CN117398481A - 一种基于乳腺癌类器官及类器官移植瘤小鼠模型的药物筛选方法 - Google Patents
一种基于乳腺癌类器官及类器官移植瘤小鼠模型的药物筛选方法 Download PDFInfo
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Abstract
本发明公开了一种基于乳腺癌类器官及类器官移植瘤小鼠模型的药物筛选方法，所述移植瘤小鼠模型的药物筛选方法包括以下步骤：步骤一：获取乳腺癌细胞，首先将乳腺癌细胞取样样本通过培育技术放置在培养基中的培养液进行培育；步骤二：乳腺癌细胞KCL22细胞移植，在超净工作台内，将KCL22细胞按0.6μL，模板cDNA10ng，加ddH2O至20.0μ,3‑actin退火温度为55℃；步骤三：小鼠模型的建立；步骤四：药物筛选，本发明一种基于乳腺癌类器官及类器官移植瘤小鼠模型的药物筛选方法，通过使用乳腺癌类器官单细胞建立的小鼠模型，该模型稳定可靠，易于重复，有利于后续使用KCL22细胞株进行的药物筛选体内实验，且药物筛选实验中对目标药物的筛选精度和反应速度最快，能够有效地加快实验速度。
Description
技术领域
本发明涉及一种基于乳腺癌类器官及类器官移植瘤小鼠模型的药物筛选方法，属于乳腺癌类器官技术领域。
背景技术
乳腺是由皮肤、纤维组织、乳腺腺体和脂肪组成的。乳腺癌(breast cancer)是指在多种致癌因素的作用下，乳腺上皮(导管或小叶上皮)组织发生增殖失控而形成的一种恶性肿瘤。乳腺癌是女性最常见的恶性肿瘤之一。早期多表现为乳房肿块、乳头和乳晕改变，晚期癌细胞可发生远处转移，引起多器官病变，严重危害患者的身心健康，当乳腺细胞生长失去控制，就会形成乳腺癌。乳腺中包含能够产生乳汁的乳腺小叶和输送乳汁的乳，腺导管。绝大部分乳腺癌都起源于导管或小叶。此外，乳腺癌还包含—一些罕见的肿瘤，例叶状肿瘤、血管肉瘤等。
类器官是人工构建的组织模型，其结构和功能与天然组织相似，可用于治疗策略的开发和评估。在乳腺癌研究中，类器官的应用主要集中在乳腺肿瘤的建模和研究方面。类器官可以通过仿真乳腺完整的结构，包括乳头，导管和腺泡等，从而能够更好地模拟生物学过程和细胞准确的互动，从而有助于研究乳腺癌提供更加准确的仿真环境。
发明内容
本发明的目的在于提供一种基于乳腺癌类器官及类器官移植瘤小鼠模型的药物筛选方法，以解决上述背景技术中提出的问题。
为实现上述目的，本发明提供如下技术方案：一种基于乳腺癌类器官及类器官移植瘤小鼠模型的药物筛选方法，所述移植瘤小鼠模型的药物筛选方法包括以下步骤：
步骤一：获取乳腺癌细胞，首先将乳腺癌细胞取样样本通过培育技术放置在培养基中的培养液进行培育；
步骤二：乳腺癌细胞KCL22细胞移植，在超净工作台内，将KCL22细胞按0.6μL，模板cDNA10 ng，加ddH2O至20.0μ,3-actin退火温度为55℃，其余条件相同，2％琼脂糖凝胶电泳鉴定PCR扩增产物，得到乳腺癌单细胞；
步骤三：小鼠模型的建立，以健康小鼠为给药对象，取步骤二中的获得的乳腺癌单细胞，通过中空纤维反应器植入健康小鼠背部，24h后建立小鼠乳腺癌肿瘤病理模型；
步骤四：药物筛选，将目标药物用于小鼠模型中的植入了乳腺癌肿瘤病理小鼠模型内，按照固定时间对小鼠模型中的小鼠进行给药，然后检测小鼠体内乳腺癌肿瘤单细胞活性，并以头乳腺癌肿瘤单细胞活性作为评价腺癌肿瘤单细胞药物的指标。
作为本发明的一种优选技术方案，所述步骤一中，使用低代次KCL22细胞在乳腺癌单细胞培养基中，于37℃,5％CO：条件下常规培养，移植前取对数生长期的细胞，用无菌PBS调整细胞浓度。
