CN110558280A - 一种肝癌动物模型的制备方法 - Google Patents
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Abstract
本发明提供一种肝癌动物模型的制备方法,包括以下具体步骤:(1)选取4周龄的ICR小鼠,观察饲养2周;将HepG2细胞在加有10%胎牛血清的DMEM培养基中培养传代扩增后用注射器注射入ICR小鼠腹腔;(2)待小鼠出现明显腹水后抽出腹水2mL,加入离心管内,离心,弃上清;加入5mL PBS溶液重悬、吹洗,再离心5分钟,弃上清;(3)用PBS溶液以步骤(2)中同样的方法重复洗2‑3遍;(4)在步骤(3)中弃上清后的沉淀内加入5mL PBS溶液吹散细胞,离心3分钟;用精密移液器将上清和沉淀上层的红细胞取出,保留下层白色沉淀;本发明制备的原发性肝癌小鼠模型的效果大大优于目前已有的原发性肝癌动物模型建立方法,较已有模型具有更加真实性、实用性、方便性、经济性。
Description
技术领域
本发明属于生物技术领域,具体涉及一种肝癌动物模型的制备方法。
背景技术
原发性肝癌是一种世界范围内恶名昭彰的癌症,以其晚发现,高死亡率而位于癌症致死率排名前列,占每年全球新增和死亡患者的54%。由于我国饮食习惯和人群易感性导致了国人肝炎高发,如果治疗不及时往往会诱发肝癌。人群的异质性、普遍的临床研究抵触情绪以及科学伦理审查的限制让临床肝癌研究诸多掣肘。因而,广大科学工作者致力于开发优良的动物模型来替代人类进行肝癌研究,并取得了诸多成果。但现存的肝癌动物模型或多或少存在着各种不足和限制,故本项目通过多年探索和改进,建立了一种新的有效的肝癌小鼠模型的构建方法。
现今采用较多的肝癌模型依据成因分为自发性、诱发性、移植性和基因修饰动物模型。自发性优点良多且符合临床病程及病理特征,但该类模型发生率低下且时间参差不齐,产生肿瘤部位以及肿瘤性状个体间差异较大,因而难以大规模应用,维持成本畸高;时下较为流行的做法是采用诱发手段产生肝癌模型,多用化学试剂诱导,如CCl4、DEN。此类试剂剧毒致癌且有挥发性,对实验者健康存在威胁。此外,此法诱导时程较长,平均9个月左右,且造模对象基数庞大,维持耗费巨大,然成模之后个体差异也十分显著,如成瘤时间、肿瘤大小、荷瘤病灶等;基因修饰肝癌动物模型制作技术要求高,价格昂贵,普通实验室无力承担,且存在着基因修饰后代偿效应而导致的成模失败的风险;移植性肿瘤模型是一种简易快捷且周期短、成瘤快的造模选择。裸鼠为移植性肝癌模型首选实验动物,但其价格高、难饲养、易被污染的不足也是研究者无法忽视的,此外,其免疫缺陷的特性也给研究结果外推增加了困难,降低了比较医学研究价值。已有报道表明用Walker-256细胞行大鼠肝内注射能够复现人肝癌的膨胀性和浸润性生长方式,但该细胞系甲胎蛋白(AFP)阴性,不同于人类肝癌,且大鼠操作难度大,占用饲养资源多、价格也相对较高。很多研究者偏爱用肿瘤组织块接种方式造模,这种方法也存在一定的不足,其一是肿瘤组织块构成复杂,并不完全是单一的肿瘤细胞,常混有其他组织。其二是肝内瘤块包埋,伤口大、出血多、对手术要求高,并且移植量较难控制。
目前已存在的各种肝癌动物模型都有一定的缺陷,无法满足目前生物医药领域的需求,因而寻求一种新的肝癌造模方式是十分必要的。
发明内容
本发明要解决的技术问题是提供一种肝癌动物模型的制备方法。
为解决上述技术问题,本发明的实施例提供一种肝癌动物模型的制备方法,其特征在于,包括以下具体步骤:
(1)选取4周龄的ICR小鼠,观察饲养2周;将HepG2细胞在加有10%胎牛血清的DMEM培养基中培养传代扩增后,PBS洗涤2~3次后,400μL PBS重悬后用注射器注射入ICR小鼠腹腔;
(2)待小鼠出现明显腹水后抽出腹水2mL,加入离心管内,离心,弃上清;加入5mL PBS溶液重悬、吹洗,再离心5分钟,弃上清;
(3)用PBS溶液以步骤(2)中同样的方法重复洗2-3遍;
(4)在步骤(3)中弃上清后的沉淀内加入5mL PBS溶液吹散细胞,离心3分钟;用精密移液器将上清和沉淀上层的红细胞取出,保留下层白色沉淀;
(5)用1mL DMEM培养基重悬细胞,计数,再加入适量DMEM培养基计数稀释成1×106个/ml的细胞悬液备用;
