产品描述

胶原酶（Collagenase）是一种蛋白酶，一种肽链内切酶，能够特异性的识别Pro-X-Gly-Pro序列（该序列高频率出现在胶原中，很少发现于其他蛋白中）并切割该序列中性氨基酸（X）和甘氨酸（Gly）之间的肽键。许多蛋白酶都能水解单链且变性的胶原多肽，但胶原酶是唯一一种可以降解具有三股超螺旋结构的天然胶原纤维的蛋白酶，这种胶原纤维广泛存在结缔组织内。

本品来源于溶组织梭菌（Clostridium histolyticum），是一种酶粗提物，不仅含有胶原酶（更准确的称法为梭菌蛋白酶A（clostridiopeptidase A）），能够降解天然胶原和网状纤维。还含有其他的一些蛋白酶，多糖酶，脂酶等，分别能够有效水解

结缔组织和上皮组织细胞外基质内的其他蛋白、多糖和脂质，使得本品非常适用于组织消化。

目前商业化提供的细菌胶原酶主要根据胶原酶活性的差异，分为四种类型：胶原酶I型，II型，III型和IV型，在应用上有所偏向：1）胶原酶I（Type I Collagenase）：含有比较均匀的各种酶活力（包括胶原酶，酪蛋白酶，梭菌蛋白酶，胰蛋白酶活性）。通常用作上皮细胞、肝、肺、脂肪和肾上腺组织细胞的制备；2）胶原酶II（Type II Collagenase）：含有更高的梭菌蛋白酶活性，通常用于心脏、骨、肌肉、胸腺和软骨等组织来源细胞的制备；3）胶原酶III（Type III Collagenase）：含有较低的蛋白酶活性，常用于乳腺细胞的制备；4）胶原酶IV（Type IV Collagenase）：含有低胰酶活性，通常用于胰岛细胞的制备，或者需要维持受体完整性的细胞制备实验。

本品为IV型胶原酶，≥125 CDU/mg solid（CDU = collagen digestion units），特别适用于胰岛组织的消化。

 

酶活力单位定义

在37℃，pH7.5 的条件下，5 h内水解胶原产生相当于1 µM L-亮氨酸的酶量定义为1个酶活力单位。

 

运输和保存方法

室温运输。4℃避光保存，2年有效。储存液-20℃避光冻存。

 

注意事项

1）为了您的安全和健康，请穿实验服并戴一次性手套操作。

2）本产品仅作科研用途！

 

使用方法

1. 胶原酶储存液的配制

向每管100 mg的胶原酶中加入1 mL的含Ca²⁺、Mg²⁺的HBSS（Hank’s平衡盐溶液，含Ca²⁺、Mg²⁺），轻轻旋涡震荡使其充分溶解，制备成100 mg/mL（即100×）的储存液。然后用低蛋白结合性的0.22 μm的滤膜过滤除菌，分装成小份量，然后于-20℃避光冻存。

使用前于冰上解冻，避免反复冻融。其用于组织和细胞分散的常用浓度为：0.5-2.5 mg/mL，用于软骨消化的常用浓度为1-2 mg/mL，需要根据特定的实验条件或者参考相应的文献资料确定所需的最佳工作浓度。

2．组织的分离

1）使用无菌手术刀或剪刀将组织切成3-4 mm大小的组织块；

2）利用含Ca²⁺、Mg²⁺的HBSS洗涤组织块数次；

3）加入足量的含Ca²⁺、Mg²⁺的HBSS，使其浸没组织块，并加入胶原酶至需要工作浓度；

4）于37℃孵育4-18 h。消化时使用水平摇床以及用3 mM的CaCl₂补充消化可以提高消化效率。

5） 已分散开的细胞可使用不锈钢或尼龙网筛筛得，收集备用。未完全解离的组织另外添加适量的新鲜胶原酶工作液于37℃继续孵育；

6）利用不含胶原酶的HBSS洗涤收集的细胞数次；

7）细胞培养液重悬上述细胞，利用自动细胞计数器或其他方法计算活细胞密度。

8）于细胞培养皿上利用合适细胞培养基接种细胞。

3． 器官灌注

1）向37℃预热的含Ca²⁺、Mg²⁺的HBSS中加入胶原酶，另添加3 mM的CaCl₂有助于提高分离效率；

2）按照已优化的速率对相应的器官灌注胶原酶工作液；

3）将上述过程中回收的灌注液流经不锈钢或尼龙网筛，从而将已解离的细胞或小片段组织块与较大团块分离开来，未充分解离的组织需利用新鲜胶原酶工作液于37℃进一步孵育；

4）利用不含胶原酶的HBSS洗涤收集的细胞数次；

5）细胞培养液重悬上述细胞，利用自动细胞计数器或其他方法计算活细胞密度。

6）于细胞培养皿上利用合适细胞培养基接种细胞。

HB220705

 

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