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疾病与药物研究

肿瘤原代细胞如何制备？消化酶的选择很重要

2019年04月28日来源：邦耀生物作者：邦耀生物责任编辑：admin

摘要：原代细胞因其更加贴近机体的生物特性，地位日渐凸显，Paper中若有了原代细胞的数据都会添色不少。但目前肿瘤细胞原代分离和培养成功率并不高，不少同学碰到从小鼠体内取出的肿瘤块，酶消化法后的细胞太少或者死亡细胞比例太高等难题。其实这中间一重要环节就是消化酶的选择，胶原酶更好？还需要加透明质酸酶和DNA酶Ⅰ吗？胶原酶用Ⅱ还是Ⅳ型？本期就为大家罗列出关于不同肿瘤组织的消化方案。

前言

原代细胞因其更加贴近机体的生物特性，地位日渐凸显，Paper中若有了原代细胞的数据都会添色不少。但目前肿瘤细胞原代分离和培养成功率并不高，不少同学碰到从小鼠体内取出的肿瘤块，酶消化法后的细胞太少或者死亡细胞比例太高等难题。其实这中间一重要环节就是消化酶的选择，胶原酶更好？还需要加透明质酸酶和DNA酶Ⅰ吗？胶原酶用Ⅱ还是Ⅳ型？本期就为大家罗列出关于不同肿瘤组织的消化方案。

一、肿瘤原代细胞培养日益重要！

原代培养也叫初代培养，是从供体取得组织后在体外进行的首次培养，原代培养的肿瘤细胞因组织刚刚离体，其生物学特性未发生很大变化，基因保留量在90%以上，因此用于药物敏感性试验及机制探究相关试验，其数据更具有说服力。同时通过原代培养技术建立相应肿瘤细胞系或株，研究其癌变、分子遗传以及转移演变机制等，对肿瘤研究与治疗起着举足轻重的作用。

从FVB/n/PyMT小鼠分离原代细胞[26]

上一期我们为大家介绍了组织分离原代的2种方法：物理方法和酶消化法。针对肿瘤组织原代细胞的分离，多采取酶消化法，此方法不仅以充分地消化组织上的瘤细胞，且尽可能减少了对成纤维细胞的消化，提高细胞纯度。

二、消化酶的分类，不止于胰酶和胶原酶

组织消化法中，大家常用到的是胶原酶和胰蛋白酶或者2者组合使用，如当消化的组织较硬，内含较多结缔组织或胶原成分时，用胰蛋白酶解离细胞的效果较差，此时可采用胶原酶。它不仅对细胞间质有消化作用且对上皮细胞影响不大，可使上皮细胞与胶原成分分离而不受损害。

如下为2种酶的区别：

同时胶原酶还分为5大类：

其实除了上述2种酶，为了获得纯度及活率更高的原代细胞，还需要搭配以下几种酶：

透明质酸酶（Hyaluronidase）：能够降低体内透明质酸活性，提高组织中液体渗透能力的酶，常与胶原酶等天然蛋白酶联合，用于解离结缔组织。

脱氧核糖核酸酶Ⅰ（Deoxyribonuclease Ⅰ,DNase Ⅰ）: 非单独使用，需配合胶原酶或透明质酸酶使用，去除细胞分离降解出的DNA，防止DNA导致细胞凝集，且不破坏细胞完整性。

中性蛋白酶（Dispase）：具有温和的蛋白酶水解活性，同时可以保持细胞膜的完整性。常作为一种二级酶与胶原酶或其他蛋白酶结合使用，其分离纤维样细胞的效率远比分离上皮样细胞效率高。

弹性蛋白酶（Elastase）：一种可以催化弹性蛋白的肽键或由中性氨基酸形成的其他肽键水解的一种丝氨酸蛋白酶。常与胰蛋白酶或者胶原酶结合用于分离含大量细胞间网状纤维的组织。

举例来说，针对癌组织一般多选用胶原酶Ⅱ或者Ⅳ，同时还需要加透明质酸酶和DNA酶才能不损伤细胞而分离到有活性的单个癌细胞。如下为肝癌的原代细胞制备时的酶配方（具体操作步骤可以参考上期内容）：

制备肝癌组织原代细胞的酶配方

三、不同肿瘤组织，消化方案均不相同

综上，我们了解到消化酶不仅仅包括胰酶和胶原酶，还有很多种可以选择。尽管这些消化酶已经普遍应用多年，但多种不确定因素的存在导致我们在消化时，依然存在较高失败率。除了酶的分类很多以外，还有诸多影响实验结果的因素，如：1、组织类型（上皮肿瘤或间质化肿瘤）2、物种来源（人源或者鼠源）3、动物年龄（如14天，6周或者老年小鼠）4、遗传改造情况（如knockout or knockin）5、所用的消化培养液（常见1640或DMEM） 6、酶的工作浓度、温度及孵育时间等（胶原酶常用剂量为最终浓度200U/ml（约为1mg/mL）或0.03%~0.3%），这一系列因素都极大的影响了消化方案的制定。

考虑到上述多种不确定因素，若是实验室没有成熟的分离方案，通过查阅文献来选择最佳组织消化酶和最适消化条件，可以起到很好的参考。我们参考《肿瘤组织消化手册》中的列表（含参考文献），按照不同的物种（如人源、小鼠及大鼠等）的不同组织（乳腺癌，前列腺癌等），列举了文献中用到的消化酶及酶溶解培养基，这样的方案，相信总有一款适合你。具体的消化时间和步骤，及分离后的细胞形态可参考文末对应文献。

1. 人源的肿瘤组织：主要列举了肺癌，结直肠癌，前列腺癌，乳腺癌等十几种常见肿瘤组织！

2. 小鼠来源的肿瘤组织：主要列举了肺癌，前列腺癌，乳腺癌等常见肿瘤组织！

3. 大鼠来源的肿瘤组织

注：以上列表的方法仅供大家参考，考虑到样本的特殊性，针对具体的样本仍需要大家反复摸索以确定最终的消化方案。

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