CN116420678A - 一种肝癌微血管侵犯动物模型的构建方法 - Google Patents
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Abstract
本发明涉及人源肿瘤异种移植技术领域,特别涉及一种在小鼠中构建肝癌微血管侵犯模型的技术方法。采集临床中MVI阳性肝癌患者的MVI组织;将其制成单细胞悬液后,通过脾静脉注射建立MVI小鼠模型;利用组织切片染色确定模型的成功构建;并进一步利用模型所得的MVI组织细胞进行细胞增殖、迁移、侵袭能力的检测。针对MVI转移灶的鉴定以在造模后取肝脏做HE染色切片检查,模型组小鼠肝脏组织中可见较多位于血管内的转移灶。造模所使用的MVI组织细胞通过CCK‑8法进行细胞增殖能力检测,造模所使用的MVI组织细胞通过Transwell法进行细胞迁移、侵袭能力的检测。
Description
技术领域
本发明涉及人源肿瘤异种移植技术领域,特别涉及一种在小鼠中构建肝癌微血管侵犯模型的技术方法。
背景技术
肝细胞肝癌(hepatocellular carcinoma, HCC)是肝脏最常见的原发性实体肿瘤,是全球第五最常见的恶性肿瘤,其肿瘤致死率排全球第二位。目前肝癌根治性治疗方法主要包括肝切除和肝移植,但约70%肝切除病人和25%肝移植病人会发生肝癌复发,而且肝癌患者的5年总体生存率仅10-20%,因此肝癌仍然是目前医学上较为棘手的难题。目前大量临床研究证实微血管侵犯(microvascular invasion, MVI)与肝癌的侵袭性生物学特性密切相关,是肝癌患者治疗预后和复发的独立危险因素。目前对于MVI的预防和治疗仍然没有较好的方法,这与MVI发生机制仍不明确有着较大的关系。目前科研中,MVI动物模型尚未有报道,这使得MVI发生机制及治疗研究带来了较大的困难,因此如果能够建立与人肝癌MVI相近的动物模型并以此研究人类肝癌MVI发生机理能,相信这会为目前肝癌的治疗提供全新的策略及方法。
小鼠在肿瘤生物学和遗传学等方面和人类十分相似,因此建立小鼠MVI模型以研究人类MVI的细胞分子生物学特性、生化免疫特征、病理生理改变、发病机制以及药物治疗和预后具有重要意义。较为常用的无胸腺裸鼠(nude mouse)是由于第VIII连锁群内裸体位点基因发生纯合而形成的先天性胸腺缺陷突变小鼠,对异源肿瘤组织排斥率较低,且无毛易于观察测量移植肿瘤。
人源肿瘤异种移植(patient derived tumor xenograft, PDX)构建动物模型的方法不需经过体外培养对肿瘤进行筛选,可以保持肿瘤原有的特性和异质性。门静脉系统是肝的机能血管集合的统称,是由肠系膜上静脉和脾静脉汇合而成,将MVI患者组织经过制备形成MVI单细胞悬液,通过脾静脉注射进入门静脉,使得MVI细胞侵犯裸鼠肝脏的微小血管,定植形成MVI,进而构成肝癌MVI小鼠模型,这样的方法目前尚无文献报道。成功构建该模型可以为后续治疗用药的筛选以及对MVI机制的探究提供适宜的实验材料。
发明内容
有鉴于此,本发明的目的在于提供一种在小鼠中肝癌微血管侵犯模型的技术方法。采用本发明构建的模型能更准确地模拟MVI的发生发展和肿瘤微环境。
为了实现上述发明目的,本发明提供以下技术方案:
采集临床中MVI阳性肝癌患者的MVI组织;将其制成单细胞悬液后,通过脾静脉注射建立MVI小鼠模型;利用组织切片染色确定模型的成功构建;并进一步利用模型所得的MVI组织细胞进行细胞增殖、迁移、侵袭能力的检测。
上述方法中,MVI小鼠模型的构建方法,包括以下步骤:
(1)在获得患者知情同意情况下,在手术后将肝癌标本取下,取癌周1cm处组织2份,1份送检病理学检测,另1份制成单细胞悬液,并镜下观察有无异核细胞;