作为本发明的一种优选技术方案，所述步骤二中，PCR反应条件：94℃预变性5min；94℃变性30s，52℃退火30s，72℃延伸30s，共30个循环；72℃再延伸5min；4℃保存2h。
作为本发明的一种优选技术方案，所述步骤三中，KCL细胞移植1周后开始每日观察小鼠模型中小鼠的一般状态，包括小鼠行为、体重、饮食、大小便和精神状况，移植4周以后，用剪尾取血法取小鼠尾静脉血10μL，加入0.39mL细胞稀释液(4％冰醋酸)中，立即混匀后取10uL进行改良牛鲍计数板充池计数乳腺癌肿瘤细胞的数量。
作为本发明的一种优选技术方案，所述步骤一中，乳腺癌肿瘤单细胞培养基的组成成分：胎牛血清5％—15％、青霉素1％—3％、神经调节蛋白2％—4％、L-谷氨酰胺0.2％—0.6％、人头蛋白5％—8％、哌嗪乙磺酸0.1％—0.3％、抑制剂0.1％—0.3％、成纤维细胞生长因子100.2％—0.6％、乙酰半胱氨酸1％—2％、抗生素0.2％—0.8％、重组人角质细胞生长因子0.1％—0.3％。
作为本发明的一种优选技术方案，所述步骤四中，取筛选的肿瘤组织剪碎，吹打，常规培养和传代于含10％小牛血清的培养液中，用生长旺盛的连续传代细胞，小鼠皮下注射2x10°细胞。
与现有技术相比，本发明的有益效果是：本发明一种基于乳腺癌类器官及类器官移植瘤小鼠模型的药物筛选方法，通过使用乳腺癌类器官单细胞建立的小鼠模型，该模型稳定可靠，易于重复，有利于后续使用KCL22细胞株进行的药物筛选体内实验，且药物筛选实验中对目标药物的筛选精度和反应速度最快，能够有效地加快实验速度。
具体实施方式
对本发明实施例中的技术方案进行清楚、完整的描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明提供了一种基于乳腺癌类器官及类器官移植瘤小鼠模型的药物筛选方法，所述移植瘤小鼠模型的药物筛选方法包括以下步骤：
步骤一：获取乳腺癌细胞，首先将乳腺癌细胞取样样本通过培育技术放置在培养基中的培养液进行培育；
步骤二：乳腺癌细胞KCL22细胞移植，在超净工作台内，将KCL22细胞按0.6μL，模板cDNA10 ng，加ddH2O至20.0μ,3-actin退火温度为55℃，其余条件相同，2％琼脂糖凝胶电泳鉴定PCR扩增产物，得到乳腺癌单细胞；
步骤三：小鼠模型的建立，以健康小鼠为给药对象，取步骤二中的获得的乳腺癌单细胞，通过中空纤维反应器植入健康小鼠背部，24h后建立小鼠乳腺癌肿瘤病理模型；
步骤四：药物筛选，将目标药物用于小鼠模型中的植入了乳腺癌肿瘤病理小鼠模型内，按照固定时间对小鼠模型中的小鼠进行给药，然后检测小鼠体内乳腺癌肿瘤单细胞活性，并以头乳腺癌肿瘤单细胞活性作为评价腺癌肿瘤单细胞药物的指标。
其中，所述步骤一中，使用低代次KCL22细胞在乳腺癌单细胞培养基中，于37℃,5％CO：条件下常规培养，移植前取对数生长期的细胞，用无菌PBS调整细胞浓度。
进一步地，所述步骤二中，PCR反应条件：94℃预变性5min；94℃变性30s，52℃退火30s，72℃延伸30s，共30个循环；72℃再延伸5min；4℃保存2h。
更进一步地，所述步骤三中，KCL细胞移植1周后开始每日观察小鼠模型中小鼠的一般状态，包括小鼠行为、体重、饮食、大小便和精神状况，移植4周以后，用剪尾取血法取小鼠尾静脉血10μL，加入0.39mL细胞稀释液(4％冰醋酸)中，立即混匀后取10uL进行改良牛鲍计数板充池计数乳腺癌肿瘤细胞的数量。
实施例一：胎牛血清5、青霉素1％、神经调节蛋白2％、L-谷氨酰胺0.2％、人头蛋白5％、哌嗪乙磺酸0.1％、抑制剂0.1％、成纤维细胞生长因子100.2％、乙酰半胱氨酸1％、抗生素0.2％、重组人角质细胞生长因子0.1％；