(6)将ICR小鼠根据50mg/kg腹腔注射戊巴比妥钠麻醉后固定;在腹部备皮,再以碘伏消毒;
(7)在小鼠剑突下沿腹白线剪开1.5cm切口,将皮肤与腹膜钝性分离,以生理盐水湿润;在腹膜上沿腹白线切开1.2cm切口,无菌纱布剪成洞巾覆盖小鼠腹部,腹撑打开腹腔,固定,用0.9%生理盐水湿润的棉签轻轻向上翻开肝脏左右叶,以无菌生理盐水浸润的纱布覆盖,暴露肝门静脉,用镊子轻轻牵拉肝门静脉并固定,用微量注射器抽取1×106×200μL细胞悬液以10-20度角注射入肝门静脉,拔出针后立即用75%酒精棉签按压至血管表面无明显渗血后,用可吸收明胶海绵颗粒封闭针孔,按压至不渗血后用无菌生理盐水冲洗暴露的肝叶及腹腔;
(8)将肝叶送回腹腔,用可吸收缝线连续缝合腹膜,间断缝合皮肤;以75%酒精擦拭切口后,肌肉注射氨苄青霉素(20mg/Kg),皮下注射缓释镇痛剂Buprinex(0.06mg/Kg);
(9)未苏醒的小鼠给予侧卧位,保持呼吸道通畅,置于37℃热台保暖,待苏醒后送回动物房,保证食水供应;
(10)4周后将小鼠处死,切开皮肤腹膜,检测小鼠肝脏和脾脏。
进一步的,所述 ICR 小鼠选取ICR昆明小鼠。
进一步的,所述步骤(2)中离心转速为1000rpm。
进一步的,所述步骤(4)中离心转速为500rpm。
本发明的上述技术方案的有益效果如下:本发明的肝癌动物模型的制备方法建立的模型小鼠肝癌发生率为100%,自然消退率多为零,可以模拟出人类肝癌病程的全过程,包括原发性肝癌的消瘦、肝脾肿大、腹水、黄疸、癌灶转移、消化道出血、肝癌肿块破裂出血等临床症状、体征以及相应的血清学检查特征;同时,本发明的制备方法很好的解决了注入的细胞悬液沿针道返流腹腔后造成模型小鼠因形成腹水癌衰竭致死而导致肿瘤种植失败的技术难题;本发明制备的原发性肝癌小鼠模型的效果大大优于目前已有的原发性肝癌动物模型建立方法,较已有模型具有更加真实性、实用性、方便性、经济性。
附图说明
图1为本发明的实施例中肝门静脉操作图;
其中,图1A为ICR小鼠麻醉后开腹,暴露肝脏操作图;图1B为用棉签暴露肝门静脉,用沾有生理盐水纱布覆盖肠和肝脏操作图;图1C为固定肝门静脉,并进行静脉内注射,明胶海绵按压止血操作图;
图2为本发明的实施例中对照组和模型组的肝脏观察图;
其中,图2A为对照组小鼠前面图;图2B为对照组小鼠肝脏脏面图;图2C为模型组小鼠前面图;图2D为模型组小鼠肝脏脏面图;
图3为本发明的实施例中对比组和模型组肝脏组织切片H&E染色图;
其中,图3A为对照组小鼠肝脏组织切片染色图(10×);图3B为对照组小鼠肝脏组织切片染色图(20×);图3C为模型组小鼠肝脏组织切片染色图(10×);图3D为模型组小鼠肝脏组织切片染色图(20×)。
具体实施方式
为使本发明要解决的技术问题、技术方案和优点更加清楚,下面将结合具体实施例进行详细描述。
一种肝癌动物模型的制备方法,包括以下具体步骤:
选取4周龄的ICR小鼠,观察饲养2周;将HepG2细胞在加有10%胎牛血清的DMEM培养基中培养传代扩增后,PBS洗涤2~3次后,400μL PBS重悬后用注射器注射入ICR小鼠腹腔;
待小鼠出现明显腹水后抽出腹水2mL,加入离心管内,离心,弃上清;加入5mL PBS溶液重悬、吹洗,再离心5分钟,弃上清;
用PBS溶液以步骤(2)中同样的方法重复洗2-3遍;
在步骤(3)中弃上清后的沉淀内加入5mL PBS溶液吹散细胞,离心3分钟;用精密移液器将上清和沉淀上层的红细胞取出,保留下层白色沉淀;
用1mL DMEM培养基重悬细胞,计数,再加入适量DMEM培养基计数稀释成1×106个/ml的细胞悬液备用;
将ICR小鼠根据50mg/kg腹腔注射戊巴比妥钠麻醉后固定;在腹部备皮,再以碘伏消毒;
在小鼠剑突下沿腹白线剪开1.5cm切口,将皮肤与腹膜钝性分离,以生理盐水湿润;在腹膜上沿腹白线切开1.2cm切口,无菌纱布剪成洞巾覆盖小鼠腹部,腹撑打开腹腔,固定,用0.9%生理盐水湿润的棉签轻轻向上翻开肝脏左右叶,以无菌生理盐水浸润的纱布覆盖,暴露肝门静脉,用镊子轻轻牵拉肝门静脉并固定,用微量注射器抽取1×106×200μL细胞悬液以10-20度角注射入肝门静脉,拔出针后立即用75%酒精棉签按压至血管表面无明显渗血后,用可吸收明胶海绵颗粒封闭针孔,按压至不渗血后用无菌生理盐水冲洗暴露的肝叶及腹腔;
