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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
| 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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