CN111778284B - 通过ctrp6介导的动脉粥样化动物模型及其建立方法 - Google Patents
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Abstract
本发明公开了一种通过CTRP6介导的动脉粥样硬化动物模型，所述动物模型通过外源注射腺病毒系统性高表达CTRP6获得。所述动物模型按照以下步骤进行：步骤1、构建腺病毒表达载体Ad‑CTRP6；步骤2、将步骤1得到的所述腺病毒载体通过尾部静脉注射到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，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CTRP6介导的动脉粥样化动物模型，还涉及上述动脉粥样化动物模型的建立方法。
背景技术
泡沫细胞的形成是引发动脉粥样硬化斑块形成非常重要的因素之一。当大动脉或中动脉的某些部位内膜增厚，沉积了大量脂蛋白，单核细胞经趋化因子MCP-1的作用，分化为巨噬细胞，巨噬细胞和少量平滑肌细胞吞噬血液中的氧化低密度脂蛋白胆固醇形成泡沫细胞，附着于血管内皮形成斑块；随着病程进展，斑块增大，斑块易损性增加，患者可表现为各种严重的临床表现，如心梗等，如果斑块破裂形成血栓，患者死亡风险增加。
C1q/肿瘤坏死因子相关蛋白6(CTRP6)是一种新发现的分泌蛋白，其结构与功能与脂联素相关。肿瘤，动脉粥样硬化和组织炎症等疾病的发病病程都与CTRP6有关，其在各种细胞的生理活动也有着重要作用。
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
本发明的目的是提供一种通过CTRP6介导的动脉粥样硬化动物模型的建立方法，缩短了动物模型构建时间。
本发明所采用的技术方案是一种通过CTRP6介导的动脉粥样硬化动物模型的建立方法，动物模型通过外源注射腺病毒系统性高表达获得。
一种通过CTRP6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、构建腺病毒表达载体Ad-CTRP6；
步骤2、将步骤1得到的所述腺病毒载体通过尾部静脉注射到ApoE-/-小鼠中，注射后采用高脂高胆固醇饲料喂养6周，第四周时再注射一次步骤1得到的腺病毒，6周后得到的小鼠即得到本发明的动物模型。
本发明所采用第二种技术方案的特点还在于，
步骤1中腺病毒表达载体具体构建步骤如下：
步骤1.1、采用HindⅢ、SalⅠ双酶切小鼠CTRP6 cDNA质粒，电洗脱回收CTRP6 cDNA片段，HindⅢ、SalⅠ双酶切pCTRP6-CMV质粒，电洗脱法回收pAdTrack-CMV HindⅢ和SalⅠ双酶切线性片段，再通过T4DNA连接酶连接CTRP6 cDNA片段与p AdTrack-CMV HindⅢ、Sal I双酶切线性片段，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；
步骤1.2、将步骤1.1所得的p AdTrack-CTRP6重组质粒经PemⅠ酶切，电洗脱回收线性片段，将酶切后的p AdTrack-CTRP6 PemⅠ酶切线性片段电转法转入含p AdEasy-1的E.coli BJ5183电感受态细胞，转化产物经Kan+LB固体培养基培养后采用碱性裂解法提取pAdEasy-CTRP6重组质粒；
步骤1.3、采用PacⅠ酶切步骤1.2所得的p AdEasy-CTRP6得到Pac I线性化重组质粒p AdEasy-CTRP6片段，采用5μg的PacⅠ线性化重组质粒p AdEasy-CTRP6片段转染HEK293细胞，转染后培养7～10天，每天观察细胞生长及绿色荧光蛋白表达，当细胞中绿色荧光蛋白表达呈“彗星”状并出现细胞病变效应时，收集细胞并涡旋震荡4次使细胞裂解，裂解后在12000r/min的条件下离心3min，收集上清液即为Ad-CTRP6病毒液，将Ad-CTRP6病毒液于-80℃保存备用；
步骤1.4、感染当天，将200μL所述步骤1.3得到的Ad-CTRP6病毒液加至1.5mL DEME完全培养液中培养90min后，加入3.5mL DMEM完全培养液培养，感染第3天，细胞出现明显细胞病变效应并伴有脱落时，收集病毒，再重复感染Ad-CTRP6病毒液4次，将所得的病毒总和进行收集即为本发明的腺病毒表达载体Ad-CTRP6。
步骤1.3中转染前1天将HEK293细胞于直径为60mm的培养皿培养，转染时细胞的细胞密度30％-50％。
步骤1.4中感染前1天，将HEK293细胞于直径为100mm的培养皿，感染时细胞的细胞密度75％-85％。
步骤2中高脂饲料的胆固醇和脂肪量为1.5％cholesterol+15％fat。
步骤2中注射入ApoE-/-小鼠的腺病毒表达载体Ad-CTRP6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，本专利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，腺病毒的转染方式有很明显的优势，不需要频繁的注射蛋白只需要每3周注射一次即可，由于CTRP6基因结构序列较大故选择腺病毒携带基因的方式最为简单方便。
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。