(2)将癌栓肿瘤组织剪切,研磨,经胰蛋白酶消化、过滤、裂解红细胞后,得到PVTT单细胞悬液;
(3)将制备的PVTT单细胞悬液行脾脏注射,完成模型的构建;
(4)术后饲养6周,将小鼠处死,收集肝脏组织标本;
(5)对标本中MVI转移灶做HE染色及免疫组化染色,鉴定造模成功;
(6)利用模型所得的MVI组织细胞进行细胞增殖、迁移、侵袭能力的检测。
上述模型构建方法中,所述制备成PVTT单细胞悬液,用组织剪将癌栓肿瘤组织剪成泥状,用大于组织量30-50倍体积的0.1%胰蛋白酶在37℃摇床上消化组织块20-60min。消化完毕后将细胞悬液通过200目孔径尼龙网过滤除掉未充分消化的组织。将已过滤的细胞悬液经1500rpm离心5min后,弃上清液,加红细胞裂解液重悬沉淀,室温作用5min,加等体积的PBS中和后,1500rpm离心5min,弃上清,用PBS洗涤一次,再用PBS重悬,得到单细胞悬液并细胞计数(细胞量:107),并镜下观察有无异核细胞。
上述模型构建方法中,所述脾脏注射,按0.01ml/g腹腔注射4%的水合氯醛麻醉裸鼠(5周大小,体重18-20g),将麻醉的裸鼠固仰卧位固定于桌面,通过皮肤腹膜切口寻找脾脏,将MVI单细胞悬液通过脾静脉注射进入麻醉的裸鼠,关闭缝合腹膜。
上述模型构建方法中,所述标本收集是在术后饲养6周,颈椎脱臼法处死裸鼠并收集肝脏组织,用含4%甲醛的PBS固定。
上述模型构建方法中,所述MVI转移灶做HE染色及免疫组化染色,是采取HE染色方法鉴定裸鼠肝脏组织中是否存在转移灶,进一步采取CD31免疫组化染色方式确定转移灶是否在血管中。
上述模型构建方法中,所述细胞增殖、迁移、侵袭能力的检测,是利用CCK-8法对MVI组织细胞进行细胞增殖分析,利用Transwell 法进行细胞迁移、侵袭能力的检测。
本发明的有益效果:针对MVI转移灶的鉴定以在造模后取肝脏做HE染色切片检查,模型组小鼠肝脏组织中可见较多位于血管内的转移灶。造模所使用的MVI组织细胞通过CCK-8法进行细胞增殖能力检测,造模所使用的MVI组织细胞通过Transwell法进行细胞迁移、侵袭能力的检测。
附图说明
图1为本发明的实施例中脾静脉注射MVI细胞的操作图。
图2为造模后裸鼠肝脏组织大体。
图3为造模后裸鼠肝脏肿瘤组织HE染色图 (50×)。
图4为MVI组织细胞的增殖能力(CCK-8法)。
图5为MVI组织细胞的迁移、侵袭能力(Transwell法),其中图A为迁移,图B为侵袭。
具体实施方式
下面结合附图1-5对本发明的具体实施方式做一个详细的说明。
实施列1:
本发明肝癌微血管侵犯小鼠模型的构建方法以及效果验证如下:
1.收集样本:上海某肝胆医院的患者宋某,年龄62岁,性别:女。
在获得患者知情同意情况下,于肝癌患者接受手术切除治疗后,利用组织剪和镊子将门静脉癌栓标本从患者肝癌组织中分离,将分离出的门静脉癌栓组织利用Hank’s平衡盐溶液漂洗3次,去除癌性坏死物质和其它组织成分,留下癌栓肿瘤组织。
2. 将MVI患者组织利用滤网制备成MVI单细胞悬液;
用组织剪将癌栓肿瘤组织剪成泥状,用大于组织量30-50倍体积的0.1%胰蛋白酶在37℃摇床上消化组织块20-60min。消化完毕后将细胞悬液通过200目孔径尼龙网过滤除掉未充分消化的组织。将已过滤的细胞悬液经1500rpm离心5min后,弃上清液,加红细胞裂解液重悬沉淀,室温作用5min,加等体积的PBS中和后,1500rpm离心5min,弃上清,用PBS洗涤一次,再用PBS重悬,得到单细胞悬液并细胞计数(细胞量:107);
3. 通过脾脏注射方法将癌栓肿瘤单细胞悬液注射入BCLC/C裸鼠门脉中;
