CN111374093A - 超级肥胖小鼠的构建与鉴定方法 - Google Patents
超级肥胖小鼠的构建与鉴定方法 Download PDFInfo
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Abstract
本发明公开了超级肥胖小鼠的构建与鉴定方法,包括雄性PPARα‑/‑小鼠、雌性OB/ob小鼠、PCR扩增仪、DNA扩增产物、2%琼脂糖凝胶电泳、鉴定依据、瘦素基因,所述PCR扩增仪的内部设置有PCR试剂盒,所述PCR试剂盒的内部设置有原料,所述原料包括DNA双链、DNA单链、引物、TapDNA聚合酶、腺嘌呤脱氧核苷酸、鸟嘌呤脱氧核苷酸、胞嘧啶脱氧核苷酸、胸腺嘧啶脱氧核苷酸,所述鉴定依据包括白色脂肪组织、棕色脂肪组织、甘油三酯、胆固醇、肝脏、脂肪肝、葡萄糖、胰岛素。本发明还公开了超级肥胖小鼠的鉴定方法。本发明的优点在于:PPARαΔob/ob超级肥胖小鼠为研究肥胖及其代谢综合症以及PPARα信号通路在肥胖发生发展过程中的作用研究提供了更加理想的模型。
Description
技术领域
本发明涉及医学技术领域,具体为超级肥胖小鼠的构建与鉴定方法。
背景技术
随着物质生活水平的提高和生活模式的改变,肥胖、高血压、高血脂、糖尿病、动脉粥样硬化等代谢综合症已严重危害了人类的健康,目前,用于研究肥胖及其代谢综合症的试验动物模型主要包括自发型、诱发型和遗传修饰型肥胖动物模型,常用的自发性肥胖小鼠模型主要有ob/ob小鼠、db/db小鼠、Yellow(Ay/a)小鼠等,其中,ob/ob小鼠肥胖的发生与6号常染色体上的瘦素基因突变有关,其特征为贪食、中等肥胖、高血糖、高胰岛素、高血脂、葡萄糖和胰岛素抵抗、代谢率低、脂肪肝等,而db/db小鼠是由位于4号常染色体上的Leptin受体基因突变诱导的自发型II型糖尿病小鼠,其特征与ob/ob小鼠相似,此外,ob/ob和db/db小鼠的肥胖特征均以常染色体隐性方式遗传,且与人类肥胖症及II型糖尿病的病理特征非常相似,Yellow(Ay/a)小鼠与位于2号常染色体上的毛色调控基因ASIP突变有关,其特征为饮食亢进、代谢率低、肥胖、癌症等,且其肥胖程度随毛色深度增加而增加,自发型肥胖小鼠模型可广泛应用于能量代谢紊乱调控机制、肥胖及糖尿病等代谢综合症的药物研发等方面的研究,但该类小鼠模型多为单基因突变引发的遗传性肥胖模型,具有随机性、不可控性等特点,而人类肥胖罕有单基因突变,多为多基因相关,且受环境因素影响较大。诱发型肥胖动物模型主要通过高能饮食诱导,如高脂、高糖日粮等,该类模型可模拟高能量饮食的环境因素,与人类肥胖具有较好的可比性,然而,动物对高能膳食诱导肥胖的易感性存在个体差异,因此,筛选并建立纯系肥胖动物模型至关重要,为此我们提出了超级肥胖小鼠的构建与鉴定方法。
发明内容
本发明所要解决的技术问题在于增加额外的播放时延并且播放流畅度较差,对于播放过程中的网络状态不能够及时向编码端传递,产生过度跳帧导致的画面质量下降的现象和缓冲区过度扩大导致的播放时延的问题。
本发明采用以下技术方案解决上述技术问题:提供了超级肥胖小鼠的构建与鉴定方法,包括雄性PPARα-/-小鼠、雌性OB/ob小鼠、PCR扩增仪、DNA扩增产物、2%琼脂糖凝胶电泳、鉴定依据、瘦素基因,所述PCR扩增仪的内部设置有PCR试剂盒,所述PCR试剂盒的内部设置有原料,所述原料包括DNA双链、DNA单链、引物、TapDNA聚合酶、腺嘌呤脱氧核苷酸、鸟嘌呤脱氧核苷酸、胞嘧啶脱氧核苷酸、胸腺嘧啶脱氧核苷酸,所述鉴定依据包括白色脂肪组织、棕色脂肪组织、甘油三酯、胆固醇、肝脏、脂肪肝、葡萄糖、胰岛素。
