CN114480250B - 构建原位原发胃癌动物模型的方法 - Google Patents
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Abstract
本发明公开了一种原位原发的胃癌肿瘤模型制备方法，属于肿瘤动物模型领域。本发明通过将小鼠胃上皮细胞以特定培养基培养成类器官，再将类器官进行遗传改造，注射回小鼠胃，使其发展成肿瘤。本发明的方法相比移植瘤动物模型，具有明确的基因突变背景，能够有效模拟人体胃癌发生发展的真实过程。相比基因工程肿瘤动物模型，本发明可以高效构建具有不同基因突变背景的胃癌小鼠模型，耗时短，成本低，成瘤率高。
Description
技术领域
本发明属于肿瘤动物模型领域。
背景技术
胃癌已成为我国发病率第二，死亡率第三的肿瘤。然而近二十年来，胃癌的临床治疗并未取得突破性进展。深入了解胃癌发生机制，找到其中的关键作用因子，进而研发更加有效的治疗手段，显得尤为迫切。肿瘤动物模型在探究肿瘤发生发展机制、研发新药物上扮演着至关重要角色。小鼠胃癌模型为研究胃癌发生与转移机制、抗肿瘤药物筛选、免疫治疗评价等方面、提供有力的工具。
目前，常用的小鼠胃癌模型，主要包括移植瘤模型和自发成瘤模型。移植瘤模型是采用的免疫缺陷小鼠(如无胸腺裸鼠、SCID小鼠)作为移植受体,将肿瘤细胞(细胞系)或肿瘤组织块移植进免疫缺陷鼠体内后形成移植肿瘤。自发成瘤模型主要包括：基因工程小鼠胃癌模型和化学诱导小鼠胃癌模型。已有报道的基因工程小鼠胃癌模型主要包括:INS-GAS小鼠、TFF-1敲除小鼠和RunnX3敲除小鼠。这些小鼠均是利用小鼠胚胎干细胞的基因同源重组，将特定基因在小鼠基因组上敲除或敲入。化学诱导小鼠胃癌模型，是利用化学致癌物质长期刺激诱发胃癌发生，常用的药物包括亚硝基化合物MNNG和MNU。
但是，目前的小鼠胃癌模型存在诸多缺点，在满足基础以及临床前研究需求都存在不足。小鼠移植瘤胃癌模型一直都饱受争议，因为移植瘤并非小鼠机体自发形成的肿瘤，不能反映体内肿瘤形成的真实过程；更重要的是，机体的免疫系统在肿瘤形成过程中十分重要，而免疫缺陷小鼠缺乏完整的免疫系统，利用移植瘤胃癌模型已经不能满足肿瘤免疫学治疗研究的需要。基于基因工程小鼠的自发成瘤模型因其类似人体内肿瘤的发生过程而得到认可，但是肿瘤的发生是多个基因累积改变的过程，传统基因工程小鼠单次只引入一至两个基因改变，若想获得多个基因突变小鼠，需要多次交配迭代；耗时长，若要同时获得大量相同基因型的肿瘤小鼠，需要更多的亲代小鼠，研究成本提高。同时，由于基因工程小鼠是生殖系突变，容易诱发多系统多器官的肿瘤，这与肿瘤实际发生情况也有差异；并且由于小鼠个体差异，自发成瘤的周期差异也较大，难以达到同步。更重要的是，基因工程小鼠构建技术门槛高，若要构建新的基因型小鼠模型周期长成本高，一般研究机构无法常规开展。对于化学诱导小鼠胃癌模型，该类模型构建时间相对较长，成瘤具有明显的个体差异，高异质性可能会造成后期数据的处理难度增加，文献报道，总体成瘤在50％左右。最重要的是该类模型缺乏明确的基因操作，不适用于发病机理研究。
发明内容
本发明的目的在于提供一种更接近胃癌生物学特性、且制备周期短的原位原发胃癌模型。
为了实现上述发明目的，本发明提供了以下技术方案：
一种构建原位原发胃癌动物模型的方法，其特征在于，包括：
将遗传改造的胃类器官注射到动物胃组织内；
所述遗传改造是指敲除抑癌基因，和/或过表达癌基因。
进一步地，所述遗传改造具体为如下任一情形：
a.敲除TP53、PTEN、SMAD4基因；
