CN112111521B - 通过igfbp5介导动脉粥样化的动物模型及建立方法 - Google Patents
通过igfbp5介导动脉粥样化的动物模型及建立方法 Download PDFInfo
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Abstract
本发明公开了通过IGFBP5介导的动脉粥样硬化动物模型及建立方法,所述动物模型按照以下步骤进行:步骤1、构建腺相关病毒表达载体AAV‑IGFBP5;步骤2、将步骤1得到的所述腺相关病毒载体通过肌肉注射到ApoE‑/‑小鼠中,注射后采用高脂饲料喂养6周即得到本发明的动物模型。本发明模型建立过程简便、成功率高、结果稳定可靠,节约了饲养实验动物的时间,还避免了模型构建失败的后果,并且免疫原性低,安全系数更高。本发明建立的动物模型在动脉粥样硬化的病因学、病理学、药物筛选、临床诊断和治疗等研究中具有重要的实际应用价值,可广泛推广。
Description
技术领域
本发明属于基础医学技术领域,具体涉及一种通过IGFBP5介导动脉粥样化的动物模型,还涉及上述动脉粥样化动物模型的建立方法。
背景技术
动脉粥样硬化是多种心血管疾病的主要原因,脂质代谢障碍为动脉粥样硬化的病变基础,其病变从血管内膜开始,一般先有脂质和复合糖类积聚、出血及血栓生成,进而纤维组织增生及钙质沉着,累及动脉中层的逐渐蜕变和钙化,导致动脉壁增厚变硬,存在于动脉内膜积聚的脂质成黄色粥样,称动脉粥样硬化。
胰岛素样生长因子结合蛋白(IGFBPs)是胰岛素的成员总科的蛋白质。在哺乳动物中,已经鉴定出6种典型的IGFBPs,IGFBP5是IGFBPs家族中最保守的蛋白,它已经被证明可以调节细胞生长、分化、迁移、侵袭和细胞代谢、影响糖尿病和胰岛素耐受性等。
在对动脉粥样硬化进行实验研究时,需要制备实验动物模型。现有的技术中,中小型动物在实验模型的构建中,一般都会使用高脂饲养以及球囊损伤的方法,需要12周去干预实验动物,耗费的时间与人力都十分庞大。
发明内容
本发明第一个目的是提供一种通过IGFBP5介导动脉粥样硬化的动物模型,缩短了动物模型的构建时间,且表达更加稳定,安全系数高。
本发明第二个目的是提供上述IGFBP5动物模型的构建方法。
本发明所采用的第一种技术方案是:通过IGFBP5介导的动脉粥样硬化动物模型,动物模型通过外源注射腺相关病毒系统性高表达IGFBP5得到。
本发明所采用的第二种技术方案是:通过IGFBP5介导的动脉粥样硬化动物模型的建立方法,按照以下步骤进行:
步骤1、构建腺相关病毒表达载体AAV-IGFBP5;
步骤2、将步骤1得到的腺相关病毒载体AAV-IGFBP5通过肌肉注射到ApoE-/-小鼠中,注射后采用高脂饲料喂养6周,6周后得到的小鼠即得到本发明的动物模型。
本发明所采用的第二种技术方案的特点还在于,
步骤1中腺相关病毒表达载体AAV-IGFBP5具体构建步骤如下:
步骤1.1、采用根据已知目的基因IGFBP5 cDNA序列设计合成扩增引物,上游引物如SEQ ID NO:1所示,下游引物如SEQ ID NO:2所示,用PCR法扩增目的基因IGFBP5,扩增条件:98℃,3min;98℃,10s;55℃,15s;72℃,1min,35个循环;72℃,10min;4℃,5min,扩增完毕用琼脂糖凝胶回收试剂盒回收PCR产物;
步骤1.2、将步骤1.1所得的目的基因PCR产物用Bam HI、Xhol内切酶双酶切,同时用这两种酶内切酶切割rAAV载体,将切割后的目的基因与rAAV载体混合,用T4 DNA连接酶在4℃的条件下连接12h,将连接得到的产物转化到E.coliDH5α感受态细胞中,经Kan+LB培养基培养后采用碱裂解法提取重组质粒,对得到的重组质粒进行阳性克隆鉴定;