优选的,所述雄性PPARα-/-小鼠与雌性OB/ob小鼠杂交得到雄性PPARα+/-/OB/ob双杂合子小鼠与雌性PPARα+/-/OB/ob双杂合子小鼠,所述雄性PPARα+/-/OB/ob双杂合子小鼠与雌性PPARα+/-/OB/ob双杂合子小鼠杂交得到PPARα∆ob/ob小鼠、PPARα野生型小鼠、杂合子小鼠、纯合子敲除小鼠、ob野生型、杂合子小鼠、纯合子敲除小鼠。
优选的,所述PPARα∆ob/ob小鼠的内部含有基因重组DNA。
优选的,所述DNA双链解离得到DNA单链,所述DNA单链通过引物与TapDNA聚合酶的作用与腺嘌呤脱氧核苷酸、鸟嘌呤脱氧核苷酸、胞嘧啶脱氧核苷酸、胸腺嘧啶脱氧核苷酸结合得到DNA扩增产物。
优选的,所述DNA扩增产物通过2%琼脂糖凝胶电泳进行检测,所述PPARα野生型小鼠显示143bp一条条带。
优选的,所述杂合子小鼠显示143 bp和280 bp两条条带,所述纯合子敲除小鼠显示280 bp一条条带。
优选的,所述ob野生型显示155 bp一条条带,所述杂合子小鼠显示55 bp、100 bp和155 bp三条条带。
优选的,所述纯合子敲除小鼠显示55 bp和100 bp两条条带。
本发明还提供了超级肥胖小鼠的构建与鉴定方法的方法,
a、正常日粮饲喂情况下,十六月龄纯合子敲除小鼠与PPARα野生型小鼠、ob野生型的体型差异不显著,而瘦素基因缺失显著促进了纯合子敲除小鼠的体脂沉积,使PPARα∆ob/ob小鼠呈现超级肥胖,六周龄之前,PPARα∆ob/ob小鼠和纯合子敲除小鼠的体重差异不显著,前者的体重略低于后者,六周龄之后,PPARα∆ob/ob小鼠小鼠的体重开始迅速增长,二十四周龄后,其体重显著高于纯合子敲除小鼠,,PPARα野生型小鼠、ob野生型与纯合子敲除小鼠的体重差异不显著,但显著低于纯合子敲除小鼠和PPARα∆ob/ob小鼠,PPARα野生型小鼠、ob野生型与纯合子敲除小鼠的采食量差异不显著,后者略低于前者,纯合子敲除小鼠的采食量显著高于PPARα野生型小鼠、ob野生型和纯合子敲除小鼠的采食量,PPARα∆ob/ob小鼠的采食量显著高于纯合子敲除小鼠的采食量,而显著低于纯合子敲除小鼠的采食量;
b、纯合子敲除小鼠呈现食欲过盛和肥胖,并伴随着高血糖症和高胰岛素血症,机体葡萄糖和胰岛素耐受性显著高于PPARα野生型小鼠、ob野生型,纯合子敲除小鼠体形正常,但血液中的葡萄糖水平降低,胰岛素水平升高,胰岛素参与调控脂肪细胞的生物学功能,而胰岛素抵抗在肥胖发生过程中发挥了重要作用,胰岛素可以促进脂肪组织从循环系统中吸收脂质蛋白,促进脂肪细胞生成脂质,当肥胖发生时,胰岛素信号通路遭到破坏,导致胰岛素抵抗;
c、葡萄糖与胰岛素耐受性结果显示,纯合子敲除小鼠显著高于PPARα野生型小鼠、纯合子敲除小鼠;
d、随着年龄增加,PPARα野生型小鼠、ob野生型、纯合子敲除小鼠和PPARα∆ob/ob小鼠的脂肪重量均增加,且纯合子敲除小鼠和PPARα∆ob/ob小鼠的白色脂肪组织、棕色脂肪组织的重量及其增长幅度显著高于PPARα野生型小鼠、ob野生型与纯合子敲除小鼠,三月龄纯合子敲除小鼠和PPARα∆ob/ob小鼠的白色脂肪组织、棕色脂肪组织的重量分别略高于PPARα野生型小鼠、ob野生型与纯合子敲除小鼠;
e、六月龄小鼠的白色脂肪组织重量分别为:PPARα野生型小鼠0.56 g,纯合子敲除小鼠1.19 g,纯合子敲除小鼠4.22 g,PPARα∆ob/ob小鼠5.87 g,纯合子敲除小鼠显著高于PPARα野生型小鼠,PPARα∆ob/ob小鼠显著高于纯合子敲除小鼠,六月龄小鼠的棕色脂肪组织重量分别为:PPARα野生型小鼠0.14 g,纯合子敲除小鼠0.24 g,纯合子敲除小鼠1.19 g,PPARα∆ob/ob小鼠小鼠1.37 g;
