結腸組織消化手冊

更新時間：2016-02-16

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Worthington組織消化指南

引言

組織消化酶主要用于組織培養和細胞生物學研究的原代細胞的分離和收集。盡管這些消化酶已經普遍應用多年，但是對其組織消化和細胞收集的作用機制卻沒有全面的理解。由此導致的結果便是選擇一種消化方法大多是隨機選擇或基于過去的經驗，而不是在*理解消化原理的基礎上選擇的消化方案，以及如何根據具體實驗目的修正消化方案以獲得更好的實驗結果。

細胞分離程序的目的是zui大程度的獲得功能活性的單個細胞，而影響結果的原因有很多，包括（不限于）以下：

1、組織類型

2、物種來源

3、動物年齡

4、遺傳改造情況（如knockouts）

5、所用的消化培養液

6、所用的酶

7、消化酶中的雜質

8、所用消化酶的工作濃度

9、溫度

10、孵育時間

通常前四個原因沒辦法選擇，而剩下的原因一般由經驗決定以獲取zui適實驗結果。

通過查閱文獻來選擇*組織消化酶和zui適消化條件的研究人員通常被互相矛盾的實驗數據搞得一頭霧水。這些巨大差異來源于組織胞外基質的復雜而動態的特性，以及過去常用的粗制、未提純的組織分離酶。另外，組織胞外基質由各種蛋白、糖蛋白、脂類和糖脂組成，這些成分的組成比例在不同物種、不同組織、甚至不同發育（年齡）之間存在明顯的差異，這些因素都極大的影響了消化方案的制定。目前通常使用的粗制酶，如鏈霉蛋白酶、NF1:250、以及含有幾種其他不同濃度蛋白酶的膠原酶、以及各種多糖酶、核酶和脂酶。

本指南總結了目前常用組織消化酶的zui適消化和細胞收集知識和方法，描述了標準的實驗步驟，以便于制定一個*的細胞分離方案，同時列舉了一些相關的參考文獻。

下表列出多種組織的適宜消化酶（含參考文獻，以組織類型和物種分類，詳見鏈接）

脂肪組織	腎上腺組織	骨組織	腦組織
軟骨組織	結腸組織	內皮組織	上皮組織
眼	心臟	腸	腎
肝	肺	淋巴結	乳腺
其它組織	其他組織	肌組織	神經組織
胰腺	腮腺	垂體	前列腺
生殖	動物鱗片	皮膚	脾臟
干細胞	胸腺	甲狀腺/甲狀旁腺	扁桃體
腫瘤