本发明是一种通过CTRP6介导的动脉粥样硬化动物模型的建立方法，所述动物模型可通过外源注射腺病毒系统性高表达CTRP6的得到，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。
本发明是一种通过CTRP6介导的动脉粥样化动物模型的构建方法，具体包括以下步骤：
步骤1、制备Ad-CTRP6基因的腺病毒表达载体
步骤1.1、采用HindⅢ、SalⅠ双酶切小鼠CTRP6 cDNA质粒，1％琼脂糖凝胶平板电泳法检测双酶切产物，电洗脱回收CTRP6cDNA片段，采用HindⅢ和SalⅠ双酶切pCTRP6-CMV质粒，电洗脱法回收pAdTrack-CMV HindⅢ、SalⅠ双酶切线性片段，再通过T4DNA连接酶连接CTRP6 cDNA片段与pAdTrack-CMV HindⅢ、SalⅠ双酶切线性片段，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得到pAdTrack-CTRP6重组质粒，通过HindⅢ、SalⅠ双酶切鉴定上述重组质粒。
步骤1.2、将步骤1.1所得的p AdTrack-CTRP6重组质粒经PemⅠ酶切，电洗脱回收线性片段，将酶切后的p AdTrack-CTRP6PemⅠ酶切线性片段电转法转入含p AdEasy-1的E.coli BJ5183电感受态细胞，转化产物经Kan+LB固体培养基培养后采用碱性裂解法提取pAdEasy-CTRP6重组质粒，分别通过BamHⅠ和PacⅠ酶切鉴定pAdEasy-CTRP6重组质粒，并对pAdEasy-CTRP6重组质粒行目的基因序列测定(Invitrogen)。
步骤1.3、采用PacⅠ酶切步骤1.2所得的pAdEasy-CTRP6得到Pac I线性化重组质粒p AdEasy-CTRP6片段，转染前1天将HEK293细胞于直径为60mm的培养皿培养，采用5μg的PacI线性化重组质粒pAdEasy-CTRP6片段转染HEK293细胞，转染时细胞密度30％-50％，转染后培养细胞7～10天，每天观察细胞生长及细胞中绿色荧光蛋白(GFP)表达情况，当GFP表达呈“彗星”状并出现细胞病变效应(CPE)时，收集细胞于15mL离心管中，采用涡旋震荡细胞4次使细胞裂解，裂解后在12000r/min的条件下离心3min，将离心后的上清液分装至1.5mL离心管中，所述上清液即为Ad-CTRP6病毒液，将Ad-CTRP6病毒液于-80℃保存备用。
步骤1.4、感染前1天，将HEK293细胞于直径为100mm的培养皿培养，感染当天，将200μL所述步骤1.3得到的Ad-CTRP6病毒液加至1.5mL DEME完全培养液中培养90min后，加入3.5mL DMEM完全培养液培养，感染第3天，细胞出现明显细胞病变效应并伴有脱落时，收集病毒，再重复感染Ad-CTRP6病毒液4次，将所得的病毒总和进行收集即为本发明的腺病毒表达载体Ad-CTRP6。
步骤2、ApoE-/-小鼠尾静脉注射步骤1.5的腺病毒表达载体Ad-CTRP6后，将ApoE-/-小鼠采用高脂饲料喂养6周，第四周时再注射一次步骤1.5的腺病毒表达载体Ad-CTRP6，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-/-小鼠尾部静脉注射时腺病毒表达载体Ad-CTRP6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(Ad-CTRP6)。
(2)ApoE-/-小鼠尾部静脉注射200μL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-/-小鼠尾静脉注射Ad-GFP腺病毒，腺病毒的病毒数量为1×1011vp/mL，将注射后的小鼠采用高脂饲料喂养6周，在第四周时再注射一次Ad-GFP，得到的小鼠即为对照组(Ad-GFP)，高脂饲料的胆固醇和脂肪量均为1.5％cholesterol+15％fat。
上述Ad-GFP腺病毒具体按照以下步骤制备：
步骤a、将pRNAT-H1.1/Adeno用PmeI进行酶切，然后通过低熔点琼脂糖凝胶电脉回收后，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pAd-GFP。
步骤b、将经PacI线性化的阳性重组质粒pAd-GFP进行苯酚/氯仿抽提，乙醇/醋酸钠共沉淀后加入40μL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。
步骤c、将步骤b得到的重组腺病毒在12000r/min的转速下离心3次后，取离心后的重组腺病毒上清液500μL，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。对重组腺病毒上清进行CsCl密度梯度离心纯化两次，并将纯化后的腺病毒采用透析液(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，采用紫外分光光度计测定。取纯化后的病毒液100μL，10％SDS 20μL、PBS 880μL，65℃热浴30min后旋涡3次，离心后测定病毒基因组DNA的OD260nm和OD280nm，据此计算病毒的颗粒数和纯度，1OD260＝1012vp/mL，若OD260/OD280>1.3表明纯度较高，病毒滴度vp/mL＝A260×dilution×1012，将所得的病毒总和进行收集即为腺病毒表达载体Ad-GFP。
