本文選題：9型腺相關(guān)病毒　切入點(diǎn)：NF-κB信號(hào)通路　出處：《新疆醫(yī)科大學(xué)》2015年碩士論文

【摘要】：目的：以重組9型腺相關(guān)病毒(recombinant adeno-associated virus serotype 9,rAAV9)為載體介導(dǎo)R65核酶基因轉(zhuǎn)導(dǎo)ApoE-/-小鼠,觀察是否能抑制主動(dòng)脈NF-κB信號(hào)通路表達(dá),進(jìn)而減輕炎癥反應(yīng),防治動(dòng)脈粥樣硬化的發(fā)生。方法：(1)8周齡ApoE-/-小鼠分別給予高脂飲食和普通飲食8,12,16,20,24周后,檢測(cè)血脂表達(dá)情況,油紅O染色觀察斑塊面積表達(dá),Western blot檢測(cè)高脂飲食組主動(dòng)脈中NF-κB信號(hào)通路P65和P50的表達(dá)。(2)8周齡ApoE-/-小鼠高脂飲食喂養(yǎng)16周后,于14,21,28,35,60天留取主動(dòng)脈組織,激光共聚焦顯微鏡和Western blot檢測(cè)主動(dòng)脈中eGFP的表達(dá)。(3)8周齡ApoE-/-小鼠尾靜脈分別注射rAAV9-eGFP和rAAV9-eGFP-R65,同時(shí)給予高脂飲食,16周后測(cè)定血脂水平,大體油紅O染色檢測(cè)斑塊面積表達(dá),Western blot檢測(cè)NF-κB信號(hào)通路P65和P50的表達(dá),ELISA檢測(cè)炎癥因子TNF-α和IL-6的表達(dá)。結(jié)果：(1)血脂檢測(cè)結(jié)果顯示高脂飲食組總膽固醇(TC)、甘油三酯(TG)和低密度脂蛋白(LDL-C)水平顯著高于普通飲食組,油紅O染色結(jié)果顯示高脂飲食組形成斑塊病變比普通飲食組更快,并可有效激活NF-κB信號(hào)通路。(2)激光共聚焦顯微鏡和Western blot結(jié)果顯示rAAV9-eGFP可有效轉(zhuǎn)染主動(dòng)脈血管,eGFP的表達(dá)高峰在第35天。(3)血清學(xué)和油紅O染色結(jié)果顯示轉(zhuǎn)導(dǎo)rAAV9-eGFP-R65可減輕血脂水平和斑塊表達(dá)。Western blot結(jié)果顯示采用高脂飲食16周后可有效激活NF-κB信號(hào)通路,且轉(zhuǎn)染rAAV9-eGFP-R65后可抑制NF-κB信號(hào)通路的活性。ELISA結(jié)果顯示,rAAV9-eGFP-R65轉(zhuǎn)染后可有效減輕炎癥因子TNF-α和IL-6表達(dá)。結(jié)論：高脂飲食可以加快動(dòng)脈粥樣硬化的病理進(jìn)程,并有效激活NF-κB信號(hào)通路,16周時(shí)可形成穩(wěn)定的動(dòng)脈粥樣硬化模型。rAAV9-eGFP可有效、持久、穩(wěn)定的轉(zhuǎn)染于動(dòng)脈粥樣硬化小鼠主動(dòng)脈中。rAAV9-eGFP-R65可減輕主動(dòng)脈中NF-κB信號(hào)通路的表達(dá)活性,有效減輕動(dòng)脈粥樣硬化病程進(jìn)展。
[Abstract]:Aim: to investigate whether recombinant adeno-associated virus serotype 9rAAV9 can inhibit the expression of NF- 魏 B signaling pathway in aorta and attenuate inflammation in ApoE-r-rAAV9 mice mediated by R65 ribozyme gene transduction. Methods to prevent and treat the occurrence of atherosclerosis. Methods ApoE-r-r-mice were given high-fat diet and normal diet for 24 weeks, respectively, and the expression of blood lipid was detected. The expression of NF- 魏 B signaling pathway p65 and p50 in aorta of high-fat diet group was detected by oil red O staining. The expression of p65 and P50 in aorta of 8-week-old ApoE-r-mice fed with high-fat diet for 16 weeks was observed. Laser confocal microscopy and Western blot were used to detect the expression of eGFP in aorta. The expression of eGFP was detected by injecting rAAV9-eGFP and rAAV9-eGFP-R65 into the caudal vein of 8-week-old ApoE-r-mice respectively. The serum lipids were measured after 16 weeks of hyperlipidemic diet. The expression of NF- 魏 B signaling pathway p65 and p50 by blot and the expression of inflammatory factors TNF- 偽 and IL-6 by Elisa. Low density lipoprotein (LDL-C) and low density lipoprotein (LDL-C) levels were significantly higher than those in the general diet group. The results of oil red O staining showed that plaque formation in high fat diet group was faster than that in general diet group. The results of laser confocal microscopy and Western blot showed that the peak expression of rAAV9-eGFP in aorta vessels was on the 35th day. The results of serology and oil red O staining showed that transduction of rAAV9-eGFP-R65 could reduce blood lipids. The level and plaque expression. Western blot showed that NF- 魏 B signaling pathway could be activated effectively after 16 weeks of high fat diet. The results of Elisa showed that rAAV9-eGFP-R65 could effectively reduce the expression of TNF- 偽 and IL-6 after transfection of rAAV9-eGFP-R65. Conclusion: high fat diet can accelerate the pathological process of atherosclerosis. The stable atherosclerosis model. RAAV9-eGFP could be effectively and persistently activated by activation of NF- 魏 B signaling pathway at 16 weeks. Stable transfection into the aorta of atherosclerotic mice could reduce the expression of NF- 魏 B signal pathway in aorta. Reduce the progression of atherosclerosis effectively.
【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R543.5
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本文編號(hào)：1683388