原代細胞因其更加貼近機體的生物特性，地位日漸凸顯，Paper中若有了原代細胞的數(shù)據(jù)都會添色不少。但目前腫瘤細胞原代分離和培養(yǎng)成功率并不高，不少同學(xué)碰到從小鼠體內(nèi)取出的腫瘤塊，酶消化法后的細胞太少或者死亡細胞比例太高等難題。其實這中間一重要環(huán)節(jié)就是消化酶的選擇，膠原酶更好？還需要加透明質(zhì)酸酶和DNA酶Ⅰ嗎？膠原酶用Ⅱ還是Ⅳ型？本期就為大家羅列出關(guān)于不同腫瘤組織的消化方案。

腫瘤原代細胞培養(yǎng)日益重要！

原代培養(yǎng)也叫初代培養(yǎng)，是從供體取得組織后在體外進行的首次培養(yǎng)，原代培養(yǎng)的腫瘤細胞因組織剛剛離體，其生物學(xué)特性未發(fā)生很大變化，基因保留量在90%以上，因此用于藥物敏感性試驗及機制探究相關(guān)試驗，其數(shù)據(jù)更具有說服力。同時通過原代培養(yǎng)技術(shù)建立相應(yīng)腫瘤細胞系或株，研究其癌變、分子遺傳以及轉(zhuǎn)移演變機制等，對腫瘤研究與治療起著舉足輕重的作用。

從FVB/n/PyMT小鼠分離原代細胞[26]

上一期我們?yōu)榇蠹医榻B了組織分離原代的2種方法：物理方法和酶消化法。針對腫瘤組織原代細胞的分離，多采取酶消化法，此方法不僅以充分地消化組織上的瘤細胞，且盡可能減少了對成纖維細胞的消化，提高細胞純度。

二、消化酶的分類，不止于胰酶和膠原酶

組織消化法中，大家常用到的是膠原酶和胰蛋白酶或者2者組合使用，如當(dāng)消化的組織較硬，內(nèi)含較多結(jié)締組織或膠原成分時，用胰蛋白酶解離細胞的效果較差，此時可采用膠原酶。它不僅對細胞間質(zhì)有消化作用且對上皮細胞影響不大，可使上皮細胞與膠原成分分離而不受損害。

如下為2種酶的區(qū)別：

同時膠原酶還分為5大類：

其實除了上述2種酶，為了獲得純度及活率更高的原代細胞，還需要搭配以下幾種酶：

透明質(zhì)酸酶（Hyaluronidase）：能夠降低體內(nèi)透明質(zhì)酸活性，提高組織中液體滲透能力的酶，常與膠原酶等天然蛋白酶聯(lián)合，用于解離結(jié)締組織。

脫氧核糖核酸酶Ⅰ（Deoxyribonuclease Ⅰ,DNase Ⅰ）: 非單獨使用，需配合膠原酶或透明質(zhì)酸酶使用，去除細胞分離降解出的DNA，防止DNA導(dǎo)致細胞凝集，且不破壞細胞完整性。

中性蛋白酶（Dispase）：具有溫和的蛋白酶水解活性，同時可以保持細胞膜的完整性。常作為一種二級酶與膠原酶或其他蛋白酶結(jié)合使用，其分離纖維樣細胞的效率遠比分離上皮樣細胞效率高。

彈性蛋白酶（Elastase）：一種可以催化彈性蛋白的肽鍵或由中性氨基酸形成的其他肽鍵水解的一種絲氨酸蛋白酶。常與胰蛋白酶或者膠原酶結(jié)合用于分離含大量細胞間網(wǎng)狀纖維的組織。

舉例來說，針對癌組織一般多選用膠原酶Ⅱ或者Ⅳ，同時還需要加透明質(zhì)酸酶和DNA酶才能不損傷細胞而分離到有活性的單個癌細胞。如下為肝癌的原代細胞制備時的酶配方（具體操作步驟可以參考上期內(nèi)容）：

制備肝癌組織原代細胞的酶配方

三、不同腫瘤組織，消化方案均不相同

綜上，我們了解到消化酶不僅僅包括胰酶和膠原酶，還有很多種可以選擇。盡管這些消化酶已經(jīng)普遍應(yīng)用多年，但多種不確定因素的存在導(dǎo)致我們在消化時，依然存在較高失敗率。除了酶的分類很多以外，還有諸多影響實驗結(jié)果的因素，如：1、組織類型（上皮腫瘤或間質(zhì)化腫瘤）2、物種來源（人源或者鼠源）3、動物年齡（如14天，6周或者老年小鼠）4、遺傳改造情況（如knockout or knockin）5、所用的消化培養(yǎng)液（常見1640或DMEM） 6、酶的工作濃度、溫度及孵育時間等（膠原酶常用劑量為最終濃度200U/ml（約為1mg/mL）或0.03%~0.3%），這一系列因素都極大的影響了消化方案的制定。

考慮到上述多種不確定因素，若是實驗室沒有成熟的分離方案，通過查閱文獻來選擇最佳組織消化酶和最適消化條件，可以起到很好的參考。我們參考《腫瘤組織消化手冊》中的列表（含參考文獻），按照不同的物種（如人源、小鼠及大鼠等）的不同組織（乳腺癌，前列腺癌等），列舉了文獻中用到的消化酶及酶溶解培養(yǎng)基，這樣的方案，相信總有一款適合你。具體的消化時間和步驟，及分離后的細胞形態(tài)可參考文末對應(yīng)文獻。

1. 人源的腫瘤組織：主要列舉了肺癌，結(jié)直腸癌，前列腺癌，乳腺癌等十幾種常見腫瘤組織！

2. 小鼠來源的腫瘤組織：主要列舉了肺癌，前列腺癌，乳腺癌等常見腫瘤組織！

3. 大鼠來源的腫瘤組織

注：以上列表的方法僅供大家參考，考慮到樣本的特殊性，針對具體的樣本仍需要大家反復(fù)摸索以確定最終的消化方案。

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來源：原創(chuàng)： 黃安玲 邦耀實驗室 如有侵權(quán)，可聯(lián)系刪除。