载脂蛋白E基因敲除(APOE-/-)小鼠基因的鉴定方法及模型应用
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摘要
背景:载脂蛋白E基因敲除小鼠是广泛应用于动脉粥样硬化研究的理想模型,也有用于其他疾病的相关报道,但是缺乏对其模型应用的总体概括。目的:系统总结载脂蛋白E基因敲除小鼠在疾病模型中的应用情况。方法:将南京大学生物模式中心所引进的SPF级载脂蛋白E基因敲除小鼠雌雄各6只饲养于SPF实验室,按照1∶1搭配进行保种繁殖,采用煮沸裂解法以提取鼠尾组织基因组DNA,采用PCR法检测其基因型。将基因鉴定为载脂蛋白E基因敲除雄性小鼠10只普通饲料喂养至8周龄,继而予以西方饮食饲料(北京华阜康公司提供,型号:H10141,脂肪21%,胆固醇0.15%)连续喂养至25周龄,同时选用8周龄C57/6 J野生型雄性小鼠10只普通饲料喂养至25周龄。结果与结论:(1)基因鉴定:琼脂糖凝胶电泳显示载脂蛋白E基因敲除小鼠PCR产物与预期的目的基因片段的大小一致;(2)相关指标变化:与野生小鼠相比,载脂蛋白E基因敲除小鼠的体质量、肝脏质量、脾脏质量、腹部脂肪质量、肝指数、血清中总胆固醇、三酰甘油、低密度脂蛋白胆固醇、高密度脂蛋白胆固醇、谷丙转氨酶及谷草转氨酶明显升高(P <0.05);(3)组织学改变:载脂蛋白E基因敲除小鼠苏木精-伊红染色主动脉弓、主动脉瓣、颈总动脉部位有明显的斑块,野生鼠无斑块;载脂蛋白E基因敲除小鼠肝脏苏木精-伊红染色有大小不等的空泡,细胞核被挤到一边,野生鼠肝脏核大居中,胞浆红染,无空泡现象;(4)结果证实,实验采用煮沸裂解法以提取DNA,采用PCR法检测成功鉴定了载脂蛋白E基因敲除小鼠的基因型。载脂蛋白E基因敲除小鼠是研究动脉粥样硬化、脂肪肝、高血脂、高胆固醇血症及肥胖等许多疾病的理想模型。
BACKGROUND: Apolipoprotein E knockout mice are an ideal model of atherosclerosis, which has been reported in the other diseases. OBJECTIVE: To systematically summarize the application of apolipoprotein E knockout mice in disease models. METHODS: Six male and six female APOE-/-mice introduced from the Biomedical Research Center of Nanjing University were bred in each cage at 1:1 in the SPF laboratory. The genomic DNA was extracted from mouse tail tissue by the method of boiling lysis, and the genotypes were detected by PCR. The APOE-/-mice were fed with normal diet for 8 weeks, and then fed with a Western diet(provided by Beijing Huafukang Bioscience Co., Ltd., No. H10141, 21% of fat, 0.15% of cholesterol) to 25 week old. At the same time, 10 8-week-old C57/6J mice were fed with a normal diet to 25 weeks old. RESULTS AND CONCLUSION: Genetic identification: agarose gel electrophoresis results indicated that the size of molecular weight of the PCR products from APOE-/-gene knockout mice was consistent with the expected target gene fragment and of different genotypes successfully. Changes of related indicators: compared with the wild type mice, the body mass, liver weight, spleen weight, abdominal fat weight, liver index, and serum levels of total cholesterol, triacylglycerol, low density lipoprotein cholesterol, high density lipoprotein cholesterol, alanine transaminase, and aspartate transaminase in the APOE-/-mice were significantly increased(P < 0.05). The histological changes: APOE-/-mice showed obvious plaques in the aortic arch, aortic valve and common carotid artery by hematoxylin-eosin staining, but no plaques in the wild type mice. The liver of APOE-/-mice had vacuoles of varying sizes, and the nucleus was pushed aside. The nucleus of wild mice liver was in the middle, the cytoplasm was red, and there was no vacuole phenomenon. These results suggest that PCR can fast and reliably identify the genotypes of apolipoprotein E knockout mice using genomic DNA extracted by boiling lysis. APOE-/-mice are ideal models for studying atherosclerosis, fatty liver, hyperlipidemia, hypercholesterolemia, obesity and many other diseases.