b.敲除TP53、APC、CDKN2A、MLL3基因；
c.敲除TP53、CDH1、ARID1A基因，过表达MYC基因。
进一步地，所述胃癌为胃腺癌。
进一步地，所述遗传改造还包括对类器官转入荧光标记基因。
进一步地，所述动物是小鼠。
前述的方法制备得到的动物模型在药物筛选、药物毒性试验或免疫治疗试验中应用。
一种胃类器官培养基，所述培养基是在DMEM/F12培养基的基础上，加上如下添加剂得到：
| 成分 | 含量 | 成分 | 含量 |
| B27 | 40～100倍稀释 | EGF | 50～200ng/ml |
| R-spondin 1 | 200～300ng/ml | Y-27632 | 5～40μM |
| Glutamax | 100倍稀释 | Gastrin | 1～10nM |
| N-acetylcysteine | 0.5～5mM | Noggin | 50～200ng/ml |
| A83-01 | 100～300nM | Nicotinamide | 5～20mM |
| WNT3a | 50～200ng/ml | N2 | 50～200倍稀释 |
。
如前述的胃类器官培养基，所述培养基是在DMEM/F12培养基的基础上，加上如下添加剂得到：
| 成分 | 含量 | 成分 | 含量 |
| B27 | 50倍稀释 | EGF | 50ng/ml |
| R-spondin 1 | 250ng/ml | Y-27632 | 10μM |
| Glutamax | 100倍稀释 | Gastrin | 1nM |
| N-acetylcysteine | 1mM | Noggin | 100ng/ml |
| A83-01 | 200nM | Nicotinamide | 10mM |
| WNT3a | 50ng/ml | N2 | 100倍稀释 |
。
一种胃类器官的培养方法，包括如下步骤：
1)胃粘膜上皮细胞混合基质胶Matrigel；
2)待Matrigel凝固，使用权利要求7或8所述培养基培养得到类器官。
进一步地，步骤1)的胃粘膜上皮细胞是原代培养的胃粘膜上皮细胞；
优选地，所述原代培养的胃细胞的制备方法如下：
a)取动物胃，洗净胃内容物，剪碎至2-3mm大小组织块；
b)用含有Y27632 10μM、EDTA 5mM的DPBS溶液，在冰上预消化30分钟后，用1ml移液枪吹打10-20次；
c)静置，待未消化组织块自然沉降，收集上清液，离心，弃上清；
d)用TrypLE消化，得胃粘膜上皮细胞的单细胞悬液。
本发明的有益效果：
本发明的肿瘤模型构建周期较基因工程动物模型大大缩短，不会导致动物成瘤前死亡，成瘤率高达100％。
本发明构建的原位原发的小鼠胃肿瘤模型，可模拟在人体内由于遗传改变导致正常细胞向肿瘤细胞转化的过程，能动态地表征肿瘤发生发展地过程，在基因层面、肿瘤微环境、肿瘤发展及病理生理等方面与肿瘤发生发展真实情况更加贴近。
总之，本发明的方法可高效率地制备得到更接近胃癌特征、符合临床研究需求的胃癌模型；该模型可以在探究胃癌发生发展机制、寻找和优化新的胃癌可能的治疗方式等研究领域提供有利工具。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1：实验例1中类器官显微观察。
图2：实验例1中类器官T7内切酶检测。
图3：实验例1中小鼠肿瘤的荧光素酶活体成像。
图4：实验例1中小鼠胃癌组织观察。
图5：实验例1中小鼠胃癌组织HE染色。
图6：实验例2中小鼠的荧光素酶活体成像。
图7：实验例2中小鼠胃癌组织HE染色。
