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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，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?zāi)康男拚桨敢垣@得更好的實(shí)驗結(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、孵育時間
通常前四個原因沒辦法選擇，而剩下的原因一般由經(jīng)驗決定以獲取zui適實(shí)驗結(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)驗步驟， 以便于制定一個*的細(xì)胞分離方案，同時列舉了一些相關(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。
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) |
| 犬 | 狗, 成年的 | Intestinal L-cells | Collagenase Type 1: 75 u/ml | HBSS | Damholt AB, Buchan AM, Kofod H: Glucagon-like-peptide-1 secretion from canine L-cells is increased by glucose-dependent-insulinotropic peptide but unaffected by glucose,Endocrinology 139, 2085-91, 1998 |
| 人類 | 人類 | Lamina propria lymphocytes | Collagenase: 20 u/ml | Medium | Ebert, Ellen.: CD2 Activation of Human Lamina Propria Lymphocytes Reduces CD3 Responsiveness., Immunology 117, 71-7, 2006 |
| 人類 | 人類 | Lamina propria mononuclear cells | Collagenase: 0.02% | HBSS | Kanai Takanori, Totsuka Teruji, Uraushihara Koji, Makita Shin, Nakamura Tetsuya, Koganei Kazutaka, Fukushima Tsuneo, Akiba Hisaya, Yagita Hideo, Okumura Ko, Machida Utako, Iwai Hideyuki, Azuma Miyuki, Chen Lieping, Watanabe Mamoru: Blockade of B7-H1 suppresses the development of chronic intestinal inflammation, J Immunol 171, 4156-63, 2003 |
| 人類 | 人類 | Intestinal epithelial | Collagenase Type 4: 72.5 u/ml | HBSS | Fahlgren, A., Hammarstrom, S., Danielsson, A. and Hammarstrom, M.: Increased Expression of Antimicrobial Peptides and Lysozyme in Colonic Epithelial Cells of Patients with Ulcerative Colitis., Clin Exp Immunol 131, 90, 2003 |
| 人類 | 人類 | Mucosal mononuclear cells | Collagenase Type 3: 0.01% | RPMI | Stallmach A, Schaffer F, Hoffmann S, Weber S, Muller-Molaian I, Schneider T, Kohne G, Ecker KW, Feifel G, Zeitz M: Increased state of activation of CD4 positive T cells and elevated interferon gamma production in pouchitis, Gut 43, 499-505, 1998 |
| Deoxyribonuclease I: 0.01% | |||||
| Soybean Trypsin Inhibitor: 0.01% | |||||
| 小鼠 | 小鼠 | Lamina propria mononuclear cells | Collagenase Type 3: 400 u/ml | HBSS | Seo, S., Kuffa, P., Kitamoto, S., Nagao-Kitamoto, H., Rousseau, J., Kim, Y., Nunez, G. and Kamada, N.: Intestinal Macrophages Arising from CCR2(+) Monocytes Control Pathogen Infection by Activating Innate Lymphoid Cells., Nat Commun 6, 8010, 2015 |
| Deoxyribonuclease I: 0.001% | |||||
| 小鼠 | 小鼠 | Lymphocytes | Collagenase Type 1: 100 u/ml | RPMI-1640 | Beura, L., Anderson, K., Schenkel, J., Locquiao, J., Fraser, K., Vezys, V., Pepper, M. and Masopust, D.: Lymphocytic Choriomeningitis Virus Persistence Promotes Effector-Like Memory Differentiation and Enhances Mucosal T cell Distribution., J Leukoc Biol 97, 217, 2015 |
| 小鼠 | 小鼠, P4 | Enteric neural crest progenitors | Neutral Protease: 0.5% | DMEM/F12 | Hotta, R., Stamp, L., Foong, J., McConnell, S., Bergner, A., Anderson, R., Enomoto, H., Newgreen, D., Obermayr, F., Furness, J. and Young, H.: Transplanted Progenitors Generate Functional Enteric Neurons in the Postnatal Colon., J Clin Invest 123, 1182, 2013 |