f、肥胖发生过程中,多余的能量首先以甘油三酯的形式储存于白色脂肪组织与棕色脂肪组织中,当白色脂肪组织与棕色脂肪组织的储存能力达到饱和时,剩余的脂肪再储存于肝脏中,,随着年龄增长,纯合子敲除小鼠肝脏内积累的脂肪逐渐增多,并且纯合子敲除小鼠肝脏脂肪变性程度高于杂合子小鼠,表现为脂滴增大,而PPARα野生型小鼠和纯合子敲除小鼠肝脏内无脂肪变性发生,此外,纯合子敲除小鼠的肝脏重量显著高于PPARα野生型小鼠和纯合子敲除小鼠,表明其肝脏发生了脂肪变性,并且纯合子敲除小鼠的肝脏重量高于纯合子敲除小鼠;
g、通过酶试剂盒检测三月龄PPARα野生型小鼠、纯合子敲除小鼠、纯合子敲除小鼠的肝脏内甘油三酯和胆固醇含量,结果显示,纯合子敲除小鼠的肝脏内甘油三酯含量显著高于PPARα野生型小鼠、纯合子敲除小鼠。
与现有技术相比,本发明提供了超级肥胖小鼠的构建与鉴定方法,具备以下有益效果:该超级肥胖小鼠的构建与鉴定方法,过氧化物酶体增殖物激活受体α(PPARα)是一类由配体激活的核转录因子,PPARα在脂肪酸氧化代谢能力强的组织中表达,如肝脏、心脏、肾脏、棕色脂肪组织、肌肉、小肠和大肠,其在调控机体脂质代谢和能量平衡过程中发挥了重要作用,因此,为研究PPARα调控的信号通路在肥胖发生过程中的作用,本发明构建了PPARα与ob双敲除(PPARα∆ob/ob)小鼠,并揭示了PPARα缺失对ob/ob小鼠肥胖综合症的影响,作为饱感激素,瘦素基因可参与调控机体的食欲、能量平衡和免疫反应,脂肪细胞内瘦素基因的水平与脂质成分和脂肪细胞体积密切相关,瘦素基因缺失型ob/ob小鼠采食量升高,能量摄入水平远远高于能量消耗水平,多余的能量以脂肪的形式储存于脂肪组织和肝脏中,最终导致棕色脂肪组织白色化和脂肪肝,而瘦素基因在调控机体脂质代谢和能量平衡过程中发挥了重要作用,其功能缺失导致脂肪酸氧化代谢体系受到破坏,从而引发脂质在脂肪组织和肝脏内堆积,本发明通过构建瘦素基因缺失型ob/ob小鼠,发现与ob/ob小鼠相比,PPARαΔob/ob小鼠的脂肪组织和肝脏内脂质沉积能力更强,呈现超级肥胖和脂肪肝,同时胰岛素和葡萄糖耐受性增加,肥胖综合症加重,因此,PPARαΔob/ob超级肥胖小鼠为研究肥胖及其代谢综合症以及PPARα信号通路在肥胖发生发展过程中的作用研究提供了更加理想的模型。
附图说明
图1为本发明流程图;
图2为本发明鉴定依据结构图。
图中:1、雄性PPARα-/-小鼠;2、雌性OB/ob小鼠;3、雄性PPARα+/-/OB/ob双杂合子小鼠;4、雌性PPARα+/-/OB/ob双杂合子小鼠;5、PPARα∆ob/ob小鼠;6、基因重组DNA;7、PCR扩增仪;8、PCR试剂盒;9、原料;10、DNA双链;11、DNA单链;12、引物;13、TapDNA聚合酶;14、腺嘌呤脱氧核苷酸;15、鸟嘌呤脱氧核苷酸;16、胞嘧啶脱氧核苷酸;17、胸腺嘧啶脱氧核苷酸;18、DNA扩增产物;19、2%琼脂糖凝胶电泳;20、鉴定依据;21、白色脂肪组织;22、棕色脂肪组织;23、甘油三酯;24、胆固醇;25、肝脏;26、脂肪肝;27、葡萄糖;28、胰岛素;29、PPARα野生型(PPARα+/+)小鼠;30、杂合子(PPARα+/-)小鼠;31、纯合子敲除(PPARα-/-)小鼠;32、ob野生型(OB/OB);33、杂合子(OB/ob)小鼠;34、纯合子敲除(ob/ob)小鼠;35、瘦素基因。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
请参阅图1-2,超级肥胖小鼠的构建与鉴定方法,包括雄性PPARα-/-小鼠1、雌性OB/ob小鼠2、PCR扩增仪7、DNA扩增产物18、2%琼脂糖凝胶电泳19、鉴定依据20、瘦素基因35,PCR扩增仪7的内部设置有PCR试剂盒8,PCR试剂盒8的内部设置有原料9,原料9包括DNA双链10、DNA单链11、引物12、TapDNA聚合酶13、腺嘌呤脱氧核苷酸14、鸟嘌呤脱氧核苷酸15、胞嘧啶脱氧核苷酸16、胸腺嘧啶脱氧核苷酸17,鉴定依据20包括白色脂肪组织21、棕色脂肪组织22、甘油三酯23、胆固醇24、肝脏25、脂肪肝26、葡萄糖27、胰岛素28。
