【作者】 冯静；

【导师】 孟晓萍；

【作者基本信息】 吉林大学 ， 内科学， 2011， 硕士

【摘要】 目的通过应用基因敲除(APOE-/-)小鼠的动脉粥样硬化斑块的模型进一步探讨免疫反应在动脉动脉粥样硬化的作用。方法把APOE基因敲除(APOE-/-)小鼠及C57BL/6小鼠分别分为两组,每组分为高脂饮食组及普通饮食组,建立不同动脉粥样硬化模型。用主动脉标本作HE切片染色及抗MMP-9、抗CD8+、及抗OX-LDL的免疫组织化学染色、拍照,并进行图片分析,通过测得抗OX-LDL、抗CD8+和抗MMP-9的免疫沉淀物灰度值作定量分析。用ELISA方法测定小鼠血浆中MMP-9与OX-LDL水平。结果动脉粥样硬化模型中的斑块大体形态：APOE-/-高脂饮食组小鼠有动脉粥样硬化斑块形成,斑块主要集中在主动脉分叉处,斑块数量中等,某些小鼠腹主动脉可见斑块。APOE-/-正常饮食组小鼠无动脉粥样硬化斑块形成。C57BL/6高脂饮食小鼠未见动脉斑块形成,但腹腔及各组织脏器表面脂肪含量明显增多,C57BL/6正常饮食组(对照组)小鼠无动脉斑块形成。HE染色标本中,APOE-/-高脂饮食组斑块程度、泡沫细胞数量较对照明显增多,C57BL/6高脂饮食组较正常对照组斑块程度、泡沫细胞数量增多,但程度较轻,APOE-/-正常饮食组与正常对照组相比,内膜光滑,无明显差别。免疫组化染色结果：APOE-/-高脂饮食组抗MMP-9、OX-LDL含量较正常对照组明显升高,抗CD8含量较正常对照组明显减少,有统计学意义(P<0.01)。C57BL/6高脂饮食组抗MMP-9、OX-LDL含量较正常对照组有升高,CD8含量较正常对照组减少,但较APOE-/-高脂饮食组程度减轻,有统计学意义(P<0.01)。APOE-/-正常饮食组,抗MMP-9含量较正常对照组升高,但无统计学意义(P>0.05), OX-LDL含量较正常对照组升高,CD8较正常对照组减少,有统计学意义(P<0.05或P<0.01)。血清检测结果：APOE-/-高脂饮食组MMP-9、OX-LDL含量较正常对照组明显曾高,有统计学意义(P<0.01)。C57BL/6高脂饮食组MMP-9、OX-LDL含量较正常对照组升高,有统计学意义(P<0.01)。APOE-/-普通饮食组,血清MMP-9含量升高,与正常对照组比较有统计学意义(P<0.05), OX-LDL含量升高,与正常对照组比较无统计学意义(P>0.05)。结论在动脉粥样硬化过程中,CD8+T淋巴细胞的表达与动脉粥样硬化斑块的数量、程度成负相关,OX -LDL和MMP-9的表达与动脉粥样硬化斑块的数量、程度成正相关,与CD8+T淋巴细胞在动脉粥样硬化中的表达程度呈相反趋势。更多还原