将肝叶送回腹腔,用可吸收缝线连续缝合腹膜,间断缝合皮肤;以75%酒精擦拭切口后,肌肉注射氨苄青霉素(20mg/Kg),皮下注射缓释镇痛剂Buprinex(0.06mg/Kg);
未苏醒的小鼠给予侧卧位,保持呼吸道通畅,置于37℃热台保暖,待苏醒后送回动物房,保证食水供应;
4周后将小鼠处死,切开皮肤腹膜,检测小鼠肝脏和脾脏。
其中,所述 ICR 小鼠选取ICR昆明小鼠。
本实施例中建立对照组和模型组,对照组采用10周龄ICR小鼠,模型组采用本发明的肝癌动物模型的制备方法制备的肝癌小鼠模型。
如图2所示,对照组和模型组这两组小鼠肝脏整体外观结果显示:对照组肝脏整体外观表面光滑,颜色红润,质地柔软;模型组肝脏表面粗糙,可见散在多个白色结节状瘤体,包膜完整,与正常肝脏组织分界清晰,颜色暗淡,质地变硬。可见小鼠肝脏和脾脏明显肿大,肝叶内有明显的白色结节。然后,取肝在福尔马林溶液中固定保存后做病理切片检查,经南通大学比较医学实验室病理检查,检查结果如图3所示,对照组和模型组这两组小鼠肝脏切片H&E染色结果显示:对照组小鼠肝脏组织中肝小叶结构完整,细胞核大而圆,居中,异染色质少而着色浅,核仁清晰,胞质丰富;模型组小鼠肝脏组织中肝小叶结构被破坏甚至消失,细胞形态大小不一,细胞核分裂现象明显增多,胞核大且深染,胞质量少且染色较浅,深染的肿瘤组织排列紊乱,异型性明显,出现腺样结构改变。
以上所述是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明所述原理的前提下,还可以作出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (4)
1.一种肝癌动物模型的制备方法,其特征在于,包括以下具体步骤:
选取4周龄的ICR小鼠,观察饲养2周;将HepG2细胞在加有10%胎牛血清的DMEM培养基中培养传代扩增后,PBS洗涤2~3次后,400μL PBS重悬后用注射器注射入ICR小鼠腹腔;
待小鼠出现明显腹水后抽出腹水2mL,加入离心管内,离心,弃上清;加入5mL PBS溶液重悬、吹洗,再离心5分钟,弃上清;
用PBS溶液以步骤(2)中同样的方法重复洗2-3遍;
在步骤(3)中弃上清后的沉淀内加入5mL PBS溶液吹散细胞,离心3分钟;用精密移液器将上清和沉淀上层的红细胞取出,保留下层白色沉淀;
用1mL DMEM培养基重悬细胞,计数,再加入适量DMEM培养基计数稀释成1×106个/ml的细胞悬液备用;
将ICR小鼠根据50mg/kg腹腔注射戊巴比妥钠麻醉后固定;在腹部备皮,再以碘伏消毒;
在小鼠剑突下沿腹白线剪开1.5cm切口,将皮肤与腹膜钝性分离,以生理盐水湿润;在腹膜上沿腹白线切开1.2cm切口,无菌纱布剪成洞巾覆盖小鼠腹部,腹撑打开腹腔,固定,用0.9%生理盐水湿润的棉签轻轻向上翻开肝脏左右叶,以无菌生理盐水浸润的纱布覆盖,暴露肝门静脉,用镊子轻轻牵拉肝门静脉并固定,用微量注射器抽取1×106×200μL细胞悬液以10-20度角注射入肝门静脉,拔出针后立即用75%酒精棉签按压至血管表面无明显渗血后,用可吸收明胶海绵颗粒封闭针孔,按压至不渗血后用无菌生理盐水冲洗暴露的肝叶及腹腔;
将肝叶送回腹腔,用可吸收缝线连续缝合腹膜,间断缝合皮肤;以75%酒精擦拭切口后,肌肉注射氨苄青霉素,皮下注射缓释镇痛剂Buprinex;
未苏醒的小鼠给予侧卧位,保持呼吸道通畅,置于37℃热台保暖,待苏醒后送回动物房,保证食水供应;
4周后将小鼠处死,切开皮肤腹膜,检测小鼠肝脏和脾脏。
2.根据权利要求1所述的一种肝癌动物模型的制备方法,其特征在于,所述ICR小鼠选取ICR昆明小鼠。
3.根据权利要求1所述的一种肝癌动物模型的制备方法,其特征在于,所述步骤(2)中离心转速为1000rpm。
4.根据权利要求1所述的一种肝癌动物模型的制备方法,其特征在于,所述步骤(4)中离心转速为500rpm。
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