雄性PPARα-/-小鼠1与雌性OB/ob小鼠2杂交得到雄性PPARα+/-/OB/ob双杂合子小鼠3与雌性PPARα+/-/OB/ob双杂合子小鼠4,雄性PPARα+/-/OB/ob双杂合子小鼠3与雌性PPARα+/-/OB/ob双杂合子小鼠4杂交得到PPARα∆ob/ob小鼠5、PPARα野生型PPARα+/+小鼠29、杂合子PPARα+/-小鼠30、纯合子敲除PPARα-/-小鼠31、ob野生型OB/OB32、杂合子OB/ob小鼠33、纯合子敲除ob/ob小鼠34;PPARα∆ob/ob小鼠5的内部含有基因重组DNA6;DNA双链10解离得到DNA单链11,DNA单链11通过引物12与TapDNA聚合酶13的作用与腺嘌呤脱氧核苷酸14、鸟嘌呤脱氧核苷酸15、胞嘧啶脱氧核苷酸16、胸腺嘧啶脱氧核苷酸17结合得到DNA扩增产物18;DNA扩增产物18通过2%琼脂糖凝胶电泳19进行检测,PPARα野生型PPARα+/+小鼠29显示143bp一条条带;杂合子PPARα+/-小鼠30显示143 bp和280 bp两条条带,纯合子敲除PPARα-/-小鼠31显示280 bp一条条带;ob野生型OB/OB32显示155 bp一条条带,杂合子OB/ob小鼠33显示55 bp、100 bp和155 bp三条条带;纯合子敲除ob/ob小鼠34显示55 bp和100bp两条条带。
工作时,将雄性PPARα-/-小鼠1与雌性OB/ob小鼠2杂交得到雄性PPARα+/-/OB/ob双杂合子小鼠3、雌性PPARα+/-/OB/ob双杂合子小鼠4,将雄性PPARα+/-/OB/ob双杂合子小鼠3与雌性PPARα+/-/OB/ob双杂合子小鼠4杂交得到PPARα∆ob/ob小鼠5、PPARα野生型PPARα+/+小鼠29、杂合子PPARα+/-小鼠30、纯合子敲除PPARα-/-小鼠31、ob野生型OB/OB32、杂合子OB/ob小鼠33、纯合子敲除ob/ob小鼠34,然后提取上述小鼠的基因重组DNA6,将基因重组DNA6与引物12、TapDNA聚合酶13、腺嘌呤脱氧核苷酸14、鸟嘌呤脱氧核苷酸15、胞嘧啶脱氧核苷酸16、胸腺嘧啶脱氧核苷酸17加入PCR扩增仪7中,基因重组DNA6中的DNA双链10在加热时解离得到DNA单链11,DNA单链11在引物12与TapDNA聚合酶13的作用下与腺嘌呤脱氧核苷酸14、鸟嘌呤脱氧核苷酸15、胞嘧啶脱氧核苷酸16、胸腺嘧啶脱氧核苷酸17互相配对,形成DNA扩增产物18,将DNA扩增产物18通过2%琼脂糖凝胶电泳19进行检测,较为实用。
综上所述,过氧化物酶体增殖物激活受体α(PPARα)是一类由配体激活的核转录因子,PPARα在脂肪酸氧化代谢能力强的组织中表达,如肝脏25、心脏、肾脏、棕色脂肪组织、肌肉、小肠和大肠,其在调控机体脂质代谢和能量平衡过程中发挥了重要作用,因此,为研究PPARα调控的信号通路在肥胖发生过程中的作用,本发明构建了PPARα与纯合子敲除(PPARα-/-)小鼠31,并揭示了PPARα缺失对ob/ob小鼠肥胖综合症的影响,作为饱感激素,瘦素基因35可参与调控机体的食欲、能量平衡和免疫反应,脂肪细胞内瘦素基因35的水平与脂质成分和脂肪细胞体积密切相关,瘦素基因35缺失型ob/ob小鼠采食量升高,能量摄入水平远远高于能量消耗水平,多余的能量以脂肪的形式储存于脂肪组织和肝脏25中,最终导致棕色脂肪组织白色化和脂肪肝,而瘦素基因35在调控机体脂质代谢和能量平衡过程中发挥了重要作用,其功能缺失导致脂肪酸氧化代谢体系受到破坏,从而引发脂质在脂肪组织和肝脏内堆积,本发明通过构建瘦素基因35缺失型ob/ob小鼠,发现与ob/ob小鼠相比,PPARαΔob/ob小鼠的脂肪组织和肝脏内脂质沉积能力更强,呈现超级肥胖和脂肪肝,同时胰岛素28与葡萄糖耐27受性增加,肥胖综合症加重,因此,PPARαΔob/ob超级肥胖小鼠为研究肥胖及其代谢综合症以及PPARα信号通路在肥胖发生发展过程中的作用研究提供了更加理想的模型。
