MicroRNA-145调控CD40参与动脉粥样硬化的作用及机制

发布时间：2018-06-16 12:21

本文选题：microRNA-145 + CD40　；参考：《天津医科大学》2017年硕士论文

【摘要】：目的探讨micro RNA(mi R) 145及CD40在动脉粥样硬化(atherosclerosis,AS)及其炎症免疫过程中的作用及机制。方法1、10只8周龄雄性野生型C57BL/6J小鼠,给予普通饮食,作为对照组;10只8周龄雄性载脂蛋白E基因敲除(ApoE~(-/-))小鼠,给予高脂饲料,以构建AS模型,作为模型组。分别喂养12周后,心脏穿刺取血检测炎症因子IL 1、IL 6和TNF α水平,全自动生化检测仪检测血脂(TC、TG、LDL、HDL)水平;处死小鼠后取主动脉标本行HE染色、免疫组织化学染色。2、体外培养小鼠巨噬细胞系RAW 264.7细胞,随机均分为mi R 145模拟物(mimic)组及其阴性对照(NC)组、mi R 145抑制剂(inhibitor)组及其阴性对照(i NC)组、沉默CD40序列(si CD40)组及其阴性对照(si NC)组,分别转染mi R 145 mimic、NC、mi R 145 inhibitor、i NC、si CD40、si NC(5ul/ml),转染6小时之后,用氧化低密度脂蛋白(ox LDL)(3.2mg/ml)刺激构建炎症免疫损伤细胞模型。应用实时荧光定量PCR(RT q PCR)和Western blot检测细胞中CD40 m RNA和蛋白的表达情况;应用酶联免疫吸附测定法(ELISA)检测细胞上清液中炎症因子IL 1、IL 6和TNF α水平。结果1、8周龄雄性ApoE~(-/-)小鼠高脂饮食喂养12周可以成功建立AS模型。ApoE~(-/-)小鼠与C57BL/6J小鼠相比,主动脉可见明显斑块形成,斑块处CD40蛋白表达水平显著升高,血中炎症因子IL 1、IL 6和TNF α水平明显升高(P0.05),血脂TC和LDL水平明显升高(P0.05),HDL水平显著下降(P0.05),TG水平未见明显差异(P0.05)。2、ox LDL刺激体外培养RAW264.7细胞24小时及48小时后可以建立成功的炎症免疫损伤细胞模型。mimic组与NC组相比,CD40 m RNA、CD40蛋白及炎症因子IL 1、IL 6、TNF α表达均显著下降(P0.05);inhibitor组与i NC组相比,CD40 m RNA、CD40蛋白及炎症因子IL 1、IL 6和TNF α表达均明显升高(P0.05);si CD40组与si NC组相比,CD40 m RNA、CD40蛋白及炎症因子IL 1、IL 6和TNF α表达均显著下降(P0.05)。结论mi R 145能够通过靶基因CD40调控AS炎症免疫过程,抑制CD40/CD40L信号通路活化,抑制炎症因子分泌,从而起到抑制AS发生发展的作用。
[Abstract]:Objective to investigate the role and mechanism of micro RNAi mi R) 145 and CD40 in atherosclerotic atherosclerosis (ASA) and its inflammatory immune process. Methods 1Ten 8-week-old male wild-type C57BL / 6J mice were given general diet and 10 8-week-old male apolipoprotein E knockout mice were given high-fat diet to construct as model group. After 12 weeks of feeding, the levels of IL 1, IL 6 and TNF 伪 were detected by cardiac puncture, and the serum lipids were measured by automatic biochemical instrument, and the aorta samples were taken for HE staining after the mice were killed. Murine macrophage cell line raw 264.7 was cultured in vitro with immunohistochemical staining. The mouse macrophage cells were randomly divided into two groups: the mi-R mimicic group and the negative control group. The silencing CD40 sequence and the negative control group were transfected with mi R ~ (145) mimici NCU mi R ~ + ~ (145) ~ (-1) ~ (-1) ~ (-1) ~ (-1) ~ (-1) I ~ (40) si ~ (40) si NC5 / ml ~ (-1) respectively. After 6 hours of transfection, the inflammatory immune injury cell model was constructed with oxidized low density lipoprotein (OLDL) oxLDLL ~ (3. 2) mg / ml. The expression of CD40 mRNA and protein was detected by real-time fluorescence quantitative PCR RT Q PCR and Western blot, and the levels of IL 1, IL 6 and TNF- 伪 in the supernatant were detected by enzyme linked immunosorbent assay (Elisa). Results (1) the atherosclerotic atherosclerosis (as) model was successfully established in 8-week-old male ApoEN / -mice by high-fat diet for 12 weeks. Compared with C57BL / 6J mice, aortic plaques were observed and the expression of CD40 protein was significantly increased in the plaques. The levels of IL 1, IL 6 and TNF- 伪 in blood increased significantly (P 0.05), and the levels of serum lipids, TC and LDL increased significantly. The levels of serum lipid, TC and LDL decreased significantly. There was no significant difference in serum levels of P0.05 and TG. The results showed that RAW264.7 cells cultured in vitro could be established after 24 hours and 48 hours of stimulation with P0. 05 and 2 oxo LDL. The expression of CD40m RNA-CD40 protein and inflammatory factor IL-1mRNA-6TNF- 伪 in the mimic group was significantly lower than that in the NC group. The expression of CD40 m RNA-CD40 protein and inflammatory cytokines IL-1, IL-6 and TNF 伪 were significantly increased in the P0.05 + inhibitor group and the I NC group compared with the I NC group, and the expression of IL-40 m RNA-CD40 protein and the inflammatory cytokines IL-1, IL-6 and TNF- 伪 were significantly increased in the mimic group compared with the NC group. The expression of CD40m RNA-CD40 protein and inflammatory cytokines IL-1, IL-6 and TNF- 伪 were significantly decreased in the group of high P0.05 and simi-CD40 compared with those in the group of si NC. Conclusion MiR-145 can regulate the inflammatory immune process of as through the target gene CD40, inhibit the activation of CD40 / CD40L signaling pathway, inhibit the secretion of inflammatory factors, and thus inhibit the occurrence and development of as.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R543.5

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