按0.01ml/g腹腔注射4%的水合氯醛麻醉裸鼠,将麻醉的裸鼠固仰卧位固定于桌面,通过皮肤腹膜切口寻找脾脏,将MVI单细胞悬液(细胞量:107)通过脾静脉注射进入麻醉的裸鼠,关闭缝合腹膜,观察裸鼠生存状态。
4. 标本收集:
术后饲养裸鼠6周,颈椎脱臼法处死裸鼠并收集肝脏组织,用含4%甲醛的PBS固定。
5. 用HE方法鉴定裸鼠转移灶,CD31免疫组化染色确定转移灶是否在血管中:
在肝组织样品用含甲醛溶液(4%)的PBS固定后,将其包埋在石蜡中,然后将石蜡包埋样品切成薄切片(5μm),然后在抗原回收前用乙醇和二甲苯进行脱蜡和再水化。接着使用含过氧化氢(0.3%)的甲醛阻断内源过氧化物酶活性30分钟,再将切片置入含有3%牛血清白蛋白和5% NGS的PBS中于37℃条件下封闭1小时,然后与抗CD31 (1:100)抗体(Abcam,Cambridge,MA)在4℃孵育过夜。接下来,切片用PBS洗涤三次,然后与等同的二级抗体在37℃孵育1小时。最终通过DAB染色。
6.细胞增殖检测:
利用CCK-8法对MVI组织细胞进行细胞增殖分析。
1) 96孔板中每孔加入100μL细胞悬液,同样的样本可以设 3个重复孔,培养至细胞贴壁充分后测定。
2) 用排枪向96孔板内每孔100μL体系中加入10μL 的CCK-8溶液,轻轻混匀。如果起始为200μL培养体积,需加入20μL(10:1)CCK-8溶液,空白对照则在空白孔加相应量细胞培养液和CCK-8溶液;
3) 在细胞培养箱内继续孵育1h,每天相同时间点使用酶标仪上检测波长550nm设置下各孔的吸光度值(OD550)。
7. 细胞迁移、侵袭能力检测:
首先将8μm 孔径聚碳酸酯膜的Transwell小室嵌套放入24孔板中,用不含血清的细胞悬液接种到Transwell小室上室内,下室孔中则加注含10%血清的培养液,上下层培养液之间以聚碳酸酯膜相隔,在膜上铺基质胶,用以模拟机体内环境的细胞外基质。膜有通透性,下室培养液中的高营养成分使得上室内的低营养环境的肿瘤细胞发生趋向运动,细胞需要先分泌基质金属蛋白酶降解基质胶,而后从上室穿聚碳酸酯膜转移到下室。最后通过计数穿膜进入下室的细胞数目即可反映细胞的侵袭能力。细胞侵袭实验时前一晚需先将 -20℃冰箱保存的 Matrigel基质胶取出置于4℃冰箱解冻过夜,待其融化后用。迁移实验中,则不需要加Matrigel胶。
1) 侵袭实验时,超净台内将冰盒内Matrigel基质胶在EP管中用无血清DMEM 培养液按1:4比例稀释(冰上操作),将Transwell培养小室嵌套取出轻轻放入BD公司的24孔板孔内,将稀释胶按100μL/孔使其均匀地包被在Transwell小室底部的聚碳脂膜上(不要产生气泡),然后37℃培养箱中放置2-3 h使基质胶凝固,再放入超净工作台内过夜干燥;
2) 第二天,将Transwell小室嵌套在紫外灯下暴露照射2h灭菌后,加入少量不含血清的DMEM培养基使其润化,并用考马斯亮兰染液随机检查一个小室有无漏孔;
3) 将转染48h后的各组细胞及对照细胞弃旧培养液,换为无血清的DMEM培养液,饥饿过夜培养;
4) 常规用0.25%胰酶液消化各组细胞后,无菌PBS清洗细胞两遍,离心后用含有10g/L BSA 的无血清培养液重悬,适当稀释调整细胞数量为1×105/ml;
5) 细胞接种:吸取100μL的细胞无血清培养混悬液,贴壁接种到Transwell小室嵌套上室中以保证分散均匀,下室加 500μL含 10%胎牛血清的 DMEM 培养基,避免小室嵌套与培养液与之间形成气泡,放入37°C、5% CO2 细胞培养箱中常规培养48h;
6) 染色:取出Transwell小室嵌套,用棉签擦尽上室面残余的Matrigel胶和未穿过的细胞,下室面则浸泡在70%甲醇液中固定15min后,再加1%结晶紫染液染色5-l0min,PBS液洗涤2遍,最后蒸馏水洗涤1遍;