注：使用Worthington公司消化酶的文獻不限于上表鏈接中所列，一般而言，Worthington公司的組織消化酶可用于大多組織類型的消化。

結腸組織消化手冊

物種	詳細來源	細胞名稱	消化用酶	培養基	參考文獻
豚鼠	豚鼠, 成年的	Enterochromaffin	Trypsin: 0.05%	DMEM	Raghupathi, R., Duffield, M., Zelkas, L., Meedeniya, A., Brookes, S., Sia, T., Wattchow, D., Spencer, N. and Keating, D.: Identification of Unique Release Kinetics of Serotonin From Guinea-pig and Human Enterochromaffin Cells., J Physiol 591, 5959-75, 2013
			Collagenase Type 1: 0.1%
豚鼠	豚鼠, 雄性, 200-250克	Myenteric ganglia	Collagenase Type 4: 0.2%	Kreb's solution	Kang, M., Maguma, H., Smith, T., Ross, G., Dewey, W. and Akbarali, H.: The Role of 尾-arrestin2 in the Mechanism of Morphine Tolerance in the Mouse and Guinea Pig Gastrointestinal Tract., J Pharmacol Exp Ther 340, 567-76, 2012
			Protease: 0.1%
人類	人類	Enterochromaffin	Trypsin: 0.05%	DMEM	Raghupathi, R., Duffield, M., Zelkas, L., Meedeniya, A., Brookes, S., Sia, T., Wattchow, D., Spencer, N. and Keating, D.: Identification of Unique Release Kinetics of Serotonin From Guinea-pig and Human Enterochromaffin Cells., J Physiol 591, 5959-75, 2013
			Collagenase Type 1: 0.1%
人類	人類	Colonic epithelial	Collagenase: 150 u/ml	Basal X media	Roig, A., Eskiocak, U., Hight, S., Kim, S., Delgado, O., Souza, R., Spechler, S., Wright, W. and Shay, J.: Immortalized Epithelial Cells Derived from Human Colon Biopsies Express Stem Cell Markers and Differentiate In Vitro., Gastroenterol 138, 1012, 2010
			Neutral Protease: 0.04 mg/ml
人類	人類	Colorectal cancer	Collagenase Type 4: 1%	HBSS	Zhou, J., Belov, L., Huang, P., Shin, J., Solomon, M., Chapuis, P., Bokey, L., Chan, C., Clarke, C., Clarke, S. and Christopherson, R.: Surface Antigen Profiling of Colorectal Cancer Using Antibody Microarrays With Fluorescence Multiplexing., J Immunol Methods 355, 40, 2010
			Deoxyribonuclease I: 0.2%
人類	人類	Colonic epithelial	Collagenase Type 4: 0.1%	not listed	Huang, E., Hynes, M., Zhang, T., Ginestier, C., Dontu, G., Appelman, H., Fields, J., Wicha, M. and Boman, B.: Aldehyde Dehydrogenase 1 is a Marker for Normal and Malignant Human Colonic Stem Cells (SC) and Tracks SC Overpopulation During Colon Tumorigenesis., Cancer Res 69, 3382-9, 2009
人類	人類	Colon cancer	Collagenase Type 1: 300 u/ml	DMEM/F12	Varnat, F., Duquet, A., Malerba, M., Zbinden, M., Mas, C., Gervaz, P. and Ruiz i Altaba, A.: Human Colon Cancer Epithelial Cells Harbour Active HEDGEHOG-GLI Signalling that is Essential for Tumour Growth, Recurrence, Metastasis and Stem Cell Survival and Expansion., EMBO Mol Med 1, 338-51, 2009
			Hyaluronidase: 100 u/ml
人類	人類	Cancer stem cell	Collagenase Type 3: 200 u/ml	RPMI-1640	Dalerba Piero, Dylla Scott J, Park In-Kyung, Liu Rui, Wang Xinhao, Cho Robert W, Hoey Timothy, Gurney Austin, Huang Emina H, Simeone Diane M, Shelton Andrew A, Parmiani Giorgio, Casli Chiara, Clarke Michael F: Phenotypic characterization of human colorectal cancer stem cells, Proc Natl Acad Sci U S A 104, 10158-63, 2007
			Deoxyribonuclease I: 100 u/ml
人類	人類	Colonic epithelial	Collagenase:	RPMI 1640	Fukushima, K. and Fiocchi, C.: Paradoxical Decrease of Mitochondrial DNA Deletions in Epithelial Cells of Active Ulcerative Colitis Patients.,Am J Physiol Gastrointest Liver Physiol Vol. 286,, , G804-13, 2004
			Neutral Protease: 0.3%
			Deoxyribonuclease I: 0.05%
人類	人類	Colonic endothelial cells	Collagenase Type 2: 0.25%	HBSS/5%FBS	Wang D., Lehman R., Donner D., Matli M., Warren R., and Welton M.: Expression and Endocytosis of VEGF and Its Receptors in Human Colonic Vascular Endothelial Cells, Am J Physiol/Gastro 282, G1088, 2002
人類	人類	Colonocytes	Collagenase:	DMEM/F12	Emenaker N, Calaf G, Cox D, Basson M and Qureshi N: Short chain fatty acids differentially modulate cellular phenotype and c-myc protein levels in primary human nonmalignant and malignant colonocytes, J Nutr 46, 96-105, 2001