本发明还提供了超级肥胖小鼠的构建与鉴定方法的方法,
a、正常日粮饲喂情况下,十六月龄纯合子敲除PPARα-/-小鼠31与PPARα野生型PPARα+/+小鼠29、ob野生型OB/OB32的体型差异不显著,而瘦素基因35缺失显著促进了纯合子敲除ob/ob小鼠34的体脂沉积,使PPARα∆ob/ob小鼠5呈现超级肥胖,六周龄之前,PPARα∆ob/ob小鼠5和纯合子敲除ob/ob小鼠34的体重差异不显著,前者的体重略低于后者,六周龄之后,PPARα∆ob/ob小鼠5小鼠的体重开始迅速增长,二十四周龄后,其体重显著高于纯合子敲除ob/ob小鼠34,,PPARα野生型PPARα+/+小鼠29、ob野生型OB/OB32与纯合子敲除PPARα-/-小鼠31的体重差异不显著,但显著低于纯合子敲除ob/ob小鼠34和PPARα∆ob/ob小鼠5,PPARα野生型PPARα+/+小鼠29、ob野生型OB/OB32与纯合子敲除PPARα-/-小鼠31的采食量差异不显著,后者略低于前者,纯合子敲除ob/ob小鼠34的采食量显著高于PPARα野生型PPARα+/+小鼠29、ob野生型OB/OB32和纯合子敲除PPARα-/-小鼠31的采食量,PPARα∆ob/ob小鼠5的采食量显著高于纯合子敲除PPARα-/-小鼠31的采食量,而显著低于纯合子敲除ob/ob小鼠34的采食量;
b、纯合子敲除ob/ob小鼠34呈现食欲过盛和肥胖,并伴随着高血糖症和高胰岛素血症,机体葡萄糖27和胰岛素28耐受性显著高于PPARα野生型PPARα+/+小鼠29、ob野生型OB/OB32,纯合子敲除PPARα-/-小鼠31体形正常,但血液中的葡萄糖27水平降低,胰岛素28水平升高,胰岛素28参与调控脂肪细胞的生物学功能,而胰岛素28抵抗在肥胖发生过程中发挥了重要作用,胰岛素28可以促进脂肪组织从循环系统中吸收脂质蛋白,促进脂肪细胞生成脂质,当肥胖发生时,胰岛素28信号通路遭到破坏,导致胰岛素28抵抗;
c、葡萄糖27与胰岛素28耐受性结果显示,纯合子敲除ob/ob小鼠34显著高于PPARα野生型PPARα+/+小鼠29、纯合子敲除PPARα-/-小鼠31;
d、随着年龄增加,PPARα野生型PPARα+/+小鼠29、ob野生型OB/OB32、纯合子敲除ob/ob小鼠34和PPARα∆ob/ob小鼠5的脂肪重量均增加,且纯合子敲除ob/ob小鼠34和PPARα∆ob/ob小鼠5的白色脂肪组织21、棕色脂肪组织22的重量及其增长幅度显著高于PPARα野生型PPARα+/+小鼠29、ob野生型OB/OB32与纯合子敲除PPARα-/-小鼠31,三月龄纯合子敲除PPARα-/-小鼠31和PPARα∆ob/ob小鼠5的白色脂肪组织21、棕色脂肪组织22的重量分别略高于PPARα野生型PPARα+/+小鼠29、ob野生型OB/OB32与纯合子敲除ob/ob小鼠34;
