CN109010379A - 慢性萎缩性胃炎动物模型的制备方法 - Google Patents
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Abstract
慢性萎缩性胃炎动物模型的制备方法，按小鼠体重灌N‑甲基‑N‑硝基‑亚硝基胍，饲养饮水使用氨水；增殖培养幽门螺杆菌，用BHI培养基调配浓度；灌胃前小鼠禁食，实验组每只小鼠灌幽门螺杆菌，灌胃后禁食禁水；抽取雌雄小鼠，进行胃黏膜病理组织学检查，并从胃粘膜分离培养幽门螺杆菌，确认幽门螺杆菌定植和炎症形成。该方法周期短、成功率高、病理改变稳定，可重复性强，具备突出的实质性特点，可为研究幽门螺杆菌感染导致的慢性萎缩性胃炎、胃癌等疾病提供良好的动物模型。
Description
技术领域
本发明属于医药领域，具体涉及慢性萎缩性胃炎动物模型的制备方法。
背景技术
慢性萎缩性胃炎(chronic atrophic gastritis,CAG)作为胃癌前期疾病的一种，其发病缓慢，病势缠绵，迁延难愈，治疗棘手，控制不好可向肠上皮化生或不典型增生发展，最后形成胃癌。关于CAG研究与治疗都备受瞩目，CAG动物模型的造模，是该疾病基础与临床研究的关键素材，良好的动物模型为将为该疾病的防治研究提供直接的体内证据。构建慢性萎缩性胃炎动物模型方法主要有化学致癌法和幽门螺杆菌感染法等，但截至目前，模型的构建还不理想，还有有很多地方值得探索，比如实验动物的选择、药物的组合及剂量的大小、造模时间的长短、操作的简便性等。N-甲基-N-硝基-亚硝基胍、氨水、脱氧胆酸钠、水杨酸钠等均是目前常用的化学方法造模物质,幽门螺杆菌是构建CAG动物的唯一病原菌，其单独及联合使用均能成功造模，但是周期比较长，而且模型不够稳定，成功率比较低。本发明采用“幽门螺杆菌+N-甲基-N-硝基-亚硝基胍+氨水”联合造模方法，探讨周期短、成功率高、病理改变稳定的造模方法，将为防治CAG奠定坚实的实验基础。
发明内容
解决的技术问题：本发明提供一种慢性萎缩性胃炎动物模型的制备方法，缩短造模周期，提高造模的成功率，稳定疾病小鼠的病理变化。
技术方案：慢性萎缩性胃炎动物模型的制备方法，步骤为：a.免疫干预：按小鼠体重5mL/kg灌100～120g/mL浓度的N-甲基-N-硝基-亚硝基胍，每天1次，连续干预7次，饲养饮水使用0.02～0.1wt.％氨水；b.细菌感染：增殖培养幽门螺杆菌3天，用BHI培养基调配浓度为1×109CFU/mL；灌胃前小鼠禁食12小时，实验组每只小鼠灌1～10×108CFU幽门螺杆菌，灌胃后禁食禁水2小时，隔天1次，连续灌5次；c.确定造模成功：分别在第1、2、3、4个月，抽取雌雄各5只小鼠，进行胃黏膜病理组织学检查，并从胃粘膜分离培养幽门螺杆菌，确认幽门螺杆菌定植和炎症形成。
优选的，上述每只小鼠灌幽门螺杆菌浓度为5×108CFU。
优选的，上述按小鼠体重5mL/kg灌120g/mL的N-甲基-N-硝基-亚硝基胍。
优选的，上述饲养饮水使用0.02wt.％氨水。
有益效果：1.本发明成功构建了小鼠CAG动物模型。2.本发明构建的小鼠CAG动物模型在3个月内成功率为90％，构建周期短，病理变化稳定，是较好的CAG动物模型构建方法。
附图说明
图1为正常小鼠胃粘膜HE染色病理图片。
图2为实验组小鼠感染后1个月胃粘膜HE染色病理图片。
图3为实验组小鼠感染后3个月胃粘膜HE染色病理图片。
图4为实验组小鼠感染后4个月胃粘膜HE染色病理图片。
图5为实验组小鼠感染后1、2、3、4个月胃粘膜幽门螺杆菌定植量。
具体实施方式
实施例1
