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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：
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。 [1]Liang S, Marti T, Dorn P, et al. Blocking the epithelial-to-mesenchymal transition pathway abrogates resistance to anti-folate chemotherapy in lung cancer[J]. Cell Death and Disease, 2015, 6(7). 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