摘要
目的
运用基因芯片技术分析动脉粥样硬化(atherosclerosis,AS)大鼠与正常大鼠动脉组织中的差异性表达基因,筛选其中与细胞外基质(Extracellular matrix,ECM)代谢相关的基因,探讨ECM与AS发病的关系。观察ECM代谢相关基因中组织蛋白酶D(cathepsinD,CathD)在AS大鼠动脉组织中的分布及表达水平,研究其在AS发病中的作用。
方法
(1)以高脂饲料(3%胆固醇、0.5%胆酸钠、5%精制糖、10%猪油及81.5%基础饲料)喂养加维生素D3腹腔注射法建立SD大鼠AS模型;
(2)实验第14周末,检测大鼠血清总胆固醇(total cholesterol,TC)及甘油三酯(triglyeride,TG),进行主动脉、冠状动脉HE及油红O染色;
(3)提取主动脉组织RNA,应用基因芯片技术分析差异性表达基因,根据基因芯片结果筛选与ECM代谢相关的基因CathD,以免疫荧光及免疫印迹法检测CathD在动脉组织中的分布情况及表达水平。
结果
(1)以高脂饮食加腹腔注射维生素D3的方法成功建立了大鼠AS模型。14周时模型组TG和TC均较对照组明显升高(P<0.01);HE染色可见模型组大鼠主动脉内皮细胞排列紊乱,部分脱落,中膜平滑肌细胞、纤维组织增生伴点状或片状钙化,弹性纤维层结构不清,形成纤维增生性动脉粥样硬化病变。模型组主动脉、冠状动脉油红O染色可见内皮下橙红色脂质沉积。对照组动脉组织未见明显异常。
(2)采用覆盖20500个大鼠基因的Oligo基因芯片,结果显示有1728个基因发生差异性表达,其中1218条基因表达下调,510基因表达上调,这些差异性表达基因分别与炎症、脂代谢、氧化应激、细胞凋亡等相关。其中可能与ECM代谢相关的基因有162条,表达上调的包括:血小板反应素、骨桥蛋白、基底膜蛋白多糖、MMp-11,CathB、L、D、Y细胞因子及炎症因子(TNF-α)等;表达下调的包括:生长因子(TGF-β),CystatinC ,MMp-2、16、23,丝氨酸蛋白酶抑制剂等。
(3)免疫荧光检测发现AS组大鼠动脉组织中红色荧光标记的CathD表达增加,主要分布在动脉内膜、中膜,在增厚的中膜中表达增高更明显。在对照组表达较少或不表达,主要分布于内膜及中膜。Western-blot检测提示:与对照组比较,AS组大鼠动脉组织CathD蛋白增加(P<0.05)。
结论
(1)通过高脂饲料加维生素D3腹腔注射的方法可以成功的复制大鼠动脉粥样硬化模型;
(2)基因芯片技术证实动脉粥样硬化是一种多因素导致的多种基因改变的复杂病变。ECM尤其是CathD可能参与了AS的发生及发展;
AIM: To explore the potential candidate genes related to extracellular matrix(ECM),especially cathepsin D, which might play roles in atherosclerosis (AS) pathogenesis by microarray technology.
METHODS: (1)Male SD rats were fed with fat diet (containing 3% cholesterol, 0.5% sodium cholate, 5% refined sugar, 10% lard, and 81.5% base feed) and injected with vitamin D3 i.p. to establish the experiment atherosclerotic model.
(2)After 14 weeks, total cholesterol (TC) and triglyeride (TG) in serum were detected. Aorta and coronary arteries were stained with HE and a solution of oil red O to visualize the lesion area.
(3)Total RNA was isolated from the aorta for microarray to explore the differential gene expression profiling.
(4)Cathepsin D expression was measured by immunofluorescence and western-blot.
RESULTS: (1)Compared with the control group, model group showed elevated TC and TG in serum (both P<0.01), and obvious aortic fibrous plaques.
(2)Of the 20500 genes explored,1218 genes were downregulated and 510 genes were upregulated. The differential expression genes were associated with inflammation, lipid metabolism, oxidative stress and cell apoptosis. The upregulated genes related to ECM remodeling included thrombospondin, osteopontin, perlecan, MMP-11, CathepsinB, CathepsinL, CathepsinD, CathepsinY, cytokines and inflammatory factors(TNF-α) etc. The downregulated ones included growth factor(TGF-β), CystatinC, MMp-2, MMp-16, MMp-23 and serine protease inhibitor, serine protease inhibitor (serpin).
(3)Immunofluorescence analysis showed that cathepsin D was mainly localized in the intima and tunica media, especially in the thicken tunica media. Further western-blot revealed that Cathepsin D protein expression was significantly increased in aorta of model group compared with control group. CONCLUSION: (1)Combining the high fat/cholesterol diets with injection of vitamin D3 successfully established the experiment atherosclerotic model in rats. (2)AS was a complex disorder resulting from a network of gene–gene and gene–environment interactions, the pathogenesis of AS was in close relation with ECM. Cathepsin D might play an important role in AS.
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