發(fā)布時間：2018-10-08 21:08

【摘要】：目的:研究過氧化物酶體增殖物激活受體-γ(peroxisome proliferator-activated receptor-γ,PPARγ)激動劑吡格列酮(pioglitazone,P io)對動脈粥樣硬化模型載脂蛋白E基因敲除(Apo E-/-)小鼠主動脈中熱休克蛋白22(heat shock protein 22,HSP22)表達的影響,并探討HSP22在其中所起的作用。方法:1、36只8~9周齡Apo E-/-雄性小鼠,隨機分為4組:對照組(n=9,普通飼料),對照干預(yù)組(n=9,普通飼料+Pio),模型組(n=9,高脂飼料)與模型干預(yù)組(n=9,高脂飼料+Pio);對照組、對照干預(yù)組喂食普通飼料12周,模型組、模型干預(yù)組喂食高脂飼料12周,即高脂飲食(high-fat diet,HFD),其中干預(yù)組從第五周開始給予Pio(20mg/kg/d)干預(yù)處理,共干預(yù)8周。2、稱量各組小鼠每周體重;檢測各組小鼠基線及干預(yù)結(jié)束的血脂水平,包括:甘油三脂(triglyceride,TG)、總膽固醇(total cholesterol,TC)、高密度脂蛋白-膽固醇(high-density lipoprotein cholesterol,HDL-C)、低密度脂蛋白-膽固醇(low-density lipoprotein cholesterol,LDL-C);檢測干預(yù)結(jié)束時小鼠的空腹血糖。3、主動脈整體油紅O染色及蘇木素-伊紅(hematoxylin and eosin,HE)染色法觀察各組小鼠動脈粥樣硬化斑塊形成情況及病理學(xué)變化。4、免疫組化法檢測小鼠主動脈HSP22表達,Western Blot法檢測PPARγ、HSP22、細(xì)胞間粘附分子1(intercellular adhesio n molecule-1,ICAM-1)、內(nèi)皮型一氧化氮合酶(endothelial nitric oxide synthase,e NOS)、p-e NOS(ser1177)表達,ELISA法檢測血清白細(xì)胞介素6(interleukin-6,IL-6)濃度。結(jié)果:1、血脂、血糖變化(1)基線血脂:各組Apo E-/-小鼠的基線血清TG、TC、HDL-C、LDL-C的水平無差異(P0.05);(2)干預(yù)結(jié)束時血脂:模型組和模型干預(yù)組小鼠血清TC、LDL-C較對照組及對照干預(yù)組明顯升高(P0.01),HDL-C顯著降低(P0.01),TG的變化無統(tǒng)計學(xué)意義。經(jīng)Pio干預(yù)后,模型干預(yù)組小鼠血清TC、LDL-C較模型組明顯降低(TC:P0.05,LDL-C:P0.01),HDL-C明顯升高(P0.01),TG的變化無統(tǒng)計學(xué)意義;對照干預(yù)組小鼠血清LDL-C較對照組顯著降低(P0.01),TG、TC、HDL-C無明顯變化。(3)干預(yù)結(jié)束時血糖:經(jīng)高脂飼料飼養(yǎng)后,模型組和模型干預(yù)組小鼠血糖較對照組及對照干預(yù)組明顯升高(P0.01);模型干預(yù)組較模型組無明顯變化。2、組織病理學(xué)觀察及分析(1)主動脈整體油紅O染色:模型組和模型干預(yù)組小鼠主動脈紅染脂質(zhì)斑塊多,其斑塊相對面積明顯高于對照組、對照干預(yù)組(P0.01),而模型干預(yù)組較模型組減少(P0.01);對照干預(yù)組較對照組有減少,但差異無統(tǒng)計學(xué)意義(P0.05)。(2)主動脈根部HE染色:與對照組、對照干預(yù)組比較,模型組及模型干預(yù)組主動脈根部可見大量動脈粥樣硬化斑塊形成,其斑塊相對面積顯著增加(P0.01),與模型組比較,模型干預(yù)組斑塊面積減少(P0.01)。3、Western Blot法檢測主動脈PPARγ蛋白的表達模型組主動脈PPARγ的表達較對照組顯著升高(P0.01);而經(jīng)Pio治療后,模型干預(yù)組PPARγ的表達較模型組進一步升高(P0.01),對照干預(yù)組PPARγ的表達較對照組進一步升高(P0.01)。4、主動脈HSP22蛋白的表達(1)Western Blot法:模型組及模型干預(yù)組主動脈HSP22的表達較對照組、對照干預(yù)組顯著升高(P0.01);而模型干預(yù)組經(jīng)Pio治療后HSP22的表達較模型組降低(P0.01);(2)免疫組織化學(xué)法:對照組及對照干預(yù)組主動脈內(nèi)棕色著色淺,HSP22表達量少;模型組主動脈斑塊及血管壁棕色著色深,HSP22大量表達;模型干預(yù)組與對照組相比,HSP22表達量也增加,但較模型組明顯較少;5、Western Blot法檢測主動脈e NOS、p-e NOS蛋白的表達模型組主動脈e NOS蛋白的表達較對照組、對照干預(yù)組顯著降低(P0.01),而模型干預(yù)組經(jīng)Pio干預(yù)后,e NOS的表達較模型組升高(P=0.027)。模型組主動脈p-e NOS蛋白的表達較對照組降低(P=0.028)、較對照干預(yù)組顯著降低(P0.01);而模型干預(yù)組p-e NOS的表達較模型組顯著升高(P0.01)。6、Western Blot法檢測ICAM-1蛋白的表達模型組及模型干預(yù)組主動脈ICAM-1的表達較對照組、對照干預(yù)組顯著升高(P0.01),給予Pio治療后模型干預(yù)組主動脈ICAM-1的表達較高脂模型組降低(P0.01)。7、血清IL-6水平模型組血清IL-6的濃度較對照組、對照干預(yù)組顯著升高(P0.01),而給予Pio干預(yù)后,模型干預(yù)組血清IL-6的濃度較模型組降低(P0.01)。結(jié)論:1、高脂飲食可上調(diào)主動脈PPARγ、HSP22的表達,下調(diào)e NOS、p-e NOS的表達,上調(diào)ICAM-1的表達,促進動脈粥樣硬化形成。2、吡格列酮可激活PPARγ,下調(diào)主動脈內(nèi)HSP22的表達,上調(diào)e NOS、p-e NOS的表達,下調(diào)ICAM-1的表達,抑制動脈粥樣硬化形成。
[Abstract]:AIM: To study the effects of pioglitazone (Pio) on the proliferation of peroxidases in the aorta of apolipoprotein E knockout mice (ApoE-/-) mice. HSP22 expression, and explore the role of HSP22 in it. Methods: 1, 36 male mice aged 8 to 9 weeks were randomly divided into 4 groups: control group (n = 9, normal feed), control intervention group (n = 9, normal feed + PIo), model group (n = 9, high fat feed) and model intervention group (n = 9, high fat feed + Pio); control group. In the control intervention group, 12 weeks of normal feed were fed, the model group and the model intervention group were fed with the high fat diet for 12 weeks, that is, the high-fat diet (HFD), among which the intervention group began to give PIo (20mg/ kg/ d) intervention treatment from the fifth week to intervene for 8 weeks. Serum lipids were detected at the end of baseline and intervention in each group, including triglyceride (TG), total cholesterol (TC), high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C); To detect the formation and pathological changes of atherosclerotic plaques in mice at the end of intervention, the formation and pathological changes of atherosclerotic plaques in mice were observed by the staining of whole oil red O and hematoxylin and eosin (HE) staining. The levels of interleukin-6 (IL-6) and interleukin-6 (IL-6) in serum were measured by Western blot. The