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发布: 2016-02-16 143阅读 107评论

通过裂解细胞来提取其中的物质是分子生物学中很常见的步骤，裂解方法也是种类繁多，可以用化学试剂、声波震荡、甚至机械压力。在整个裂解过程中，细胞膜并不是什么阻碍，细胞壁（cell wall）才是最顽强、最难打破的。什么情况下用什么方法更好，确实是一个很值得讨论的话题。在说到细胞裂解法之前，我们先来了解一下细胞壁分哪几种。

细胞壁是细胞的保护层，存在于除了动物细胞外几乎所有生物细胞的外层。与有些生物书中卡通图展示的平滑的细胞壁不同，现实的细胞壁应该是凹凸不平、甚至是崎岖陡峭的外表层。根据组成成分或者结构的不同，细胞壁可以分为五种。

植物细胞壁

主要成分：纤维素（cellulose）、果胶（pectin）和木质素（lignin）

植物细胞壁分为初生层和可能存在的第二层。初生壁中主要含有纤维素、半纤维素和果胶基质 （如图1所示）。纤维素是重复单元为二糖的多糖链，在细胞壁中形成微管结构，就像房屋的钢结构一样，给细胞壁一个整体的稳定性。同时半纤维素将各种纤维素微管串联在一起，整体结构浸润在果胶基质中。植物细胞壁，抗张能力强，是帮助对抗渗透作用和维持植物细胞稳定结构的基础。

4

图1. 植物细胞壁分子结构

藻类细胞壁

主要成分：纤维素（cellulose）和多糖（ploysaccharide）

藻类细胞和植物细胞类似，除了含有纤维素以外，还包含一定量的多糖，比如说甘露聚糖（mannan）和木聚糖（xylan）。甘露聚糖是由甘露糖（mannose）聚合而成（β(1-4) 糖苷键），木聚糖是由木糖（xylose）聚合而成。甘露聚糖和木聚糖的组成比例是决定藻类种类的一个因素。

真菌细胞壁

主要成分：甲壳素/几丁质（chitin）

与纤维素少有不同，几丁质的糖链中多了氮源（图2，纤维素和几丁质的对比）。几丁质也出现在一些节肢动物的外壳之中形成坚硬的外骨骼。

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图2. 纤维素和几丁质的区别

细菌细胞壁

主要成分：肽聚糖（peptidoglycan）

说到细菌细胞壁，不得不提的是革兰氏染色。我们定义细胞壁由较多的（~90%）肽聚糖组成的细菌为革式阳性菌（gram-positive），染色后显深蓝或紫色；而细胞壁由较少（~10%）肽聚糖组成的细菌为革氏阴性菌（gram-negative），染色后显粉色。肽聚糖是连接着氨基酸的聚糖组成的，它们组成网状结构环绕在细胞表面维持细胞结构。因为肽聚糖细胞壁只发现存在于细菌细胞壁中，所以对于自身免疫系统和免疫治疗来说，细菌细胞壁是很好的抗原标靶。

古生菌细胞壁

主要成分：假肽聚糖（pseudo-peptidoglycan）

古生菌细胞壁并没有被科学家们充分研究过，只知道它们的细胞壁是由类似于细菌胞壁的肽聚糖组成的。同时，古生菌的细胞壁也有其它成分，用以对抗恶劣的生存环境。

现在我们知道了一些细胞壁是什么样的，那么现在就来聊聊如何破坏细胞壁进而裂解细胞。 细胞裂解是分离纯化细胞内非分泌类物质的基础，算是十分常见的分子生物学技术。细胞裂解大致可以分为两类：机械性裂解和非机械性裂解（如下表所示）。

表一：裂解方法与描述

分类

 裂解方法 描述

机械裂解

 手动研磨法

(Mortar and pestle）

 非常老派的裂解细胞法。一般用于液氮速冻过的植物细胞上，直接使用杵和研钵，手动研磨就可破坏细胞，释放细胞质。
滚珠搅拌法

(Bead beating)

 玻璃或陶瓷珠在与细胞悬浊液混合后搅拌旋转，珠子在液体中旋转碰撞产生的切变力（shear force）十分小，但足以破细胞而且不伤害到细胞器。
超声波破碎法

(Sonication)

 超声波产生震荡穿过整个细胞悬浊液，细胞壁因为周围压力的改变而解体，超声波法十分方便也容易操作。要注意的是，裂解过程最好在冰浴中发生，否则超声波震荡产生大量的热可能会对所需的产物产生破坏。
高压匀浆破碎法

(Homogenization)

 强制细胞高速通过窄小空间而后撞击停止，会产生巨大的液相剪切力，使得细胞外层被强制破碎。工业上常使用多次循环高压匀浆，保证细胞裂解得彻底。
非机械裂解 反复冻融破碎法

(Freeze-thaw)

 反复冷冻解冻细胞，方法十分简单，唯一的不好就是比较费时间，而且破裂率也无法保证。
高温裂解法

(High temperature)

 升高温度，细胞破裂。不足的一点是可能对要收集的蛋白有负面的影响（变性失活）。

酶溶裂解法

(Enzyme lysis)

 一些天然存在的酶就有分解细胞壁的作用。根据你要处理的细胞的细胞壁类型，也有不同的酶可以使用。比如处理细菌细胞的肽聚糖细胞壁，可以使用溶菌酶(lysozyme)。

化学破碎法

(Chemical treatment)

一些有机溶剂可以渗透过细胞壁和细胞膜，特别是我们在提取憎水分子时，使用有机溶剂会特别方便。再比如乙二胺四乙酸（EDTA），作为强力的金属离子螯合物，可以抢走用于稳定细胞壁的Mg2+ 和 Ca2+，使得细胞裂解，EDTA特别常见于裂解革氏阴性菌上。

虽然有这样或那样的方法可以用来裂解细胞，但在实际试验中也要因情况而定，甚至多种方法同时使用，以保证裂解得顺利并高效地获得我们想要的细胞内物质。仅仅知道方法是肯定不够的，小编认为更需要一个正确的逻辑去分析、决定用哪些方法去裂解细胞获得产物，更多的时候也是不断尝试才能改进方法。相信每个人都有自己的思考方法和实验要求，小编就不在这里献丑自己的逻辑了。就像解数学题一样，只要能解出答案，哪样的思路都是好的。但小编真心要推荐一个网站，是EMBL编写的一般性细胞裂解—蛋白提纯的方法和思路，其中也有介绍到不同的缓冲液使用情况。

https://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/index.html

如有新的细胞裂解方法或裂解心得，欢迎留言交流，置评可否！

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