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，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(Ad-GFP)小鼠动物模型剥离的主动脉，图1右侧为实验组(Ad-CTRP6)小鼠动物模型剥离的主动脉，由图1对比可知，从实验组(Ad-CTRP6)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(Ad-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(Ad-GFP)和实验组(Ad-CTRP6)的编号；纵坐标表示小鼠总损伤面积的大小，单位长度为10mm2，计量单位为平方毫米；可以看到两组对照组的总损伤面积约为14mm2，其损伤面积小于实验组所构建的动物模型所产生的总损伤面积约为31mm2。说明本发明通过CTRP6加速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病毒进行干预，为了确定不是腺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Ad-CTRP6做对比，可以排除腺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病毒成功感染了实验动物。
本发明是一种体外注射腺病毒携带CTRP6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CTRP6在动脉粥样硬化斑块实验动物模型构建中的作用，利用CTRP6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，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。
Claims (5)
1.一种通过CTRP6介导的动脉粥样硬化动物模型的建立方法，其特征在于，按照以下步骤进行：
步骤1、构建腺病毒表达载体Ad-CTRP6；
腺病毒表达载体具体构建步骤如下：
步骤1.1、采用HindⅢ、SalⅠ双酶切小鼠CTRP6 cDNA质粒，电洗脱回收CTRP6 cDNA片段，HindⅢ、SalⅠ双酶切pCMV-CTRP6质粒，电洗脱法回收pAdTrack-CMV HindⅢ和SalⅠ双酶切线性片段，再通过T4DNA连接酶连接CTRP6 cDNA 片段与pAdTrack-CMV HindⅢ、SalI双酶切线性片段，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；
步骤1.2、将步骤1.1所得的pAdTrack-CTRP6重组质粒经PemⅠ酶切，电洗脱回收线性片段，将酶切后的pAdTrack-CTRP6 PemⅠ酶切线性片段电转法转入含pAdEasy-1的E.coliBJ5183电感受态细胞，转化产物经Kan+LB固体培养基培养后采用碱性裂解法提取pAdEasy-CTRP6重组质粒；
步骤1.3、采用PacⅠ酶切步骤1.2所得的pAdEasy-CTRP6得到Pac I线性化重组质粒pAdEasy-CTRP6片段，采用5μg的 Pac Ⅰ线性化重组质粒p AdEasy-CTRP6片段转染HEK293细胞，转染后培养细胞7～10天，每天观察细胞生长及绿色荧光蛋白表达，当细胞中绿色荧光蛋白表达呈“彗星”状并出现细胞病变效应时，收集HEK293细胞并涡旋震荡4次使细胞裂解，裂解后在12000r/min的条件下离心3 min，收集上清液即为Ad-CTRP6病毒液，将Ad-CTRP6病毒液于-80℃保存备用；
步骤1.4、感染当天，将200μL所述步骤1.3得到的Ad-CTRP6病毒液加至1.5 mL DEME完全培养液中培养90min后，加入3.5 mLDMEM完全培养液培养，感染第3天，细胞出现明显细胞病变效应并伴有脱落时，收集病毒，再重复感染Ad-CTRP6病毒液4次，将所得的病毒总和进行收集即为腺病毒表达载体Ad-CTRP6；
步骤2、将步骤1得到的所述腺病毒载体Ad-CTRP6通过尾部静脉注射到ApoE-/-小鼠中，注射后采用高脂饲料喂养6周，第四周时再注射一次腺病毒表达载体Ad-CTRP6，6周后得到的小鼠即得到动物模型。
2.根据权利要求1所述的一种通过CTRP6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60mm的培养皿培养，转染时细胞的细胞密度30%-50%。
3.根据权利要求1所述的一种通过CTRP6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1.4中感染前1天，将HEK293细胞于直径为100mm的培养皿，感染时细胞密度75%-85%。
4.根据权利要求1所述的一种通过CTRP6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。
5.根据权利要求1所述的一种通过CTRP6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-/-小鼠的腺病毒表达载体Ad-CTRP6的浓度是1×1011vp/mL。
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