实施例二：胎牛血清10％、青霉素2％、神经调节蛋白3％、L-谷氨酰胺0.4％、人头蛋白5％—8％、哌嗪乙磺酸0.2％、抑制剂0.2％、成纤维细胞生长因子100.4％、乙酰半胱氨酸1.5％、抗生素0.5％、重组人角质细胞生长因子0.2％；
实施例三：胎牛血清15％、青霉素1％、神经调节蛋白2％、L-谷氨酰胺0.2％、人头蛋白5％、哌嗪乙磺酸0.1％、抑制剂0.1％、成纤维细胞生长因子100.2％、乙酰半胱氨酸1％、抗生素0.2％、重组人角质细胞生长因子0.1％；
其中，所述步骤四中，取筛选的肿瘤组织剪碎，吹打，常规培养和传代于含10％小牛血清的培养液中，用生长旺盛的连续传代细胞，小鼠皮下注射2x10°细胞。
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。
Claims (6)
1.一种基于乳腺癌类器官及类器官移植瘤小鼠模型的药物筛选方法，其特征在于，所述移植瘤小鼠模型的药物筛选方法包括以下步骤：
步骤一：获取乳腺癌细胞，首先将乳腺癌细胞取样样本通过培育技术放置在培养基中的培养液进行培育；
步骤二：乳腺癌细胞KCL22细胞移植，在超净工作台内，将KCL22细胞按0.6μL，模板cDNA10 ng，加ddH2O至20.0μ,3-actin退火温度为55℃，其余条件相同，2％琼脂糖凝胶电泳鉴定PCR扩增产物，得到乳腺癌单细胞；
步骤三：小鼠模型的建立，以健康小鼠为给药对象，取步骤二中的获得的乳腺癌单细胞，通过中空纤维反应器植入健康小鼠背部，24h后建立小鼠乳腺癌肿瘤病理模型；
步骤四：药物筛选，将目标药物用于小鼠模型中的植入了乳腺癌肿瘤病理小鼠模型内，按照固定时间对小鼠模型中的小鼠进行给药，然后检测小鼠体内乳腺癌肿瘤单细胞活性，并以头乳腺癌肿瘤单细胞活性作为评价腺癌肿瘤单细胞药物的指标。
2.根据权利要求1所述的一种基于乳腺癌类器官及类器官移植瘤小鼠模型的药物筛选方法，其特征在于：所述步骤一中，使用低代次KCL22细胞在乳腺癌单细胞培养基中，于37℃,5％CO：条件下常规培养，移植前取对数生长期的细胞，用无菌PBS调整细胞浓度。
3.根据权利要求1所述的一种基于乳腺癌类器官及类器官移植瘤小鼠模型的药物筛选方法，其特征在于：所述步骤二中，PCR反应条件：94℃预变性5min；94℃变性30s，52℃退火30s，72℃延伸30s，共30个循环；72℃再延伸5min；4℃保存2h。
4.根据权利要求1所述的一种基于乳腺癌类器官及类器官移植瘤小鼠模型的药物筛选方法，其特征在于：所述步骤三中，KCL细胞移植1周后开始每日观察小鼠模型中小鼠的一般状态，包括小鼠行为、体重、饮食、大小便和精神状况，移植4周以后，用剪尾取血法取小鼠尾静脉血10μL，加入0.39mL细胞稀释液(4％冰醋酸)中，立即混匀后取10uL进行改良牛鲍计数板充池计数乳腺癌肿瘤细胞的数量。
5.根据权利要求1所述的一种基于乳腺癌类器官及类器官移植瘤小鼠模型的药物筛选方法，其特征在于：所述步骤一中，乳腺癌肿瘤单细胞培养基的组成成分：胎牛血清5％—15％、青霉素1％—3％、神经调节蛋白2％—4％、L-谷氨酰胺0.2％—0.6％、人头蛋白5％—8％、哌嗪乙磺酸0.1％—0.3％、抑制剂0.1％—0.3％、成纤维细胞生长因子100.2％—0.6％、乙酰半胱氨酸1％—2％、抗生素0.2％—0.8％、重组人角质细胞生长因子0.1％—0.3％。
6.根据权利要求1所述的一种基于乳腺癌类器官及类器官移植瘤小鼠模型的药物筛选方法，其特征在于：所述步骤四中，取筛选的肿瘤组织剪碎，吹打，常规培养和传代于含10％小牛血清的培养液中，用生长旺盛的连续传代细胞，小鼠皮下注射2x10°细胞。
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