結(jié)腸組織消化手冊(cè)

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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(yǎng)和細(xì)胞生物學(xué)研究的原代細(xì)胞的分離和收集。盡管這些消化酶已經(jīng)普遍應(yīng)用多年，但是對(duì)其組織消化和細(xì)胞收集的作用機(jī)制卻沒有全面的理解。由此導(dǎo)致的結(jié)果便是選擇一種消化方法大多是隨機(jī)選擇或基于過去的經(jīng)驗(yàn)，而不是在*理解消化原理的基礎(chǔ)上選擇的消化方案，以及如何根據(jù)具體實(shí)驗(yàn)?zāi)康男拚桨敢垣@得更好的實(shí)驗(yàn)結(jié)果。

細(xì)胞分離程序的目的是zui大程度的獲得功能活性的單個(gè)細(xì)胞，而影響結(jié)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(dòng)物年齡

4、遺傳改造情況（如knockouts）

5、所用的消化培養(yǎng)液

6、所用的酶

7、消化酶中的雜質(zhì)

8、所用消化酶的工作濃度

9、溫度

10、孵育時(shí)間

通常前四個(gè)原因沒辦法選擇，而剩下的原因一般由經(jīng)驗(yàn)決定以獲取zui適實(shí)驗(yàn)結(jié)果。

通過查閱文獻(xiàn)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(shí)驗(yàn)數(shù)據(jù)搞得一頭霧水。這些巨大差異來源于組織胞外基質(zhì)的復(fù)雜而動(dòng)態(tài)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(zhì)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(fā)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(jié)了目前常用組織消化酶的zui適消化和細(xì)胞收集知識(shí)和方法，描述了標(biāo)準(zhǔn)的實(shí)驗(yàn)步驟，以便于制定一個(gè)*的細(xì)胞分離方案，同時(shí)列舉了一些相關(guān)的參考文獻(xiàn)。

下表列出多種組織的適宜消化酶（含參考文獻(xiàn)，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(jié)腸組織	內(nèi)皮組織	上皮組織
眼	心臟	腸	腎
肝	肺	淋巴結(jié)	乳腺
其它組織	其他組織	肌組織	神經(jīng)組織
胰腺	腮腺	垂體	前列腺
生殖	動(dòng)物鱗片	皮膚	脾臟
干細(xì)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(xiàn)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(jié)腸組織消化手冊(cè)

物種	詳細(xì)來源	細(xì)胞名稱	消化用酶	培養(yǎng)基	參考文獻(xiàn)
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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