CN110547249A - 一种升主动脉瘤动物模型的制备方法 - Google Patents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
本发明涉及一种升主动脉瘤动物模型的制备方法,包括如下步骤:(1)在鼠升主动脉根部采用外径为1.0~1.6 mm的垫针进行结扎;(2)对进行结扎手术的鼠喂食含有盐酸曲马多胶囊的口服补液盐溶液。本发明的制备方法简单易行,能广泛推广应用,成瘤率高,成瘤时间短。
Description
技术领域
本发明具体涉及一种升主动脉瘤动物模型的制备方法。
背景技术
在过去的30年,随着人口老龄化的增加,胸主动脉瘤(TAA)的患病率也有所提高。65岁以上人群的TAA发病率大约为3-4%,其中60%的患者为升主动脉瘤(ATAA),使患者易患危及生命的并发症,如主动脉夹层或破裂。目前研究表明,TAA与腹主动脉瘤(AAA)的形成机制有所不同,胸主动脉瘤大多数是退行性的病变。在病理学上,TAA表现出基质变性,血管平滑肌丢失,基质弹力纤维破坏,蛋白聚糖沉积和主动脉壁中炎性细胞(巨噬细胞和T淋巴细胞)浸润。在此过程中,伴随微核糖核酸表达的改变、单核细胞趋化因子-1(MCP-1)、血管细胞粘附分子-1(VCAM-1)、细胞间黏附分子-1(ICAM-1)、基质金属蛋白酶-2(MMP-2)和基质金属蛋白酶-9(MMP-9)的活性增加。其中MCP-1、VCAM-1和ICAM-1在炎性细胞募集中起重要作用,而浸润的炎性细胞释放许多炎性细胞因子促进MMP-2和MMP-9的产生,MMP-2和MMP-9可以降解细胞外基质和基质弹力纤维,从而破坏弹力纤维并使血管平滑肌排列紊乱。TAA的病因主要分为先天性(马凡综合征和主动脉瓣二瓣化畸形)和非先天性(如主动脉粥样硬化、感染(梅毒)和血流动力学的改变)疾病。
目前,治疗升主动脉瘤主要使用他汀类药物、血管紧张素II受体1拮抗剂药(氯沙坦)、血管紧张素转换酶抑制药(培哚普利)、β受体阻滞药(普萘洛尔)。这些药主要是通过减少动脉粥样硬化的产生,降低血压、降低心肌收缩力,从而降低血管壁张力,对血管壁起到保护的作用。药物治疗在升主动脉瘤形成早期有一定的治疗效果,但是随着时间的推移主动脉会逐渐增宽,其发生破裂的机率增大。若患者升主动脉瘤超过5.5 cm,瘤体破裂的倾向就会更大,若超过8 cm至10 cm,几乎是100%破裂。所以升主动脉瘤患者一定要接受手术治疗,通过手术治疗患者完全可以痊愈,但创伤大,围手术期并发症高。为了避免手术治疗的缺点,也可以使用介入方法,即用支架加固血管壁或封闭夹层起源。虽然创伤小,但不能消除主动脉增宽、有时支架无法放置。因此,如何提高早期升主动脉瘤的诊断和药物治疗,防止早期升主动脉瘤的形成,瘤体增宽及彻底治愈主动脉瘤,已成降低升主动脉瘤死亡率的关键。
科研工作者们长期从事于研究ATAA的发病机制和治疗药物的开发。药物在应用于临床实验之前必需先经动物实验验证,而适宜的动物模型是决定实验能否顺利进行以及能否获得可靠结果的前提和基础。目前用于制作ATAA动物模型的方法较少,有利用弹性蛋白酶损伤和CaCl2侵蚀对升主动脉直接破坏或激活内源性蛋白酶间接破坏作用。这种方法剂量过大时易侵蚀过度造成动物死亡;剂量较小时对升主动脉外膜有破坏作用,但对升主动脉的内膜和中膜作用较小,侵蚀过程历时长,成瘤时间长(一般要2-5个月)且成瘤率较低。除此之外,弹性蛋白酶和CaCl2破坏性作用诱导升主动脉局部扩张,在一定程度上影响了内源性病因学研究。此外,还有研究者在幼鼠升主动脉根部周围放置与主动脉直径相同柔性聚乙烯管,3-5个月后升主动脉直径仅增加44.26%,且成瘤率低(66.67%)。这极大地延长了实验周期和实验数据的稳定性。综上,目前用于制作ATAA动物模型的方法所制作的动物模型成瘤率低且成瘤时间长,影响实验的顺利进行及最终结果的可靠性。
发明内容
本发明所要解决的技术问题是提供一种成瘤时间短的升主动脉瘤动物模型的制备方法。
为解决以上技术问题,本发明采用如下技术方案:
本发明提供一种升主动脉瘤动物模型的制备方法,包括如下步骤:
(1)在鼠升主动脉根部采用外径为1.0~1.6 mm的垫针进行结扎;
(2)对进行结扎手术的鼠喂食含有盐酸曲马多胶囊的口服补液盐溶液。