图8：实验例2中小鼠胃癌组织T7内切酶检测。
图9：实验例3中小鼠的荧光素酶活体成像。
图10：实验例3中小鼠胃癌组织HE染色。
图11：实验例3中小鼠胃癌组织T7内切酶检测(左图)和蛋白免疫印迹检测(右图)。
图12：实验例4中小鼠的荧光素酶活体成像。
图13：实验例4中小鼠肿瘤重量。
具体实施方式
本发明中的部分英文缩写解释如下：
DMEM：是一种应用十分广泛的培养基，可用于许多哺乳动物细胞培养，购买自GIBCO公司。
DMEM/F12：是F12培养基和DMEM培养基按照1：1结合，称为DMEM/F12培养基。综合了F12含有较丰富的成分和DMEM含有较高浓度养分的优点。购买自GIBCO公司。
Matrigel，从富含胞外基质蛋白的EHS小鼠肿瘤中分离出，其主要成分由层粘连蛋白，Ⅳ型胶原，巢蛋白，硫酸肝素糖蛋白等组成，还包含生长因子和基质金属蛋白酶等。购买自BD公司。
B27，即B27补充剂，市售产品，可用于配制培养基。B27补充剂以50倍浓度(使用时进行50倍稀释)的液体浓缩液提供，除其它成分外其包含生物素、胆固醇、亚油酸、亚麻酸、黄体酮、腐胺、视黄醇、视黄醇乙酸酯、亚硒酸钠、三碘甲状腺原氨酸(T3)、DL-α-生育酚(维生素E)、白蛋白、胰岛素以及转铁蛋白。购自Life Technologies公司。N-acetylcysteine：N-乙酰半胱氨酸，购买自Sigma公司。
EGF，表皮生长因子，市售产品，购买自R&D公司。
Noggin，细胞生长蛋白成分，市售产品，购买自Peprotech公司。
R-spondin 1，人细胞生长编码蛋白，市售产品，购买自Peprotech公司。
A83-01，TGF-β抑制剂，购买自Tocris Bioscience公司。
FGF10，成纤维细胞生长因子，购买自Peprotech公司。
Nicotinamide，烟酰胺，购买自Sigma公司。
Y-27632，ROCK特异性通路阻断剂。购买自Abmole Bioscience公司。
WNT3a，WNT激动剂，细胞中激活TCF/LEF-介导的转录的因子，购买自PeproTech公司。
Glutamax，市售细胞培养添加剂，购自：GIBCO公司。
N2，N2补充剂以100倍的液体浓缩液提供，其包含500μg/ml人转铁蛋白，500μg/ml。
Gastrin，胃泌素，购买自Sigma公司。
TrypLE，用于解离贴壁哺乳动物细胞的重组消化酶，购买自GIBCO公司。
实施例1本发明小鼠胃癌模型的构建及验证
(1)利用类器官培养技术，体外培养胃类器官
取C57BL/6小鼠胃，用预冷的DPBS溶液洗净胃内容物，剪碎至2-3mm大小的组织块，用含有Y27632 10μM、EDTA 5mM的DPBS溶液，在冰上预消化30分钟后，用1ml移液枪吹打10-20次，观察可见大量胃黏膜上皮腺体组织被释放出来，静置，待未消化的组织块自然沉降，收集上清液于15ml BD管，350g离心5min，弃上清，加入10ml细胞消化液TrypLE，37摄氏度消化15min，得到小鼠胃粘膜上皮细胞单细胞悬液，进行台盼蓝计数，以30000个细胞/30ul基质胶Matrigel的密度接种于48孔板中，放入培养箱10分钟，待Matrigel凝固，加入胃类器官培养基在37℃、5％CO2的细胞培养箱中培养，每2-3天更换新的胃类器官培养基，每7-9天进行传代。
上述胃类器官培养基，以DMEM/F12培养基为基础，加如下成分进行配置：
| 成分 | 含量 | 成分 | 含量 |
| B27 | 50倍稀释 | EGF | 50ng/ml |