e、六月龄小鼠的白色脂肪组织21重量分别为:PPARα野生型PPARα+/+小鼠290.56 g,纯合子敲除PPARα-/-小鼠311.19 g,纯合子敲除ob/ob小鼠344.22 g,PPARα∆ob/ob小鼠55.87 g,纯合子敲除PPARα-/-小鼠31显著高于PPARα野生型PPARα+/+小鼠29,PPARα∆ob/ob小鼠5显著高于纯合子敲除ob/ob小鼠34,六月龄小鼠的棕色脂肪组织22重量分别为:PPARα野生型PPARα+/+小鼠290.14 g,纯合子敲除PPARα-/-小鼠310.24 g,纯合子敲除ob/ob小鼠341.19 g,PPARα∆ob/ob小鼠5小鼠1.37 g;
f、肥胖发生过程中,多余的能量首先以甘油三酯23的形式储存于白色脂肪组织21与棕色脂肪组织22中,当白色脂肪组织21与棕色脂肪组织22的储存能力达到饱和时,剩余的脂肪再储存于肝脏中,,随着年龄增长,纯合子敲除ob/ob小鼠34肝脏25内积累的脂肪逐渐增多,并且纯合子敲除ob/ob小鼠34肝脏脂肪变性程度高于杂合子PPARα+/-小鼠30,表现为脂滴增大,而PPARα野生型PPARα+/+小鼠29和纯合子敲除PPARα-/-小鼠31肝脏25内无脂肪变性发生,此外,纯合子敲除ob/ob小鼠34的肝脏25重量显著高于PPARα野生型PPARα+/+小鼠29和纯合子敲除PPARα-/-小鼠31,表明其肝脏25发生了脂肪变性,并且纯合子敲除ob/ob小鼠34的肝脏25重量高于纯合子敲除PPARα-/-小鼠31;
g、通过酶试剂盒检测三月龄PPARα野生型PPARα+/+小鼠29、纯合子敲除PPARα-/-小鼠31、纯合子敲除ob/ob小鼠34的肝脏25内甘油三酯23和胆固醇24含量,结果显示,纯合子敲除ob/ob小鼠34的肝脏25内甘油三酯23含量显著高于PPARα野生型PPARα+/+小鼠29、纯合子敲除PPARα-/-小鼠31。
需要说明的是,在本文中,诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来,而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且,术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含,从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素,而且还包括没有明确列出的其他要素,或者是还包括为这种过程、方法、物品或者设备所固有的要素。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
Claims (9)
1.超级肥胖小鼠的构建与鉴定方法,包括雄性PPARα-/-小鼠(1)、雌性OB/ob小鼠(2)、PCR扩增仪(7)、DNA扩增产物(18)、2%琼脂糖凝胶电泳(19)、鉴定依据(20)、瘦素基因(35),其特征在于;所述PCR扩增仪(7)的内部设置有PCR试剂盒(8),所述PCR试剂盒(8)的内部设置有原料(9),所述原料(9)包括DNA双链(10)、DNA单链(11)、引物(12)、TapDNA聚合酶(13)、腺嘌呤脱氧核苷酸(14)、鸟嘌呤脱氧核苷酸(15)、胞嘧啶脱氧核苷酸(16)、胸腺嘧啶脱氧核苷酸(17),所述鉴定依据(20)包括白色脂肪组织(21)、棕色脂肪组织(22)、甘油三酯(23)、胆固醇(24)、肝脏(25)、脂肪肝(26)、葡萄糖(27)、胰岛素(28)。
2.根据权利要求1所述的超级肥胖小鼠的构建与鉴定方法,其特征在于:所述雄性PPARα-/-小鼠(1)与雌性OB/ob小鼠(2)杂交得到雄性PPARα+/-/OB/ob双杂合子小鼠(3)与雌性PPARα+/-/OB/ob双杂合子小鼠(4),所述雄性PPARα+/-/OB/ob双杂合子小鼠(3)与雌性PPARα+/-/OB/ob双杂合子小鼠(4)杂交得到PPARα∆ob/ob小鼠(5)、PPARα野生型(PPARα+/+)小鼠(29)、杂合子(PPARα+/-)小鼠(30)、纯合子敲除(PPARα-/-)小鼠(31)、ob野生型(OB/OB)(32)、杂合子(OB/ob)小鼠(33)、纯合子敲除(ob/ob)小鼠(34)。