BACKGROUND: Apolipoprotein E knockout mice are an ideal model of atherosclerosis, which has been reported in the other diseases. OBJECTIVE: To systematically summarize the application of apolipoprotein E knockout mice in disease models. METHODS: Six male and six female APOE-/-mice introduced from the Biomedical Research Center of Nanjing University were bred in each cage at 1:1 in the SPF laboratory. The genomic DNA was extracted from mouse tail tissue by the method of boiling lysis, and the genotypes were detected by PCR. The APOE-/-mice were fed with normal diet for 8 weeks, and then fed with a Western diet(provided by Beijing Huafukang Bioscience Co., Ltd., No. H10141, 21% of fat, 0.15% of cholesterol) to 25 week old. At the same time, 10 8-week-old C57/6J mice were fed with a normal diet to 25 weeks old. RESULTS AND CONCLUSION: Genetic identification: agarose gel electrophoresis results indicated that the size of molecular weight of the PCR products from APOE-/-gene knockout mice was consistent with the expected target gene fragment and of different genotypes successfully. Changes of related indicators: compared with the wild type mice, the body mass, liver weight, spleen weight, abdominal fat weight, liver index, and serum levels of total cholesterol, triacylglycerol, low density lipoprotein cholesterol, high density lipoprotein cholesterol, alanine transaminase, and aspartate transaminase in the APOE-/-mice were significantly increased(P < 0.05). The histological changes: APOE-/-mice showed obvious plaques in the aortic arch, aortic valve and common carotid artery by hematoxylin-eosin staining, but no plaques in the wild type mice. The liver of APOE-/-mice had vacuoles of varying sizes, and the nucleus was pushed aside. The nucleus of wild mice liver was in the middle, the cytoplasm was red, and there was no vacuole phenomenon. These results suggest that PCR can fast and reliably identify the genotypes of apolipoprotein E knockout mice using genomic DNA extracted by boiling lysis. APOE-/-mice are ideal models for studying atherosclerosis, fatty liver, hyperlipidemia, hypercholesterolemia, obesity and many other diseases.
引文
[1]Huang Y,Mahley RW.Apolipoprotein E:structure and function in lipid metabolism,neurobiology,and Alzheimer’s diseases.Neurobiol Dis.2014;72 Pt A:3-12.
[2]Tani M,MateraR,Horvath KV,et al.The influence of apo E-deficiency and LDL-receptor-deficiency on the HDLsubpopulation profile in mice and in humans.Atherosclerosis.2014;233(1):39-44.
[3]梁璐,洪瑞云,周敏,等.APOR基因敲除小鼠动脉粥样硬化模型构建[J].赣南医学院学报,2017,37(3):337-338.
[4]许增祥,谢闵,黄小梅.ApoE-/-小鼠颈总动脉斑块Fractalkine与TLR4的表达[J].中国实验动物学报,2018,23(1):76-79.
[5]熊敏,琪陈瑜,张腾.三七皂苷R1抑制ApoE基因敲除小鼠肝脏脂质沉积的作用及其机制研究[J].上海中医药杂志,2018,52(5):65-70.
[6]朱景平,卫欣妤,许晓乐.二氢杨梅素对高脂喂养Apoe(-/-)小鼠体内胆固醇逆向转运的影响[J].中国药理学通报,2018(11):1610-1616.
[7]耿涛,房玉涛,张云,等.新活络效灵丹对ApoE基因敲除早期动脉粥样硬化小鼠Cav-1、SR-BI的影响[J].中国中医基础医学杂志,2018,24(9):1212-1215.
[8]吴卫东.ApN/TNF-α信号途径在有氧运动防治ApoE-/-小鼠动脉粥样硬化过程中的抗炎症作用[J].中国老年学杂志,2018,38(16):3987-3989.
[9]欧海龙,张礼林,何晓兰,等.ApoE(-/-)小鼠动脉粥样硬化模型的建立[J].生命科学研究,2015,19(02):141-144.
[10]张晓明,王鹏,罗绍忠,等.普洱熟茶茶多糖对ApoE-/-小鼠动脉粥样硬化的作用研究[J].中国民族民间医药,2018,27(8):27-30.
[11]韩世飞,牟丽娜,陆芳.白藜芦醇在高脂饮食诱导ApoE基因敲除小鼠动脉粥样硬化形成中的作用及其相关机制研究[J].临床心血管病杂志,2016,32(5):505-508.
[12]Moore KJ,Sheedy FJ,Fisher EA.Macrophages in atherosclerosis:a dynamic balance.Nat Rev Immunol.2013;13(10):709-721.
[13]刘玉晖,侯贝贝,游宇,等.补阳还五汤稳定APOE-/-小鼠动脉粥样硬化易损斑块的作用机制[J].中国实验方剂学杂志,2018,24(15):112-114.
[14]张晓明,王鹏,罗绍忠,等.邓老冠心方对ApoE-/-小鼠动脉粥样硬化不稳定斑块NOX4的影响[J].广州中医药大学学报,2018,35(6):1065-1069.
[15]李振华,张英砂,周应毕,等.碱性裂解液提取DAN法在环氧化酶亚型基因敲除小鼠基因型鉴定中的应用[J].中国医学创新,2014,11(6):6-7.