| R-spondin 1 | 250ng/ml | Y-27632 | 10μM |
| Glutamax | 100倍稀释 | Gastrin | 1nM |
| N-acetylcysteine | 1mM | Noggin | 100ng/ml |
| A83-01 | 200nM | Nicotinamide | 10mM |
| WNT3a | 50ng/ml | N2 | 100倍稀释 |
上述用于胃类器官的培养的组织，包括但不限于C57BL/6小鼠，基因工程小鼠(如)，以及手术及活检采集的人体胃标本。
(2)病毒载体构建、包装以及体外验证
利用基因过表达技术过表达癌基因，利用CRISPR/Cas9基因敲除技术敲除抑癌基因，具体如下：
以pMIG-Luci载体出发，通过常规酶切、酶连操作，将目标癌基因构建到pMIG-Luci载体上，该载体上同时携带有荧光素酶报告基因Luciferase和红色荧光标记基因。
以V2TC空载质粒出发，通过常规酶切、酶连操作，引入目标抑癌基因的gRNA，Cas9蛋白来源于LentiCRISPRv2质粒。
上述目标癌基因，包括但不限于以下基因：ERBB2、CDK12、MYC、CCNE1、RARA、CDK6、KRAS、EGFR、ZNF217、IKZF3、CCND1、FGF19。
上述目标抑癌基因，包括但不限于以下基因：TP53、ARID1A、LRP1B、KMT2D、KMT2C、FAT4、CDH1、KMT2B、BRCA2、PTEN、PTPRD、CDKN2A、CDKN2B、PRKN、MTAP、FHIT、SMAD4、IRF2。
将构建好的上述病毒载体及包装质粒共转染293T细胞，转染后8小时培养基，分别在24h更换为DMEMF12基础培养基(不含FBS)，分别在30h36h 42h和48h收集病毒上清，0.45过滤除去细胞碎片后，用病毒液体外感染3T3工具细胞，提取RNA和蛋白，用QPCR和WesternBlot的方法检测目标癌基因的扩增效果；提取DNA，直接测序或者T7E1或者酶切鉴定目标抑癌基因的突变情况。选取突变效果最佳的gRNA进行后续实验。
(3)小鼠胃类器官感染、基因敲除验证
取培养3代以内的小鼠胃类器官，用TrypLE消化至单细胞，以0.5ml新鲜病毒液重悬，加入助转染试剂Polybrene(最终工作浓度2μg/ml)以及Y27632(最终工作浓度10μM)，放入24孔板，31摄氏度2000转/分离心60min，放入37孵箱中孵育12h，取出用10ml DPBS溶液稀释病毒液，300g离心10min，去上清，用基质胶Matrigel重悬细胞，接种于48孔板中，加入胃类器官培养基，在37℃、5％CO2的细胞培养箱中培养。取培养5-7天的胃类器官提取RNA和蛋白，用QPCR和Western Blot的方法检测目标癌基因的扩增效果；提取DNA，直接测序或者T7E1或者酶切鉴定目标抑癌基因的突变情况。
成功转染病毒的类器官如图1所示，左图为非荧光观察，右图为荧光观察；从右图可见红色荧光，表明病毒成功转染。
(4)类器官原位移植
取传代后5-7天的胃类器官，制成胃类器官DPBS悬液，显微镜下计数。取6-8周C57BL/6小鼠作为移植受体小鼠，氯胺酮麻醉后，腹部剃毛，聚维酮碘消毒，在距离小鼠腹中线1cm处剪开腹部，将胃拖出，在胃下三分之一靠近幽门处，用胰岛素针注射胃类器官悬液至小鼠胃壁黏膜下层。
上述作为移植受体的小鼠，包括但不限于具有正常免疫的小鼠(如C57BL/6，基因工程小鼠等)，免疫缺陷小鼠(如无胸腺裸鼠、SCID小鼠、NSG小鼠)，人源化小鼠(如huNSG、huNOG)。