7) 拍照:在倒置显微镜下随机选取高倍镜视野(×200)。
8.结果:
本实施例中,针对MVI转移灶的鉴定以在造模后取肝脏做HE染色切片检查,由图2所示模型组小鼠肝脏组织中可见较多位于血管内的转移灶。造模所使用的MVI组织细胞通过CCK-8法进行细胞增殖能力检测,其生长增殖曲线如图4所示。造模所使用的MVI组织细胞通过Transwell法进行细胞迁移、侵袭能力的检测,如图5的A和B分别展示了MVI组织细胞的迁移、侵袭能力。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
Claims (6)
1.一种肝癌微血管侵犯动物模型的构建方法,采集临床中MVI阳性肝癌患者的MVI组织;将其制成单细胞悬液后,通过脾静脉注射建立MVI小鼠模型;利用组织切片染色确定模型的成功构建;并进一步利用模型所得的MVI组织细胞进行细胞增殖、迁移、侵袭能力的检测;其特征在于:上述方法中,MVI小鼠模型的构建方法,包括以下步骤:
(1)、在获得患者知情同意情况下,在手术后将肝癌标本取下,取癌周1cm处组织2份,1份送检病理学检测,另1份制成单细胞悬液,并镜下观察有无异核细胞;
(2)、将癌栓肿瘤组织剪切,研磨,经胰蛋白酶消化、过滤、裂解红细胞后,得到PVTT单细胞悬液;
(3)、将制备的PVTT单细胞悬液行脾脏注射,完成模型的构建;
(4)、术后饲养6周,将小鼠处死,收集肝脏组织标本;
(5)、对标本中MVI转移灶做HE染色及免疫组化染色,鉴定造模成功;
(6)、利用模型所得的MVI组织细胞进行细胞增殖、迁移、侵袭能力的检测。
2.根据权利要求1所述的一种肝癌微血管侵犯动物模型的构建方法,其特征在于所述的模型构建方法中,所述制备成PVTT单细胞悬液,用组织剪将癌栓肿瘤组织剪成泥状,用大于组织量30-50倍体积的0.1%胰蛋白酶在37℃摇床上消化组织块20-60min;消化完毕后将细胞悬液通过200目孔径尼龙网过滤除掉未充分消化的组织;将已过滤的细胞悬液经1500rpm离心5min后,弃上清液,加红细胞裂解液重悬沉淀,室温作用5min,加等体积的PBS中和后,1500rpm离心5min,弃上清,用PBS洗涤一次,再用PBS重悬,得到单细胞悬液并细胞计数(细胞量:107),并镜下观察有无异核细胞。
3.根据权利要求1所述的一种肝癌微血管侵犯动物模型的构建方法,其特征在于所述的模型构建方法中,所述脾脏注射,按0.01ml/g腹腔注射4%的水合氯醛麻醉裸鼠(5周大小,体重18-20g),将麻醉的裸鼠固仰卧位固定于桌面,通过皮肤腹膜切口寻找脾脏,将MVI单细胞悬液通过脾静脉注射进入麻醉的裸鼠,关闭缝合腹膜。
4.根据权利要求1所述的一种肝癌微血管侵犯动物模型的构建方法,其特征在于所述的模型构建方法中,所述标本收集是在术后饲养6周,颈椎脱臼法处死裸鼠并收集肝脏组织,用含4%甲醛的PBS固定。
5.根据权利要求1所述的一种肝癌微血管侵犯动物模型的构建方法,其特征在于所述的模型构建方法中,所述MVI转移灶做HE染色及免疫组化染色,是采取HE染色方法鉴定裸鼠肝脏组织中是否存在转移灶,进一步采取CD31免疫组化染色方式确定转移灶是否在血管中。
6.根据权利要求1所述的一种肝癌微血管侵犯动物模型的构建方法,其特征在于所述的模型构建方法中,所述细胞增殖、迁移、侵袭能力的检测,是利用CCK-8法对MVI组织细胞进行细胞增殖分析,利用Transwell法进行细胞迁移、侵袭能力的检测。
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