优选地,步骤(1)的具体方法为:用非吸收性缝线从升主动脉与肺动脉之间穿过,把外径为1.3~1.5 mm的垫针放置到升主动脉上,然后在升主动脉根部迅速结扎,抽去垫针。
进一步优选地,所述的非吸收性缝线采用0号丝线编织的非吸收性缝线。
优选地,喂食所述的含有盐酸曲马多胶囊的口服补液盐溶液2~4天。
优选地,所述的含有盐酸曲马多胶囊的口服补液盐溶液中,所述的含有盐酸曲马多胶囊的口服补液盐溶液中,所述的盐酸曲马多的浓度为0.7~0.9 mg/mL,所述的口服补液盐的浓度为27.5~28.5 mg/mL。
优选地,进行步骤(1)后,禁食1~2天后进行所述的步骤(2)。
优选地,所述的鼠为大鼠。
根据一种具体且优选地实施方式,所述的制备方法的具体步骤为:
(1)将鼠禁食1~2天,正常饮水;
(2)对鼠进行麻醉后,将鼠的毛发脱掉;
(3)将鼠固定在手术台上,口腔连通呼吸机;
(4)对皮肤消毒后,开胸暴露升主动脉;
(5)在鼠升主动脉根部结扎,然后关胸;
(6)待鼠自然苏醒,然后喂食含有盐酸曲马多胶囊的口服补液盐溶液2~4天,同时术后禁食1~2天,而后正常饮水、喂食。
具体地,采用质量百分比为8~12%的水合氯醛对所述的鼠进行腹腔注射麻醉,用量为2~4 mL/kg。
具体地,所述的呼吸机设定的呼吸比为1:1~2,频率为80~90次/min,潮气量为28~32 mL/kg。
由于上述技术方案的实施,本发明与现有技术相比具有如下优点:
本发明的制备方法简单易行,能广泛推广应用,成瘤率高,成瘤时间短,且老鼠死亡率低,存活率高。
附图说明
图1为大鼠升主动脉及主动脉弓部动脉的正常与升主动脉瘤超声检测图,其中,(a)正常升主动脉(b)升主动脉瘤;
图2为大鼠升主动脉及主动脉弓部动脉的正常与升主动脉瘤实体图,其中(a)为正常升主动脉,(b)为升主动脉瘤;
图3为采用不同直径垫针的大鼠成瘤率和死亡率统计图,其中,(a)成瘤率(b)死亡率;
图4为术后禁食,喂加入盐酸曲马多胶囊(0.8 mg/mL)的口服补液盐溶液和灭菌自来水的存活率统计图。
具体实施方式
为了使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解,下面结合具体实施例,进一步阐述本发明。
下面将结合实施例对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可从商业途径获得。
实施例1:
外科手术进行升主动脉缩窄,通过增加管壁压力和血流剪切力,构建升主动脉瘤模型。实验中,通过使用不同管径的垫针(1.0 mm、1.2 mm、1.4 mm和1.6 mm)对大鼠升主动脉进行缩窄,增加管壁压力和剪切力。实验步骤如下:
(1)购买苏州大学实验动物中心提供的8周,230-250g的雌性Sprague Dawley大鼠,术前禁食12小时,饮水正常。并对其称重,标记。
(2)10%的水合氯醛腹腔注射麻醉大鼠(3 mL/kg),用脱毛膏将大鼠胸骨上窝至胸部的毛发脱掉。
(3)75%酒精对手术操作台消毒,将大鼠固定在手术台上,选用16号的动脉穿刺套管针进行口腔气管插管,并连通动物呼吸机;呼吸比:1:1.5,频率:85次/min,潮气量:28-32mL/kg。
(4)75%酒精擦拭切口处皮肤,沿胸骨上窝用眼科剪刀纵向剪开。
(5)剪开胸骨前壁的肌肉层,暴露胸骨,使用组织剪沿胸骨上窝正中方向剪开胸骨至第二肋肋骨平胸骨水平位置。
(6)撑开器暴露胸腔纵膈位置,无齿镊子沿正中位置分离胸腺组织,暴露升主动脉。
(7)用钝性弯镊将0号丝线编织非吸收性缝线从升主动脉与肺动脉之间穿过,将不同管径的垫针放置到升主动脉上,然后在升主动脉根部迅速结扎,抽去垫针,剪掉结扎线头。观察结扎后的心率情况,待恢复正常后,将胸腺复位。
(8)用3号丝线编织非吸收性缝线间断缝合的方法进行逐层关胸(分别为胸骨、肌层及皮肤层)。
(9)拔出气管插管,将大鼠放置在电热毯上待其自然苏醒。术后禁食1天,喂加入盐酸曲马多胶囊(0.8 mg/mL)的口服补液盐溶液(27.9 mg/mL)3天,而后正常饮水,喂食。
(10)对照组只用钝性弯镊将0号丝线编织非吸收性缝线从升主动脉与肺动脉之间穿过,不结扎。其它操作与实验组相同。