步骤1.3、采用骤1.2鉴定正确的质粒转化E.coliDH5α感受态细胞,挑取单克隆、摇菌和质粒提取,所提取的质粒转染到HEK293细胞,培养72h后将细胞离心收集,在4℃的条件下,于53 000r/min高速离心30h,收集富含rAAV的细胞上清液;
步骤1.4、将步骤1.3中得到的细胞上清液加入树脂柱进行柱纯化,收集最终得到的纯化后的病毒,于-80℃保存。
步骤1.3中转染前1天将HEK293细胞于直径为100mm的培养皿,转染时细胞密度75%-85%。
步骤2中高脂饲料的胆固醇和脂肪量均为1.5%cholesterol+15%fat。
步骤2中注射入ApoE-/-小鼠的腺相关病毒表达载体AAV-IGFBP5的浓度是1×1011vp/mL。
本发明的有益效果是:与现有动物模型的建立方法相比,12周的实验动物模型构建时间太长并且消耗人力物力都十分巨大,本发明所提到的动物模型构建方法所需时间只有6周就可以构建所需的动脉粥样硬化模型。与一般的注射外源性蛋白方法相比,腺相关病毒的转染效率高,携带目的基因表达时间长,因此不需要频繁的注射蛋白,只需要注射一次即可。并且该载体扩散性更好,免疫原性低,安全系数高,在体内表达更稳定。
附图说明
图1为本发明动物模型与对照组通过油红O染色后显微镜拍照主动脉弓斑块时的对比示意图;
图2为本发明通过计算各组斑块面积后所得到的统计对比图。
具体实施方式
下面结合附图和具体实施方式对本发明进行详细说明。
本发明通过IGFBP5介导动脉粥样硬化的动物模型,所述动物模型可通过外源注射腺相关病毒系统性高表达IGFBP5得到,本发明制备的动物模型的造模时间相比于现有的造模时间明显缩短,操作简单,只需注射一次药物,并且表达更加安全稳定。
本发明通过IGFBP5介导动脉粥样化的动物模型的构建方法,具体包括以下步骤:
步骤1、构建腺相关病毒表达载体AAV-IGFBP5
步骤1.1、采用根据已知目的基因IGFBP5 cDNA序列设计合成上、下游引物:
上游引物Forward primer:CATCTGAATCCTCCTTGCC
下游引物Reverse primer:GTGTCTCCTTAGCCCTTGC
用PCR法扩增目的基因IGFBP5,扩增条件为:98℃,3min;98℃,10s;55℃,15s;72℃,1min。35个循环;72℃,10min;4℃,5min。扩增完毕用琼脂糖凝胶回收试剂盒回收PCR产物。
步骤1.2、将步骤1.1所得的目的基因IGFBP5用Bam HI、Xhol内切酶双酶切,同时用这两种酶内切酶切割腺相关病毒载体rAAV,将切割后的目的基因片段与rAAV载体混合,混合后用T4 DNA连接酶于4℃条件下连接12h,将连接得到的产物转化到E.coliDH5α感受态细胞中,经Kan+LB培养基培养后采用碱裂解法提取重组质粒,对得到的重组质粒进行阳性克隆鉴定。
步骤1.3、采用骤1.2鉴定正确的重组质粒转化E.coliDH5α感受态细胞,挑取单克隆、摇菌,进行大规模质粒提取,转染前一天,将HEK293细胞于直径为100mm的培养皿培养,所提取的质粒转染到HEK293细胞中,培养72h后将细胞离心收集,于4℃的条件下53 000r/min高速离心30h,收集富含rAAV的细胞上清液。
步骤1.4、将步骤1.3中得到的细胞上清液加入树脂柱进行柱纯化,收集最终得到的纯化后的病毒表达载体AAV-IGFBP5,于-80℃保存,并对病毒标准稀释品进行滴度测定。
步骤2、ApoE-/-小鼠肌肉注射步骤1.4的腺相关病毒表达载体AAV-IGFBP5后,将ApoE-/-小鼠采用高脂饲料喂养6周,得到的小鼠即为本发明的动物模型。高脂饲料的胆固醇和脂肪量均为1.5%cholesterol+15%fat。
上述ApoE-/-小鼠肌肉注射时腺相关病毒表达载体AAV-IGFBP5的浓度是1×1011vp/mL。
模型验证:
1.实验对象
(1)选用本发明制备好的动物模型为试验组(AAV-IGFBP5)。