1.动物分组：随机选220只6周龄的昆明小鼠，分为PBS对照组、幽门螺杆菌组、N-甲基-N-硝基-亚硝基胍组、氨水组、幽门螺杆菌组+基-N-硝基-亚硝基胍组+氨水组联合造模组，雌雄各半。
2.免疫干预：实验组按小鼠体重5mL/kg灌120g/mL浓度的N-甲基-N-硝基-亚硝基胍，每天1次，连续干预7次，饲养饮水使用0.02wt.％氨水。对照组：不用任何干预，正常饲养。
3.细菌感染：采用临床分离的幽门螺杆菌菌株，经革兰氏染色、尿素酶试验、氧化酶试验、过氧化氢酶试验及16S鉴定为幽门螺杆菌，菌株CagA和VagA为阳性，增殖培养该幽门螺杆菌3天，用BHI培养基调配浓度为1×109CFU/mL；灌胃前小鼠禁食12小时，实验组每只小鼠灌5×108CFU幽门螺杆菌，灌胃后禁食禁水2小时，隔天1次，连续灌5次。对照组每只灌0.5mLBHI培养基，同样灌5次。
4.确定造模成功：分别在第1、2、3、4个月，从实验组和对照组中随机抽取雌雄各5只小鼠，进行胃黏膜病理组织学检查，并从胃粘膜分离培养幽门螺杆菌，确认幽门螺杆菌定植和炎症形成。
5.实验结果：联合造模组小鼠感染后第一个月，全部小鼠均发生急性胃炎，尚未发现有慢性萎缩性胃炎小鼠，如图2；第二个月与第一个月对比没有明显差异；第三个月90％(9/10)出现轻度慢性萎缩性胃炎，如图3，第4个月小鼠由轻度慢性萎缩性胃炎变为中度慢性萎缩性胃炎，如图4。实验组小鼠感染后1-4个月每克胃粘膜均有1×104-5CFU定植，组间无显著性差异，如图5和表1，幽门螺杆菌验证如表2。PBS对照组、幽门螺杆菌组、N-甲基-N-硝基-亚硝基胍组、氨水组小鼠未见有慢性萎缩性胃炎。
6.实验结论：“幽门螺杆菌+N-甲基-N-硝基-亚硝基胍+氨水”组合方法构建慢性萎缩性胃炎3个月的成功率是90％，4个月病理变化更明显。
表1慢性萎缩性胃炎造模结果
表2造模小鼠分离培养物验证
实施例2
采用上述实施例1的方法，幽门螺杆菌按每只小鼠灌1×108CFU，N-甲基-N-硝基-亚硝基胍按小鼠体重5mL/kg灌100g/mL浓度的灌胃，每天1次，连续干预7次，饲养饮水使用0.1wt.％氨水，实验结果见表2。
表2慢性萎缩性胃炎造模结果
实施例3
采用上述实施例1的方法，幽门螺杆菌按每只小鼠灌1×109CFU，N-甲基-N-硝基-亚硝基胍按小鼠体重5mL/kg灌100g/mL浓度的灌胃，每天1次，连续干预7次，饲养饮水使用0.1wt.％氨水，实验结果见表3。
表3慢性萎缩性胃炎造模结果
Claims (4)
1.慢性萎缩性胃炎动物模型的制备方法，其特征在于步骤为：
a. 免疫干预：按小鼠体重5 mL/kg灌100～120 g/mL浓度的N-甲基-N-硝基-亚硝基胍，每天1次，连续干预7次，饲养饮水使用0.02～0.1 wt.%氨水；
b. 细菌感染：增殖培养幽门螺杆菌3天，用BHI培养基调配浓度为1×109CFU/mL；灌胃前小鼠禁食12小时，实验组每只小鼠灌1～10×108CFU幽门螺杆菌，灌胃后禁食禁水2小时，隔天1次，连续灌5次；
c. 确定造模成功：分别在第1、2、3、4个月，抽取雌雄各5只小鼠，进行胃黏膜病理组织学检查，并从胃粘膜分离培养幽门螺杆菌，确认幽门螺杆菌定植和炎症形成。
2.根据权利要求1所述慢性萎缩性胃炎动物模型的制备方法，其特征在于所述每只小鼠灌幽门螺杆菌浓度为5×108CFU。
3.根据权利要求1所述慢性萎缩性胃炎动物模型的制备方法，其特征在于所述按小鼠体重5 mL/kg灌120 g/mL的N-甲基-N-硝基-亚硝基胍。
4.根据权利要求1所述慢性萎缩性胃炎动物模型的制备方法，其特征在于所述饲养饮水使用0.02wt.%氨水。
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