本文選題：microRNA-145 + CD40　； 參考：《天津醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的探討micro RNA(mi R) 145及CD40在動脈粥樣硬化(atherosclerosis,AS)及其炎癥免疫過程中的作用及機(jī)制。方法1、10只8周齡雄性野生型C57BL/6J小鼠,給予普通飲食,作為對照組;10只8周齡雄性載脂蛋白E基因敲除(ApoE~(-/-))小鼠,給予高脂飼料,以構(gòu)建AS模型,作為模型組。分別喂養(yǎng)12周后,心臟穿刺取血檢測炎癥因子IL 1、IL 6和TNF α水平,全自動生化檢測儀檢測血脂(TC、TG、LDL、HDL)水平;處死小鼠后取主動脈標(biāo)本行HE染色、免疫組織化學(xué)染色。2、體外培養(yǎng)小鼠巨噬細(xì)胞系RAW 264.7細(xì)胞,隨機(jī)均分為mi R 145模擬物(mimic)組及其陰性對照(NC)組、mi R 145抑制劑(inhibitor)組及其陰性對照(i NC)組、沉默CD40序列(si CD40)組及其陰性對照(si NC)組,分別轉(zhuǎn)染mi R 145 mimic、NC、mi R 145 inhibitor、i NC、si CD40、si NC(5ul/ml),轉(zhuǎn)染6小時之后,用氧化低密度脂蛋白(ox LDL)(3.2mg/ml)刺激構(gòu)建炎癥免疫損傷細(xì)胞模型。應(yīng)用實(shí)時熒光定量PCR(RT q PCR)和Western blot檢測細(xì)胞中CD40 m RNA和蛋白的表達(dá)情況;應(yīng)用酶聯(lián)免疫吸附測定法(ELISA)檢測細(xì)胞上清液中炎癥因子IL 1、IL 6和TNF α水平。結(jié)果1、8周齡雄性ApoE~(-/-)小鼠高脂飲食喂養(yǎng)12周可以成功建立AS模型。ApoE~(-/-)小鼠與C57BL/6J小鼠相比,主動脈可見明顯斑塊形成,斑塊處CD40蛋白表達(dá)水平顯著升高,血中炎癥因子IL 1、IL 6和TNF α水平明顯升高(P0.05),血脂TC和LDL水平明顯升高(P0.05),HDL水平顯著下降(P0.05),TG水平未見明顯差異(P0.05)。2、ox LDL刺激體外培養(yǎng)RAW264.7細(xì)胞24小時及48小時后可以建立成功的炎癥免疫損傷細(xì)胞模型。mimic組與NC組相比,CD40 m RNA、CD40蛋白及炎癥因子IL 1、IL 6、TNF α表達(dá)均顯著下降(P0.05);inhibitor組與i NC組相比,CD40 m RNA、CD40蛋白及炎癥因子IL 1、IL 6和TNF α表達(dá)均明顯升高(P0.05);si CD40組與si NC組相比,CD40 m RNA、CD40蛋白及炎癥因子IL 1、IL 6和TNF α表達(dá)均顯著下降(P0.05)。結(jié)論mi R 145能夠通過靶基因CD40調(diào)控AS炎癥免疫過程,抑制CD40/CD40L信號通路活化,抑制炎癥因子分泌,從而起到抑制AS發(fā)生發(fā)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 偽 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- 偽 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- 偽 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- 偽 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 偽 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- 偽 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- 偽 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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R543.5

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