3.根据权利要求1-2任意一条所述的超级肥胖小鼠的构建与鉴定方法其特征在于:所述PPARα∆ob/ob小鼠(5)的内部含有基因重组DNA(6)。
4.根据权利要求1所述的超级肥胖小鼠的构建与鉴定方法其特征在于:所述DNA双链(10)解离得到DNA单链(11),所述DNA单链(11)通过引物(12)与TapDNA聚合酶(13)的作用与腺嘌呤脱氧核苷酸(14)、鸟嘌呤脱氧核苷酸(15)、胞嘧啶脱氧核苷酸(16)、胸腺嘧啶脱氧核苷酸(17)结合得到DNA扩增产物(18)。
5.根据权利要求1-2任意一条所述的超级肥胖小鼠的构建与鉴定方法其特征在于:所述DNA扩增产物(18)通过2%琼脂糖凝胶电泳(19)进行检测,所述PPARα野生型(PPARα+/+)小鼠(29)显示143bp一条条带。
6.根据权利要求1-2任意一条所述的超级肥胖小鼠的构建与鉴定方法,其特征在于:所述杂合子(PPARα+/-)小鼠(30)显示143 bp和280 bp两条条带,所述纯合子敲除(PPARα-/-)小鼠(31)显示280 bp一条条带。
7.根据权利要求1-2任意一条所述的超级肥胖小鼠的构建与鉴定方法,其特征在于:所述ob野生型(OB/OB)(32)显示155 bp一条条带,所述杂合子(OB/ob)小鼠(33)显示55 bp、100 bp和155 bp三条条带。
8.根据权利要求1-2任意一条所述的超级肥胖小鼠的构建与鉴定方法,其特征在于:所述纯合子敲除(ob/ob)小鼠(34)显示55 bp和100 bp两条条带。
9.根据权利要求1-8所述的超级肥胖小鼠的构建与鉴定方法,其特征在于:
a、正常日粮饲喂情况下,十六月龄纯合子敲除(PPARα-/-)小鼠(31)与PPARα野生型(PPARα+/+)小鼠(29)、ob野生型(OB/OB)(32)的体型差异不显著,而瘦素基因(35)缺失显著促进了纯合子敲除(ob/ob)小鼠(34)的体脂沉积,使PPARα∆ob/ob小鼠(5)呈现超级肥胖,六周龄之前,PPARα∆ob/ob小鼠(5)和纯合子敲除(ob/ob)小鼠(34)的体重差异不显著,前者的体重略低于后者,六周龄之后,PPARα∆ob/ob小鼠(5)小鼠的体重开始迅速增长,二十四周龄后,其体重显著高于纯合子敲除(ob/ob)小鼠(34),,PPARα野生型(PPARα+/+)小鼠(29)、ob野生型(OB/OB)(32)与纯合子敲除(PPARα-/-)小鼠(31)的体重差异不显著,但显著低于纯合子敲除(ob/ob)小鼠(34)和PPARα∆ob/ob小鼠(5),PPARα野生型(PPARα+/+)小鼠(29)、ob野生型(OB/OB)(32)与纯合子敲除(PPARα-/-)小鼠(31)的采食量差异不显著,后者略低于前者,纯合子敲除(ob/ob)小鼠(34)的采食量显著高于PPARα野生型(PPARα+/+)小鼠(29)、ob野生型(OB/OB)(32)和纯合子敲除(PPARα-/-)小鼠(31)的采食量,PPARα∆ob/ob小鼠(5)的采食量显著高于纯合子敲除(PPARα-/-)小鼠(31)的采食量,而显著低于纯合子敲除(ob/ob)小鼠(34)的采食量;
b、纯合子敲除(ob/ob)小鼠(34)呈现食欲过盛和肥胖,并伴随着高血糖症和高胰岛素血症,机体葡萄糖(27)和胰岛素(28)耐受性显著高于PPARα野生型(PPARα+/+)小鼠(29)、ob野生型(OB/OB)(32),纯合子敲除(PPARα-/-)小鼠(31)体形正常,但血液中的葡萄糖(27)水平降低,胰岛素(28)水平升高,胰岛素(28)参与调控脂肪细胞的生物学功能,而胰岛素(28)抵抗在肥胖发生过程中发挥了重要作用,胰岛素(28)可以促进脂肪组织从循环系统中吸收脂质蛋白,促进脂肪细胞生成脂质,当肥胖发生时,胰岛素(28)信号通路遭到破坏,导致胰岛素(28)抵抗;