人類	人類	Epithelial and mucosal lymphocytes	Neutral Protease: 0.1%	RPMI 1640	Hisamatsu, T., Watanabe, M., Ogata, H., Ezaki, T., Hozawa, S., Ishii, H., Kanai, T. and Hibi, T.: Interferon-Inducible Gene Family 1-8U Expression in Colitis-Associated Colon Cancer and Severely Inflamed Mucosa in Ulcerative Colitis., Cancer Res 59, 5927-31, 1999
			CLSPA: 0.02%
			Deoxyribonuclease I: 0.01%
人類	人類	Colonic epithelial	Neutral Protease: 1.2 u/ml	HBSS	Gibson, P., Rosella, O., Wilson, A., Mariadason, J., Rickard, K., Byron, K. and Barkla, D.: Colonic Epithelial Cell Activation and the Paradoxical Effects of Butyrate.,Carcinogenesis 20, 539, 1999
			Collagenase Type 4: 50 u/ml
人類	人類	Lamina propria lymphocytes	Collagenase: 25 u/ml	HBSS	Ueyama H, Kiyohara T, Sawada N, Isozaki K, Kitamura S, Kondo S, Miyagawa J, Kanayama S, Shinomura Y, Ishikawa H, Ohtani T, Nezu R, Nagata S, Matsuzawa Y:High Fas ligand expression on lymphocytes in lesions of ulcerative colitis, Gut 43, 48-55, 1998
小鼠	小鼠	Lamina propria	Collagenase Type 2: 0.1%	RPMI 1640	Atarashi, K., Nishimura, J., Shima, T., Umesaki, Y., Yamamoto, M., Onoue, M., Yagita, H., Ishii, N., Evans, R., Honda, K. and Takeda, K.: ATP Drives Lamina Propria T(H)17 Cell Differentiation., Nature 455, 808, 2008
			Neutral Protease: 0.1%
			Deoxyribonuclease I: 0.004%
小鼠	小鼠, 6-8 周	Cancer stem cell	Collagenase Type 3: 200 u/ml	RPMI-1640	Dalerba Piero, Dylla Scott J, Park In-Kyung, Liu Rui, Wang Xinhao, Cho Robert W, Hoey Timothy, Gurney Austin, Huang Emina H, Simeone Diane M, Shelton Andrew A, Parmiani Giorgio, Casli Chiara, Clarke Michael F: Phenotypic characterization of human colorectal cancer stem cells, Proc Natl Acad Sci U S A 104, 10158-63, 2007
			Deoxyribonuclease I: 100 u/ml
小鼠	小鼠	Lamina propria mononuclear cells	Collagenase: 0.05%	HBSS	Weigmann Benno, Tubbe Ingrid, Seidel Daniel, Nicolaev Alex, Becker Christoph, Neurath Markus F: Isolation and subsequent analysis of murine lamina propria mononuclear cells from colonic tissue, Nat Protoc 2, 2307-11, 2007
			Deoxyribonuclease I: 0.05%
			Neutral Protease: 0.3%
小鼠	小鼠	Dentritic	Collagenase: 300 u/ml	RPMI 1640	Abe, K., Nguyen, K., Fine, S., Mo, J., Shen, C., Shenouda, S., Corr, M., Jung, S., Lee, J., Eckmann, L. and Raz, E.: Conventional Dendritic Cells Regulate the Outcome of Colonic Inflammation Independently of T Cells., Proc Natl Acad Sci U S A 104, 17022, 2007
			Deoxyribonuclease I: 0.002%
小鼠	小鼠	Lympocytes	Collagenase/Dispase: 100 u/ml	RPMI 1640	Annacker O, Coombes JL, Malmstrom V, Uhlig HH, Bourne T, Johansson-Lindbom B, Agace WW, Parker CM, Powrie F.: Essential role for CD103 in the T cell-mediated regulation of experimental colitis, J Exp Med 202, 1051, 2005
小鼠	小鼠, 6-8 周	Lamina propria lymphocytes	Collagenase Type 1: 0.2%	HBSS	Totsuka T, Kanai T, Uraushihara K, Iiyama R, Yamazaki M, Akiba H, Yagita H, Okumura K, Watanabe M: Therapeutic effect of anti-OX40L and anti-TNF-alpha MAbs in a murine model of chronic colitis, Am J Physiol/Gastro 284, G595-603, 2003
			Deoxyribonuclease I: 0.01%
小鼠	小鼠, 6-8 周	Lamina propria mononuclear cells	Collagenase Type 2: 0.015%	RPMI	Wirtz S., Becker C., Blumberg R., Galle P., and Neurath M.: Treatment of T cell-dependent experimental colitis in SCID mice by local administration of an adenovirus expressing IL-18 antisense mRNA, J Immunol 168(1), 411, 2002
			Deoxyribonuclease I: 0.01%
大鼠	大鼠, 成年的	Colon smooth muscle	Soybean Trypsin Inhibitor: 0.01%	DMEM	Somara, S., Bashllari, D., Gilmont, R. and Bitar, K.: Real-Time Dynamic Movement of Caveolin-1 During Smooth Muscle Contraction of Human Colon and Aged Rat Colon Transfected with Caveolin-1 cDNA., Am J Physiol Gastrointest Liver Physiol 300, G1022, 2011
			Collagenase Type 2: 0.1%

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