9型腺相关病毒导R65核酶基因靶向转导干预动脉粥样硬化的研究

发布时间：2018-03-29 23:35

  本文选题：9型腺相关病毒　切入点：NF-κB信号通路　出处：《新疆医科大学》2015年硕士论文

【摘要】：目的：以重组9型腺相关病毒(recombinant adeno-associated virus serotype 9,rAAV9)为载体介导R65核酶基因转导ApoE-/-小鼠,观察是否能抑制主动脉NF-κB信号通路表达,进而减轻炎症反应,防治动脉粥样硬化的发生。方法：(1)8周龄ApoE-/-小鼠分别给予高脂饮食和普通饮食8,12,16,20,24周后,检测血脂表达情况,油红O染色观察斑块面积表达,Western blot检测高脂饮食组主动脉中NF-κB信号通路P65和P50的表达。(2)8周龄ApoE-/-小鼠高脂饮食喂养16周后,于14,21,28,35,60天留取主动脉组织,激光共聚焦显微镜和Western blot检测主动脉中eGFP的表达。(3)8周龄ApoE-/-小鼠尾静脉分别注射rAAV9-eGFP和rAAV9-eGFP-R65,同时给予高脂饮食,16周后测定血脂水平,大体油红O染色检测斑块面积表达,Western blot检测NF-κB信号通路P65和P50的表达,ELISA检测炎症因子TNF-α和IL-6的表达。结果：(1)血脂检测结果显示高脂饮食组总胆固醇(TC)、甘油三酯(TG)和低密度脂蛋白(LDL-C)水平显著高于普通饮食组,油红O染色结果显示高脂饮食组形成斑块病变比普通饮食组更快,并可有效激活NF-κB信号通路。(2)激光共聚焦显微镜和Western blot结果显示rAAV9-eGFP可有效转染主动脉血管,eGFP的表达高峰在第35天。(3)血清学和油红O染色结果显示转导rAAV9-eGFP-R65可减轻血脂水平和斑块表达。Western blot结果显示采用高脂饮食16周后可有效激活NF-κB信号通路,且转染rAAV9-eGFP-R65后可抑制NF-κB信号通路的活性。ELISA结果显示,rAAV9-eGFP-R65转染后可有效减轻炎症因子TNF-α和IL-6表达。结论：高脂饮食可以加快动脉粥样硬化的病理进程,并有效激活NF-κB信号通路,16周时可形成稳定的动脉粥样硬化模型。rAAV9-eGFP可有效、持久、稳定的转染于动脉粥样硬化小鼠主动脉中。rAAV9-eGFP-R65可减轻主动脉中NF-κB信号通路的表达活性,有效减轻动脉粥样硬化病程进展。
[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- 伪 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- 伪 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.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R543.5
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