[16]沈伟,刘湘绪,施海明,等.一种新型apoE-/-小鼠动脉粥样硬化和心肌梗死双模型的建立[J].中西医结合心脑血管病杂志,2018,16(17):2473-2478.
[17]Nagashima M,Watanabe T,Shiraishi Y,et al.Chronic infusion of salusin-alpha and-beta exerts opposite effects on atherosclerotic lesion development in apolipoprotein E-deficient mice.Atherosclerosis.2010;212(1):70-77.
[18]Raygani A V,Rahimi Z,Kharazi H,et al.Association between apolipoprotein E polymorphism and serum lipid and apolipoprotein levels with Alzheimer's disease.Neurosci Lett.2006;408(1):68-72.
[19]谢雪姣,徐佳,李亚梅.等.加味二陈汤对ApoE-/-小鼠脂质代谢及肝细胞形态的影响[J].中国比较医学杂志,2015,25(4):44-47.
[20]刘海红.二氢杨梅素对高脂诱导ApoE-/-小鼠肝脏损伤的保护作用及机制[J].中成药,2017,39(12):2448-2953
[21]Rademakers T,van der Vorst EP,et al.Adventitial lymphatic capillary expansion impacts on plaque T cell accumulation in atherosclerosis.Sci Rep.2017;7:45263.
[22]陈依楚,李亮,衡先培,等.丹瓜方对ApoE-/-糖尿病模型小鼠脑组织Caspase-3蛋白及Bcl-2、Bax mRNA表达水平的影响[J].中国中西医结合杂志,2017,37(12):1476-1481.
[23]王热华.白藜芦醇对糖尿病ApoE-/-小鼠动脉粥样硬化的影响及机制研究[D].广州:南方医科大学,2017.
[24]冷蔚玲.达格列净对糖尿病ApoE-/-小鼠动脉粥样硬化的作用及其机制研究[D].重庆:第三军医大学,2017.
[25]张彦,马双陶,杨永健.糖尿病合并动脉粥样硬化小鼠模型的建立[J].中国动脉硬化杂志,2014,22(2):186-189.
[26]杨小惠.ApoE4在阿尔茨海默病中的研究进展[J].临床与病理杂志,2017,37(3):627-631.
[27]侯江淇,张欣,龙倩,等.高脂血症与Aβ的协同作用促进引发阿尔茨海默症[J].中国药理学通报,2017,33(4):498.
[28]刘钧天,崔莹雪,黄玉海,等.艾烟与香烟对载脂蛋白E基因敲除小鼠学习记忆功能与海马β淀粉样蛋白沉淀的影响[J].环球中医药,2015,8(5):527-531.
[29]Chapman S,Michaelson DM.Spacific neurochemical derangements of brain projecting neurons in apolipoprotein E-deficient mice.J Neurochem.1998;70(2):708-714.
[30]杨秀丽,张文高,郑广娟.脂欣康胶囊改善ApoE基因敲除小鼠学习记忆行为障碍的水迷宫观察[J].世界中西医结合杂志,2007,2(5):274-276.
[31]张宁,倪普,徐凌凡,等.ApoE基因敲除小鼠海马神经元内β淀粉样蛋白的表达变化[J].安徽医科大学学报,2011,46(6):531-534.
[2]Tani M,MateraR,Horvath KV,et al.The influence of apo E-deficiency and LDL-receptor-deficiency on the HDLsubpopulation profile in mice and in humans.Atherosclerosis.2014;233(1):39-44.
[3]梁璐,洪瑞云,周敏,等.APOR基因敲除小鼠动脉粥样硬化模型构建[J].赣南医学院学报,2017,37(3):337-338.
[4]许增祥,谢闵,黄小梅.ApoE-/-小鼠颈总动脉斑块Fractalkine与TLR4的表达[J].中国实验动物学报,2018,23(1):76-79.
[5]熊敏,琪陈瑜,张腾.三七皂苷R1抑制ApoE基因敲除小鼠肝脏脂质沉积的作用及其机制研究[J].上海中医药杂志,2018,52(5):65-70.
[6]朱景平,卫欣妤,许晓乐.二氢杨梅素对高脂喂养Apoe(-/-)小鼠体内胆固醇逆向转运的影响[J].中国药理学通报,2018(11):1610-1616.
[7]耿涛,房玉涛,张云,等.新活络效灵丹对ApoE基因敲除早期动脉粥样硬化小鼠Cav-1、SR-BI的影响[J].中国中医基础医学杂志,2018,24(9):1212-1215.
[8]吴卫东.ApN/TNF-α信号途径在有氧运动防治ApoE-/-小鼠动脉粥样硬化过程中的抗炎症作用[J].中国老年学杂志,2018,38(16):3987-3989.