(11)在术后2周使用Vevo 2100小动物超声成像系统检测大鼠的升主动脉直径,升主动脉增宽1.5倍及以上,即可判断升主动脉瘤动物模型制作成功。
术后2周,用Vevo 2100小动物超声成像系统检测大鼠的升主动脉直径。将大鼠用3%异氟烷与 0.5-1.0 L/min100%氧气混合进行诱导麻醉,1.5%异氟烷维持麻醉。再把大鼠固定在检测台上,连接温度及心率检测设备,使用脱毛膏除去胸部毛发,检测台水平右倾斜45°,将超声探头放置在胸骨左缘1cm处,定位出升主动脉的位置,在B模式下检测升主动脉直径。如图1,术后2周,与正常组相比,结扎组大鼠的升主动脉直径增宽>1.5倍,证明升主动脉瘤动物建模成功。检测完毕,撤去麻醉,大鼠放置在加热毯上待其自然复苏后放入于鼠笼中,正常饮水、喂食。
统计2周不同缩窄直径的大鼠成瘤率和死亡率,如图3,缩窄直径为1.0 mm和1.2mm的成瘤率很高,同时死亡率也比较高。然而缩窄直径为1.6 mm时,死亡率虽然较低,但与其它缩窄直径相比成瘤率较低。可缩窄直径为1.4 mm时,成瘤率较高,同时死亡率较低。所以制造大鼠(230-250 g)升主动脉瘤模型最佳的缩窄直径为1.4 mm。
实施例2
采用直径为1.4mm的垫针进行手术,手术步骤与实施例1相同,不同之处在于:给药组在术后给老鼠喂加入盐酸曲马多胶囊(0.8 mg/mL)的口服补液盐溶液(27.9 mg/mL),而不给药组老鼠喂灭菌自来水,统计术后5天老鼠的存活率,结果如图4,可见,老鼠喂加入盐酸曲马多胶囊的口服补液盐溶液,可以显著提高老鼠存活率。
申请人声明,本发明通过上述实施例来说明本发明的详细方法,但本发明并不局限于上述详细方法,即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了,对本发明的任何改进,对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等,均落在本发明的保护范围和公开范围之内。
Claims (10)
1.一种升主动脉瘤动物模型的制备方法,其特征在于:包括如下步骤:
(1)在鼠升主动脉根部采用外径为1.0~1.6 mm的垫针进行结扎;
(2)对进行结扎手术的鼠喂食含有盐酸曲马多胶囊的口服补液盐溶液。
2.根据权利要求1所述的升主动脉瘤动物模型的制备方法,其特征在于:步骤(1)的具体方法为:用非吸收性缝线从升主动脉与肺动脉之间穿过,把外径为1.3~1.5 mm的垫针放置到升主动脉上,然后在升主动脉根部迅速结扎,抽去垫针。
3.根据权利要求2所述的升主动脉瘤动物模型的制备方法,其特征在于:所述的非吸收性缝线采用0号丝线编织的非吸收性缝线。
4.根据权利要求1所述的升主动脉瘤动物模型的制备方法,其特征在于:喂食所述的含有盐酸曲马多胶囊的口服补液盐溶液2~4天。
5.根据权利要求1所述的升主动脉瘤动物模型的制备方法,其特征在于:所述的含有盐酸曲马多胶囊的口服补液盐溶液中,所述的盐酸曲马多的浓度为0.7~0.9 mg/mL,所述的口服补液盐的浓度为27.5~28.5 mg/mL。
6.根据权利要求1所述的升主动脉瘤动物模型的制备方法,其特征在于:进行步骤(1)后,禁食1~2天后进行所述的步骤(2)。
7.根据权利要求1所述的升主动脉瘤动物模型的制备方法,其特征在于:所述的鼠为大鼠。
8.根据权利要求1所述的升主动脉瘤动物模型的制备方法,其特征在于:所述的制备方法的具体步骤为:
(1)将鼠禁食1~2天,正常饮水;
(2)对鼠进行麻醉后,将鼠的毛发脱掉;
(3)将鼠固定在手术台上,口腔连通呼吸机;
(4)对皮肤消毒后,开胸暴露升主动脉;
(5)在鼠升主动脉根部结扎,然后关胸;
(6)待鼠自然苏醒,然后喂食含有盐酸曲马多胶囊的口服补液盐溶液2~4天,同时术后禁食1~2天,而后正常饮水、喂食。
9.根据权利要求8所述的升主动脉瘤动物模型的制备方法,其特征在于:采用质量百分比为8~12%的水合氯醛对所述的鼠进行腹腔注射麻醉,用量为2~4 mL/kg。
10.根据权利要求8所述的升主动脉瘤动物模型的制备方法,其特征在于:所述的呼吸机设定的呼吸比为1:1~2,频率为80~90次/min,潮气量为28~32 mL/kg。
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