Ox-LDL and its receptor insurface of macrophagecould induce a series of incidents inner cells, such as generating inflamm cytokinesand magnifing inflamm responses. Although Ox-LDL had some immunogenicityssimilar to foreign proteins caused, its functions focued on that it could cause thehighly express of CD40/CD40L insurface of blood vessel endothelium cell. Howeverthere was no clear machanism of Ox-LDL to dendritic cells.It's well known that wecould use statins to cure CHD. And its pharmaco-mechanism is that statins couldcompetitively inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reducingenzyme. So that the synthesis of cholesterol decreased because of mevalonate reduced.Atorvastatin is the newest HMG-CoA reducing enzyme inhibitor. Several serious ofclinical researches discovered that atorvastatin has ideal effect on balancing blood-fatdisorders. Our studies will maily be focus on observing the immune function changes ofdendritic cells excited with Ox-LDL in the level of cytobiology and molecule immue.Then to discuss how the Ox-LDL influences the immunological regulation ofdendritic cells which were the special antigen presenting cell with most capability. Sothat some immune theorys in our studies could offer a suppourt to the clinialprevention and cure CHD.Objective1. To determine whether Ox-LDL could influence DC maturing and immunefunction.2. To investigate whether Atorvastatin could regulate adaptive immunity whichwas elicited by Ox-LDLSubjects and Method1,SubjectsTwenty healthy persons is verification by medical examination that include twelvemails and 8 femails.Their mean age is 40.5±8.7 years.2,Method2.1 Preparation of Ox-LDL and DiI-labelledWe separated and quantitatified the LDL from blood of healthy individuals by Superhigh density gradient centrifugation, and adjusted it to a final concentration of500ug/mL,then incubated with 10mmol/mL CuSO4.The oxidation was stopped with100μmol/L EDTA. Finally, we labeled the Ox-LDL with DiI.2.2 Culture and interference of DCPeriperil blood mononuclear cell (PBMC) were isolated by density gradientcentrifugation, and cultured with RPMI-1640(containing 1% FCS, rhGM-CSF1,000,000 U/L, rhIL-4,500,000U/L)at an atmosphere of 5%CO2 for 5 days.Afterbeing taken into several groups, with different interferences and culturing for another 2h, DC were collected. Culture changes in cell morphology were observed under aninverted microscope.2.3 Groups(1) control group: DC were cultured without any treatment(2) Ox-LDL group : DC were cultured with Ox-LDL for 48h(3) Atorvastatin group: DC were cultured both with Atorvastatin and Ox-LDL2.4 Proliferation of lymphocytes assayProliferation of allogeneic T lymphocyte were evaluated after incubation withDC which served as a stimulant.2.5 Flow cytometry analysisDC were collected and the expression of relative differentiation antigens on thesurface of DC were analyzed on a flow meter.2.6 Detection of IL-12Double mAb sandwich ELISA was applied in quantitation of IL-12.3,Statistical analysis: SPSS 10.0 statistical soft was used.The data was shown as((?)±s), The compared of two groups was tested by paired sample t-test,Results1,Observation on morphology of DCSeveral phenomenons were observed under an inverted microscope: (1)control group: There were distinct dendritic structures and abundant cytoplasm in DCafter cultured for 5 days. (2)Ox-LDL group :The volume of DC grew bigger andbigger with more abundant cytoplasm, dyed red, and became irregular morphous withmany pseudopodia. (3) Atorvastatin group: The number of DC whichphagocytosed Ox-LDL diminished, and the dyeing of DC became lighter afterincubating with both Atorvastatin and Ox-LDL libeled with DiI.2,Proliferation of lymphocytes assay Differences in proliferation of T lymphocytes between treated with Ox-LDLgroup (3.9±0.8) and the control group (1.0±0.4) were found to be statisticallysignificant (P＜0.05) .And the same result (P＜0.05) also occurred between the grouptreated both with Atorvastatin and Ox-LDL(1.3±0.2)and treated with Ox-LDL.3,Influence of phenotypeCollected the suspension cells treated for 48h, and detected themolecules expressed on the cell surface by FACS analysis. The results showedthat, compared with the negative control and PBS group, there was higherexpression of CD1a and HLA-DR on the cell surface of Ox-LDLtreated group,both of which could stimulate the proliferation of T lymphocytes. Differencebetween the control and treated group (35.3±10.1 vs 69.8±9.1) and (11.3 vs. 70.5±92.3±5.0) was statistically significant (P＜0.05); although compared with the controlgroup,there was mild expression of CD80 and CD86 (63.5±7.7 vs 88.0±7.9) and(10.2 vs. 61.3±80.84±1.12 P＜0.01),difference was not statistically significant (P＞0.05). Compared with Ox-LDL treated group, there was a significant decrese on theexpression of CDla (69.8±9.1 vs 55.5±12.4) and HLA-DR (92.3±5.0 vs66.8±5.8) on DC treated with atorvastatin.The difference was statistically significant(P＜0.05).Conclusion1. The adherent cells of PBMC in peripheral blood could be induced to be DCafter culturing with GM-CSF or IL-4 for 7 days.2. After incubated with Ox-LDL, there was high expression of MHC classⅡmolecules on the cell surface of DC, which could enhance its capacity to uptake ofantigens, and activate T cells and initial immune response.3. Atorvastatin can inhibit immunocompetence of DC obviously, by inhibitingbiological effects of Ox-LDL and probably by participating the intracellular signal transduction. This presumption is a new mechanism of immune modulationtherapy of coronary heart diseases.
|
PDF Full Text Request