(5)小鼠的监测和病理鉴定
移植后实时观察小鼠状态，利用荧光素酶活体成像技术连续监测小鼠胃类器官生长情况，注射后4周左右小鼠开始发病，主要表现为消瘦、活动减少、进食减少。解剖后取出小鼠胃，探查小鼠脑、肝、肺是否有转移。取部分胃肿瘤组织液氮冷冻保存，取部分胃肿瘤组织继续培养胃肿瘤类器官，取部分胃肿瘤组织按常规病理检测方法进行固定、脱水、包埋、切片及HE染色处理来检测组织的病理特征，确定通过移植编辑后胃类器官形成的胃癌模型属于胃癌。
以下用实验例的形式对本发明的有益效果做进一步说明。
实验例1 TP53/PTEN/SMAD4敲除的原发、原位小鼠胃癌模型构建
1.方法
取正常免疫完全C57BL/6小鼠的胃组织，使用实施例1的方法依次进行类器官培养、传代、遗传改造、基因敲除验证，并将所得遗传改造的类器官移植到小鼠胃，通过活体成像技术监测肿瘤的大小和位置，移植100天后，牺牲小鼠，对胃肿瘤进行直接观察、HE染色及免疫组织化学染色观察。
所述遗传改造是利用CRISPR/Cas9基因敲除技术，敲除抑癌基因TP53、PTEN、SMAD4。
2.结果
2.1基因敲除验证
利用T7核酸内切酶检测遗传改造后类器官中TP53、PTEN、SMAD4的突变情况如图2所示，可见TP53、PTEN、SMAD4都被成功敲除。
2.2肿瘤活体成像
原位移植100天后，利用活体荧光成像系统检测小鼠成瘤情况，结果显示小鼠胃的大体解剖部位呈现明显的强阳性信号，表明原位移植的编辑后类器官在体内能够生长增殖(图3)。
2.3小鼠胃形态观察
如图4所示，上图为小鼠胃大体形态，箭头所示处可见明显新生物。下图为小鼠胃沿胃大弯剖开图，虚线所示为异常胃黏膜表型，具备典型胃癌特征。
2.4HE染色
小鼠胃切片HE染色，显示为胃壁明显增厚，肿瘤浸润胃壁全层。癌细胞表现不同程度的异型性，大小不一，形态各异，可见病理性核分裂像(图5)。
本实验例得到的胃癌模型属于胃腺癌模型，6只小鼠在100天内全部成功发展成胃癌。
实验例2构建TP53/APC/CDKN2A/MLL3敲除的小鼠胃癌模型
1.方法
取转基因小鼠TP53-/-,Cas9小鼠胃(TP53基因缺失，表达Cas9蛋白)组织，使用实施例1的方法依次进行类器官培养、传代、遗传改造，并将所得遗传改造的类器官移植到裸鼠胃(免疫缺陷小鼠)，通过活体成像技术监测肿瘤的大小和位置，移植45天后，牺牲小鼠，对胃组织进行HE染色及T7核酸内切酶基因敲除验证。
所述遗传改造是利用CRISPR/Cas9基因敲除技术敲除抑癌基因APC、CDKN2A、MLL3(因小鼠本身为TP53基因缺失，此处就不再对TP53进行敲除)。
2.结果
2.1肿瘤活体成像
移植40天后，利用活体荧光成像系统检测小鼠成瘤情况，结果显示小鼠胃的大体解剖部位呈现明显的强阳性信号(图6)。
2.2HE染色
HE染色结果显示，部分癌细胞排列成腺管样结构，部分小群的癌细胞和癌性腺体浸润胃壁，癌间纤维组织增加，呈现中到低分化腺癌特征(图7)。
2.3基因敲除验证
T7核酸内切酶检测显示，肿瘤组织中APC、CDKN2A、MLL3都被成功敲除(图8)。
本实验例的5只小鼠中，均在45天内成功发展成胃癌。
实验例3构建TP53/CDH1/ARID1A敲除，MYC过表达的小鼠胃癌模型
1.方法
取转基因小鼠TP53-/-,Cas9小鼠胃(TP53基因缺失，表达Cas9蛋白)组织，使用实施例1的方法依次进行类器官培养、传代、遗传改造，并将所得遗传改造的类器官移植到小鼠胃，通过活体成像技术监测肿瘤的大小和位置，移植45天后，牺牲小鼠，对胃组织进行HE染色及T7核酸内切酶基因敲除验证。