c、葡萄糖(27)与胰岛素(28)耐受性结果显示,纯合子敲除(ob/ob)小鼠(34)显著高于PPARα野生型(PPARα+/+)小鼠(29)、纯合子敲除(PPARα-/-)小鼠(31);
d、随着年龄增加,PPARα野生型(PPARα+/+)小鼠(29)、ob野生型(OB/OB)(32)、纯合子敲除(ob/ob)小鼠(34)和PPARα∆ob/ob小鼠(5)的脂肪重量均增加,且纯合子敲除(ob/ob)小鼠(34)和PPARα∆ob/ob小鼠(5)的白色脂肪组织(21)、棕色脂肪组织(22)的重量及其增长幅度显著高于PPARα野生型(PPARα+/+)小鼠(29)、ob野生型(OB/OB)(32)与纯合子敲除(PPARα-/-)小鼠(31),三月龄纯合子敲除(PPARα-/-)小鼠(31)和PPARα∆ob/ob小鼠(5)的白色脂肪组织(21)、棕色脂肪组织(22)的重量分别略高于PPARα野生型(PPARα+/+)小鼠(29)、ob野生型(OB/OB)(32)与纯合子敲除(ob/ob)小鼠(34);
e、六月龄小鼠的白色脂肪组织(21)重量分别为:PPARα野生型(PPARα+/+)小鼠(29)0.56 g,纯合子敲除(PPARα-/-)小鼠(31)1.19 g,纯合子敲除(ob/ob)小鼠(34)4.22 g,PPARα∆ob/ob小鼠(5)5.87 g,纯合子敲除(PPARα-/-)小鼠(31)显著高于PPARα野生型(PPARα+/+)小鼠(29),PPARα∆ob/ob小鼠(5)显著高于纯合子敲除(ob/ob)小鼠(34),六月龄小鼠的棕色脂肪组织(22)重量分别为:PPARα野生型(PPARα+/+)小鼠(29)0.14 g,纯合子敲除(PPARα-/-)小鼠(31)0.24 g,纯合子敲除(ob/ob)小鼠(34)1.19 g,PPARα∆ob/ob小鼠(5)小鼠1.37 g;
f、肥胖发生过程中,多余的能量首先以甘油三酯(23)的形式储存于白色脂肪组织(21)与棕色脂肪组织(22)中,当白色脂肪组织(21)与棕色脂肪组织(22)的储存能力达到饱和时,剩余的脂肪再储存于肝脏中,,随着年龄增长,纯合子敲除(ob/ob)小鼠(34)肝脏(25)内积累的脂肪逐渐增多,并且纯合子敲除(ob/ob)小鼠(34)肝脏脂肪变性程度高于杂合子(PPARα+/-)小鼠(30),表现为脂滴增大,而PPARα野生型(PPARα+/+)小鼠(29)和纯合子敲除(PPARα-/-)小鼠(31)肝脏(25)内无脂肪变性发生,此外,纯合子敲除(ob/ob)小鼠(34)的肝脏(25)重量显著高于PPARα野生型(PPARα+/+)小鼠(29)和纯合子敲除(PPARα-/-)小鼠(31),表明其肝脏(25)发生了脂肪变性,并且纯合子敲除(ob/ob)小鼠(34)的肝脏(25)重量高于纯合子敲除(PPARα-/-)小鼠(31);
g、通过酶试剂盒检测三月龄PPARα野生型(PPARα+/+)小鼠(29)、纯合子敲除(PPARα-/-)小鼠(31)、纯合子敲除(ob/ob)小鼠(34)的肝脏(25)内甘油三酯(23)和胆固醇(24)含量,结果显示,纯合子敲除(ob/ob)小鼠(34)的肝脏(25)内甘油三酯(23)含量显著高于PPARα野生型(PPARα+/+)小鼠(29)、纯合子敲除(PPARα-/-)小鼠(31)。
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