[9]欧海龙,张礼林,何晓兰,等.ApoE(-/-)小鼠动脉粥样硬化模型的建立[J].生命科学研究,2015,19(02):141-144.
[10]张晓明,王鹏,罗绍忠,等.普洱熟茶茶多糖对ApoE-/-小鼠动脉粥样硬化的作用研究[J].中国民族民间医药,2018,27(8):27-30.
[11]韩世飞,牟丽娜,陆芳.白藜芦醇在高脂饮食诱导ApoE基因敲除小鼠动脉粥样硬化形成中的作用及其相关机制研究[J].临床心血管病杂志,2016,32(5):505-508.
[12]Moore KJ,Sheedy FJ,Fisher EA.Macrophages in atherosclerosis:a dynamic balance.Nat Rev Immunol.2013;13(10):709-721.
[13]刘玉晖,侯贝贝,游宇,等.补阳还五汤稳定APOE-/-小鼠动脉粥样硬化易损斑块的作用机制[J].中国实验方剂学杂志,2018,24(15):112-114.
[14]张晓明,王鹏,罗绍忠,等.邓老冠心方对ApoE-/-小鼠动脉粥样硬化不稳定斑块NOX4的影响[J].广州中医药大学学报,2018,35(6):1065-1069.
[15]李振华,张英砂,周应毕,等.碱性裂解液提取DAN法在环氧化酶亚型基因敲除小鼠基因型鉴定中的应用[J].中国医学创新,2014,11(6):6-7.
[16]沈伟,刘湘绪,施海明,等.一种新型apoE-/-小鼠动脉粥样硬化和心肌梗死双模型的建立[J].中西医结合心脑血管病杂志,2018,16(17):2473-2478.
[17]Nagashima M,Watanabe T,Shiraishi Y,et al.Chronic infusion of salusin-alpha and-beta exerts opposite effects on atherosclerotic lesion development in apolipoprotein E-deficient mice.Atherosclerosis.2010;212(1):70-77.
[18]Raygani A V,Rahimi Z,Kharazi H,et al.Association between apolipoprotein E polymorphism and serum lipid and apolipoprotein levels with Alzheimer's disease.Neurosci Lett.2006;408(1):68-72.
[19]谢雪姣,徐佳,李亚梅.等.加味二陈汤对ApoE-/-小鼠脂质代谢及肝细胞形态的影响[J].中国比较医学杂志,2015,25(4):44-47.
[20]刘海红.二氢杨梅素对高脂诱导ApoE-/-小鼠肝脏损伤的保护作用及机制[J].中成药,2017,39(12):2448-2953
[21]Rademakers T,van der Vorst EP,et al.Adventitial lymphatic capillary expansion impacts on plaque T cell accumulation in atherosclerosis.Sci Rep.2017;7:45263.
[22]陈依楚,李亮,衡先培,等.丹瓜方对ApoE-/-糖尿病模型小鼠脑组织Caspase-3蛋白及Bcl-2、Bax mRNA表达水平的影响[J].中国中西医结合杂志,2017,37(12):1476-1481.
[23]王热华.白藜芦醇对糖尿病ApoE-/-小鼠动脉粥样硬化的影响及机制研究[D].广州:南方医科大学,2017.
[24]冷蔚玲.达格列净对糖尿病ApoE-/-小鼠动脉粥样硬化的作用及其机制研究[D].重庆:第三军医大学,2017.
[25]张彦,马双陶,杨永健.糖尿病合并动脉粥样硬化小鼠模型的建立[J].中国动脉硬化杂志,2014,22(2):186-189.
[26]杨小惠.ApoE4在阿尔茨海默病中的研究进展[J].临床与病理杂志,2017,37(3):627-631.
[27]侯江淇,张欣,龙倩,等.高脂血症与Aβ的协同作用促进引发阿尔茨海默症[J].中国药理学通报,2017,33(4):498.
[28]刘钧天,崔莹雪,黄玉海,等.艾烟与香烟对载脂蛋白E基因敲除小鼠学习记忆功能与海马β淀粉样蛋白沉淀的影响[J].环球中医药,2015,8(5):527-531.
[29]Chapman S,Michaelson DM.Spacific neurochemical derangements of brain projecting neurons in apolipoprotein E-deficient mice.J Neurochem.1998;70(2):708-714.
[30]杨秀丽,张文高,郑广娟.脂欣康胶囊改善ApoE基因敲除小鼠学习记忆行为障碍的水迷宫观察[J].世界中西医结合杂志,2007,2(5):274-276.
[31]张宁,倪普,徐凌凡,等.ApoE基因敲除小鼠海马神经元内β淀粉样蛋白的表达变化[J].安徽医科大学学报,2011,46(6):531-534.
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