expression of interleukin-6 (IL-6) was detected by ELISA. Results: There was no difference in serum TG, TC, HDL-C and LDL-C in the baseline serum TG, TC, HDL-C and LDL-C in all groups of Aso E-/-mice (P0.05). LDL-C was significantly higher than that in control group (P0.01), HDL-C decreased significantly (P <0.01), and TG had no statistical significance. After PIO intervention, the serum TC and LDL-C of the model intervention group were significantly lower than that in the model group (TC: P0.05, LDL-C: P0.01), HDL-C increased significantly (P <0.01), TG did not change significantly, and the serum LDL-C of the control group was significantly lower than that in the control group (P0.01), TG, TC and HDL-C had no obvious change. (3) At the end of the intervention, the blood glucose in the model group and the model intervention group was significantly higher than that in the control group (P0.01). Histopathological observation and analysis (1) The whole aorta oil red O staining: the model group and the model intervention group were significantly higher than the control group and the control intervention group (P0.01), while the model intervention group was lower than that in the model group (P0.01). There was no significant difference between control intervention group and control group (P0.05). (2) HE staining of the aortic root: Compared with the control group and the control intervention group, the model group and the model intervention group showed a large number of atherosclerotic plaques in the aortic root, and the relative area of the plaque increased significantly (P0.01). Compared with the model group, the plaque area of the model intervention group decreased (P0.01). Compared with the control group (P0.01), the expression of CD44v6 in the model intervention group was higher than that in the control group (P0.01). The expression of HSP22 in aorta and the expression of HSP22 in the model group and the model intervention group were significantly higher than that in the control group (P0.01). The expression of HSP22 in the model intervention group was lower than that of the model group (P0.01). In the control group and the control intervention group, the brown coloration of the aorta was shallow and the expression of HSP22 was small; the model group aortic plaque and blood vessel wall brown staining depth, HSP22 were expressed in large numbers; the model intervention group also increased the expression level of HSP22 compared with the control group, but the model group was less obvious; 5, The expression of NOS, p-e NOS protein in aorta was lower than that in control group (P = 0.01), and the expression of e NOS in the model group was higher than that in control group (P = 0. 01). The expression of p-e NOS protein in the model group was lower than that in the control group (P = 0.028), but the expression of p-e NOS in the model group was significantly higher than that in the control group (P0.01). The expression model of ICAM-1 and the expression of ICAM-1 in the model intervention group were higher than that in the control group (P0.01). The expression of ICAM-1 in the model intervention group was lower than that in the control group (P0.01). The serum IL-6 concentration in serum IL-6 group was higher than that in control group (P0.01), and the concentration of IL-6 in serum IL-6 group was lower than that in control group (P0.01). Conclusion: 1. High fat diet can upregulate the expression of ENOS, p-e NOS, regulate the expression of ICAM-1, regulate the expression of ICAM-1 and promote the formation of atherosclerosis. Down-regulates the expression of ICAM-1 and inhibits the formation of atherosclerosis.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R543.5

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8 游潔冰;PPARγ激動劑、胰島素通過上調(diào)負(fù)性炎性因子TIPE2的表達抑制高糖、Aβ1-40引起的炎性反應(yīng)及神經(jīng)細(xì)胞調(diào)亡[D];山東大學(xué);2015年

9 劉常為;CTGF、COL-I、PPARγ在卵巢細(xì)胞外基質(zhì)的表達及與多囊卵巢綜合征的關(guān)系[D];暨南大學(xué);2015年

10 曹小潔;TLR4通過PPARγ下調(diào)ABCG1表達促進血管平滑肌細(xì)胞內(nèi)炎癥反應(yīng)及脂質(zhì)沉積[D];第三軍醫(yī)大學(xué);2015年

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