所述遗传改造是利用CRISPR/Cas9基因敲除技术敲除抑癌基因CDH1、ARID1A(因小鼠本身为TP53基因缺失，此处就不再对TP53进行敲除)，过表达MYC基因。
2.结果
2.1肿瘤活体成像
图9显示了移植后第7天和第30天的活体成像情况，可见随着时间增加，小鼠胃部荧光信号逐渐增强。
2.2HE染色
图10显示在移植后第45天，取小鼠胃进行HE染色，进行病理分析，部分癌细胞排列成腺管样结构，部分小群的癌细胞和癌性腺体浸润胃壁，癌间纤维组织增加，可见部分角化癌细胞及细胞间桥，提示为中到低分化腺癌(含磷癌成分)。
2.3基因敲除和基因过表达验证
基因敲除验证如图11左图所示，可见CDH1、ARID1A都被成功敲除。
基因过表达验证如图11右图所示，可见肿瘤组织相对于正常组织过表达MYC。
本实验例中，3只小鼠均在45天内成功发展成胃癌。
实验例4利用本发明模型进行体内药物实验
利用该模型进行体内药物试验的具体步骤如下。
(1)将本项目构建的胃癌小鼠进行配对分组：分为不同浓度给药组和溶剂组。
(2)用腹腔注射或灌胃等方式给药。
(3)于不同时间点通过荧光素酶活体成像系统，观察并统计小鼠肿瘤负荷的情况。
本实验例以5-氟尿嘧啶为例进行说明。
利用本方法构建的模型小鼠，观察5-氟尿嘧啶的体内治疗作用。
1.方法
取3只小鼠作为实验组，采用腹腔注射方式，注射剂量5-氟尿嘧啶15mg/kg，每日1次；取3只小鼠作为对照组，采用腹腔注射方式，注射与实验组相等体积的生理盐水，每日1次。
连续给药4周后，通过荧光素酶活体成像系统，观察并统计小鼠肿瘤负荷的情况。
2.结果
结果如图12、13所示。
图12显示了荧光素酶活体成像系统观察到的小鼠荷瘤情况，可见相比于对照组，实验组在给药4周后，肿瘤负荷明显降低。
图13显示了两组小鼠肿瘤的重量，可见相比于对照组，实验组肿瘤重量明显减小。
本实验例证实得到胃癌模型对5-氟尿嘧啶治疗敏感，表明本发明的模型可用于抗肿瘤药物的筛选。
综上，本发明的方法可高效率地制备得到更接近胃癌特征、符合临床研究需求的胃癌模型；该模型可以在探究胃癌发生发展机制、寻找和优化新的胃癌可能的治疗方式等研究领域提供有利工具。
Claims (7)
1.一种构建原位原发胃癌动物模型的方法，其特征在于，包括：
将遗传改造的小鼠胃类器官注射到小鼠胃组织内；
所述遗传改造是指敲除抑癌基因，和/或过表达癌基因；
所述遗传改造具体为如下任一情形：
a.敲除TP53、APC、CDKN2A、MLL3基因；
b.敲除TP53、CDH1、ARID1A基因，过表达MYC基因；
所述胃癌为胃腺癌。
2.如权利要求1所述的方法，其特征在于：
所述遗传改造还包括对类器官转入荧光标记基因。
3.权利要求1～2任一所述的方法制备得到的动物模型在药物筛选、药物毒性试验中应用。
4.如权利要求1所述的方法，其特征在于：
所述胃类器官的培养基是在DMEM/F12培养基的基础上，加上如下添加剂得到：
。
5.如权利要求4所述的方法，其特征在于：所述培养基是在DMEM/F12培养基的基础上，加上如下添加剂得到：
。
6.一种如权利要求1-4中胃类器官的培养方法，其特征在于，包括如下步骤：
1)取小鼠胃粘膜上皮细胞混合基质胶Matrigel；
2)待Matrigel凝固，使用权利要求4或5所述培养基培养得到类器官。
7.如权利要求6所述的培养方法，其特征在于：
步骤1)的胃粘膜上皮细胞是原代培养的胃粘膜上皮细胞。
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