【Abstract】 Background and purpose:With the improvement of living standards and the change of social labor, the morbidity and mortality rates of atherosclerosis increase year by year, which has become the top killer to human health, affecting people’s quality of life seriously, and causing great mental burden and economic burden. Although medical level and treatment improved, it is difficult to cure the disease. Recently, it was reported that there are a lot of T lymphocytes in atheromatous plaques, and since then, the relationship between atherosclerosis and immune mechanism were more and more concerned. There are few reports about the (?)ole of CD8+T lymphocytes in atherosclerosis. The trend of expression of CD8+T lymphocytes in atherosclerosis remains absurd.This research mainly explores the role of CD8+T,MMP-9, and OX-LDL in atherosclerosis through testing their expression in atherosclerosis process. In recent years, it was found that OX-LDL play a very important role in process of atherosclerosis formation, especially in ApoE gene knockout mice atherosclerosis model, the OX-LDL promote atherosclerosis formation obviously. MMP-9 is an important member of the matrix metalloproteinase family, which play key roles in damage reactions and atherosclerosis (AS) formation process. They could biodegrade basement membrane collagen and lead to the occurrence of acute coronary syndrome.Methods:Establish atherosclerosis sclerosis mice model.28 Laboratory mice,16 of them for Apoe gene knockout (Apoe-/-) mice,10 of which were randomly divided into a high-fat diet group (Ah), and the rest 6 mice were normal diet group (An). Another 12 C57BL/6 mice were homologous to knockout mice. Six of them were randomly divided into a high-fat diet group (Ch) and 6 mice were normal diet group (Cn). Ah group and Ch mice were given high-fat diet for 12 weeks. Then, we picked eye to take some blood serum from empty stomach mice, then killed them and get aorta to observe atheromatous plaque formation conditions. We observed pathological change under light-microscopy after Aortic specimen for HE dyed, and detected the concentration of MMP-9, CD8, OX-LDL in atheromatous plaque using immunohistochemical method, measured the mean gray degree of the immune deposits of MMP-9, CD8, OX-LDL in aortic endometrial and measured MMP-9 and ox-LDL level in the plasma of mice using the method of ELISA.Results:General form of Atherosclerosis plaques in gross:Ah mice atherosclerotic plaque formation is mainly concentrated in arterial plaque bifurcation place. The quantity of plaques is medium. There are patches on the abdominal aorta in some of the mice. There is no atherosclerotic plaque formation in An and Cn group mouse. Neither in Ch group mice, but the fat content in peritoneal and organ surface are significantly increased. The result of HE stain showed the plaques formation and foam cells in Ah groups are more than Cn group. The plaques formation and foam cells in Ch groups are more than normal group, but in a lesser degree. Endometrium is smooth in An group which is exactly the same as the normal group. Immunohistochemical stains show that in An group: the expression of MMP-9 is more than control group, but no statistical significance,P>0.05. MMP-9 expression in Ch group is more than control group, P<0.01. MMP-9 expression in Ah group is more than control group, P<0.01. The result of comparing MMP-9 expression in different groups is Cn<An<Ch<Ah. The expressions of CD8+T cells in Ah, Ch and An group are significantly less than Cn group, which is statistically significant, P<0.01. Comparing of CD8+expression in different groups is:Ah< Ch< An< Cn. The expression of OX-LDL in An group is more than Cn group, P<0.05. The expression of OX-LDL in Ch group is more than Cn group significantly, P<0.01. Also Ah group is obviously higher than Cn group, P<0.01. It was statistically significant in all above comparing. Comparing of OX-LDL expression in different groups is:An<Ch<cn<Ah. The results of concentrations of OX-LDL and MMP-9 in plasma by ELISA:The concentration of MMP-9 in An group is more than Cn group P<0.05, which is statistically significant. Also in Ch group is more than Cn group, P< 0.01, which is statistically significant. The concentration of MMP-9 in Ah mice is more than Cn group, P<0.01, which is statistically significant. Comparison of MMP-9 concentration in different groups:Ah>Ch>An>Cn. The concentration of OX-LDL in An group is more than Cn group, which is no statistically significant, P>0.05. OX-LDL level in Ch group is more than Cn group, P<0.01, statistically significant. Comparing of OX-LDL level in different groups is:Ah>Ch>An>Cn. It was statistically significant between these groups.Conclusion:In the model of mice atherosclerosis, with the patch quantity and foam cells increasing, CD8+T lymphocytes are diminished.In mice models of atherosclerosis, MMP-9 is positive related with progression of atherosclerosis degree. In mice models of atherosclerosis, OX-LDL promotes the progression of atherosclerosis degree.更多还原