(2)ApoE-/-小鼠肌肉注射200L生理盐水后采用高脂饲料喂养6周及得到空白对照组(saline)。
(3)ApoE-/-小鼠肌肉注射AAV-GFP腺相关病毒,腺相关病毒的病毒数量为1×1011vp/mL,将注射后的小鼠采用高脂饲料喂养6周,得到的小鼠即为对照组(AAV-GFP),高脂饲料的胆固醇和脂肪量均为1.5%cholesterol+15%fat。
上述AAV-GFP腺相关病毒具体按照以下步骤制备:
步骤a、用PmlⅠ和BsmBⅠ双酶切rAAV质粒,用琼脂糖凝胶电泳分离和回收含rAAV左右的4.1kb片段,用Klenow fragment补平后回收,再用碱性磷酸酶CIAP去磷酸化、苯酚/氯仿抽提后,转化入含有腺相关病毒骨架载体pAdeasy-1的超感受态BJ5183中,通过细菌内同源重组法获得的阳性重组质粒命名为pAAV-GFP。
步骤b、将经PacI线性化的阳性重组质粒pAAV-GFP进行苯酚/氯仿抽提,乙醇/醋酸钠共沉淀后加入40L消毒三蒸水溶解,待完全溶解后加入等体积的阳离子脂质体Lipofectamine2000,于37℃孵育4h后,由阳离子脂质体Lipofectamine2000介导转染HEK293细胞细胞,置37℃、5%CO2的培养箱中孵育14~20天后即可观察到病毒蚀斑的出现,说明病毒已成功转染细胞。
步骤c、将步骤b得到的重组腺相关病毒在12000r/min的转速下离心3次后,取离心后的重组腺相关病毒上清液500L,加入90%融合的HEK293细胞中,于37℃、5%CO2培养箱中培养,显微镜下观察到95%~100%细胞出现细胞病变后,离心收集细胞,并将细胞于-80℃和37℃之间反复冻融3次,以3000rpm/min离心5min收集上清液。对重组腺相关病毒上清进行CsCl密度梯度离心纯化两次,并将纯化后的病毒采用透析(10mmol/LTris-HCI,1mmol/LMgCl2,10%甘油)4℃透析两次,每次24h,将透析后的病毒液进行滴度测定。DNase I及蛋白酶K反应液消化rAAV病毒样品,用PCR缓冲液将稀释标准品稀释成不同拷贝梯度105、106、107、108、109、1010。QPCR测定,反应体系为2X mix 4.8μl,引物-F 0.4μl,引物-R0.4μl,模板1μl,水3μl。经一次95℃180s预变性后,94℃30s变性,62℃30s退火,72℃30s延伸,进行40个循环,利用统计软件数据分析循环后的结果是否正确。
2.验证步骤:
饲喂6周后,对空白对照组、对照组和实验组均腹腔注射1%戊巴比妥钠安乐处死,分离其各组主动脉冷冻保存,将冷冻保存的三组主动脉置于室温解冻30min,然后放于4%的多聚甲醛中固定15min,在置于双蒸水中两次(一次一分钟),取出后放在60%的异丙醇中5min,之后放于油红O染液中30min,染色结束依次放入60%的异丙醇与双蒸水中各1min,之后用水溶性封片剂封片,并且置于显微镜下计算斑块面积,如图1所示,图1左侧为对照组(saline)的小鼠动物模型剥离的主动脉,图1中间为对照组(AAV-GFP)小鼠动物模型剥离的主动脉,图1右侧为实验组(AAV-IGFBP5)小鼠动物模型剥离的主动脉,由图1对比可知,从实验组(AAV-IGFBP5)小鼠动物模型剥离的主动脉上斑块面积大于空白对照组(saline)和对照组(AAV-GFP)小鼠动物模型剥离的主动脉上斑块面积,由图2可知:横坐标表示空白对照组(saline),对照组(AAV-GFP)和实验组(AAV-IGFBP5)的编号;纵坐标表示小鼠总损伤面积的大小所占的比例;可以看到两组对照组损伤面积明显小于实验组,且无明显统计学差异。说明本发明通过IGFBP5加重动脉粥样硬化形成的动物模型制备成功,且表型明显。
由于与正常的动物模型的建立相比本实验需要使用腺相关病毒进行干预,为了确定不是腺相关病毒导致实验动物出现明显的动脉粥样硬化过程,故使用GFP荧光蛋白与AAV-IGFBP5做对比,可以排除腺相关病毒对实验动物的影响,此外还可以通过观察荧光反应确定腺相关病毒成功感染了实验动物。
本发明是一种体外注射腺相关病毒携带IGFBP5蛋白基因通过循环系统去快速制备动脉粥样硬化实验动物模型的方法。通过观察小鼠主动脉上斑块的形成量,从而去检测IGFBP5在动脉粥样硬化斑块实验动物模型构建中的作用,利用IGFBP5的功能快速制备动脉粥样硬化的实验动物模型。本发明模型建立过程简便、成功率高、结果稳定可靠,节约了饲养实验动物的时间,还避免了模型构建失败的后果,并且安全系数更高。本发明建立的动物模型在动脉粥样硬化的病因学、病理学、药物筛选、临床诊断和治疗等研究中具有重要的实际应用价值,可广泛推广。
序列表
<110> 西安医学院
<120> 通过IGFBP5介导动脉粥样化的动物模型及建立方法
<130> 2020
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
catctgaatc ctccttgcc 19
<210> 2
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gtgtctcctt agcccttgc 19
Claims (4)
1.通过IGFBP5介导的动脉粥样硬化动物模型的建立方法,其特征在于,所述动物模型通过外源注射腺相关病毒系统性高表达IGFBP5得到;
按照以下步骤进行:
步骤1、构建腺相关病毒表达载体AAV-IGFBP5;具体构建步骤如下:
步骤1.1、根据目的基因IGFBP5 cDNA序列设计合成扩增引物,上游引物如SEQ ID NO:1所示,下游引物如SEQ ID NO:2 所示,用PCR法扩增目的基因IGFBP5,扩增条件:98℃,3min;98℃,10s;55℃,15s;72℃,1min,35个循环;72℃,10min;4℃,5min,扩增完毕用琼脂糖凝胶回收试剂盒回收PCR产物;
步骤1.2、将步骤1.1所得的目的基因PCR产物用Bam HI、Xhol内切酶双酶切,同时用这两种内切酶切割rAAV载体,将切割后的目的基因与rAAV载体混合,用T4 DNA连接酶在4℃的条件下连接12h,将连接得到的产物转化到E.coliDH5α感受态细胞中,经Kan+LB培养基培养后采用碱裂解法提取重组质粒,对得到的重组质粒进行阳性克隆鉴定;
步骤1.3、采用骤1.2鉴定正确的质粒转化E.coliDH5α感受态细胞,挑取单克隆、摇菌和质粒提取,所提取的质粒转染到HEK293细胞,培养72h后将细胞离心收集,在4℃的条件下,于53000r/min高速离心30h,收集富含rAAV的细胞上清液;
步骤1.4、将步骤1.3中得到的细胞上清液加入树脂柱进行柱纯化,收集最终得到的纯化后的病毒,于-80℃保存;
步骤2、将步骤1得到的所述腺相关病毒载体AAV-IGFBP5通过肌肉注射到ApoE-/-小鼠中,注射后采用高脂饲料喂养6周,6周后得到的小鼠即为所述动物模型。
2.根据权利要求1所述的通过IGFBP5介导的动脉粥样硬化动物模型的建立方法,其特征在于,所述步骤1.3中转染前1天将HEK293细胞于直径为100mm的培养皿培养,转染时细胞密度75%-85%。
3.根据权利要求1所述的通过IGFBP5介导的动脉粥样硬化动物模型的建立方法,其特征在于,所述步骤2中高脂饲料的胆固醇和脂肪量为1.5% cholesterol + 15% fat。
4.根据权利要求1所述的通过IGFBP5介导的动脉粥样硬化动物模型的建立方法,其特征在于,所述步骤2中注射入ApoE-/-小鼠的腺相关病毒表达载体AAV-IGFBP5的浓度是1×1011vp/mL。
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