| Collagenase animal free: 0.05% | |||||
| 小鼠 | 小鼠 | colon tumor organoids | Collagenase Type 4: 200 u/ml | DMEM | Xue, X. and Shah, Y.: In Vitro Organoid Culture of Primary Mouse Colon Tumors., J Vis Exp , e50210, 2013 |
| Neutral Protease: 0.01% | |||||
| 小鼠 | 小鼠 | Intestinal organoids | Collagenase Type 1: 800 u/ml | DMEM | Barthel, E., Speer, A., Levin, D., Sala, F., Hou, X., Torashima, Y., Wigfall, C. and Grikscheit, T.: Tissue Engineering of the Intestine in a Murine Model., J Vis Exp 70, e4279, 2012 |
| Neutral Protease: 0.013% | |||||
| 小鼠 | 小鼠 | Dendritic, macrophages | Collagenase: 0.15% | HBSS | Geem, D., Medina-Contreras, O., Kim, W., Huang, C. and Denning T.: Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine., J Vis Exp 63, e4040, 2012 |
| Deoxyribonuclease I: 0.004% | |||||
| 小鼠 | 小鼠, 新生的及成年的 | Myenteric plexus | Collagenase Type 4: 0.025-0.1% | HBSS | Joseph, N., He, S., Quintana, E., Kim, Y., Nunez, G. and Morrison, S.: Enteric Glia are Multipotent in Culture but Primarily Form Glia in the Adult Rodent Gut., J Clin Invest 121, 3398, 2011 |
| Papain: 10 u/ml | |||||
| Deoxyribonuclease I: 100 u/ml | |||||
| 小鼠 | 小鼠 | Lamina propria lymphocytes | Collagenase Type 1: 0.3% | RPMI 1640 | Ito, Y., Kanai, T., Totsuka, T., Okamoto, R., Tsuchiya, K., Nemoto, Y., Yoshioka, A., Tomita, T., Nagaishi, T., Sakamoto, N., Sakanishi, T., Okumura, K., Yagita, H. and Watanabe, M.:Blockade of NKG2D Signaling Prevents the Development of Murine CD4+ T Cell-Mediated Colitis., Am J Physiol Gastrointest Liver Physiol 294, G199, 2008 |
| Deoxyribonuclease I: 0.01% | |||||
| 小鼠 | 小鼠 | Intestinal mononuclear | Collagenase Type 4: 0.1% | RPMI 1640 | Forbes, E., Groschwitz, K., Abonia, J.P., Brandt, E., Cohen, E., Blanchard, C., Ahrens, R., Seidu, L., McKenzie, A., Strait, R., Finkelman, F., Foster, P., Matthaei, K., Rothenberg, M. and Hogan, S.: IL-9- and Mast Cell-Mediated Intestinal Permeability Predisposes to Oral Antigen Hypersensitivity., J Exp Med 205, 897, 2008 |
| 小鼠 | 小鼠, 南非乳鼠 | Enterochromaffin cells | Collagenase: 0.025% | HBSS | Kidd, M., Modlin, I., Eick, G. and Champaneria, M.: Isolation, Functional Characterization, and Transcriptome of Mastomys Ileal Enterochromaffin Cells., Am J Physiol Gastrointest Liver Physiol 291, G778-91, 2006 |
| Pronase E: 0.07% | |||||
| 小鼠 | 小鼠, 7 周 | Interstitial cells | Collagenase: 0.04% | see reference | Goto Kazunori, Matsuoka Satoshi, Noma Akinori: Two types of spontaneous depolarizations in the interstitial cells freshly prepared from the murine small intestine, J Physiol 559, 411-22, 2004 |
| Trypsin: 0.02% | |||||
| 小鼠 | 小鼠, 9-13 天 | Interstitial cells of Cajal | Collagenase Type 2: 0.13% | HBSS | Ordag Tamas, Redelman Doug, Miller Lisa J, Horvath Viktor J, Zhong Qiao, Almeida-Porada Graca, Zanjani Esmail D, Horowitz Burton, Sanders Kenton M: Purification of interstitial cells of Cajal by fluorescence-activated cell sorting, Am J Physiol Cell Physiol 286, C448-56, 2004 |
| 小鼠 | 小鼠, 0-15 天 | Interstitial cells of Cajal | Collagenase Type 2: 0.13% | Hanks | Lee Young Mee, Kim Byung Joo, Kim Hyun Jin, Yang Dong Ki, Zhu Mei Hong, Lee Kyu Pil, So Insuk, Kim Ki Whan: TRPC5 as a candidate for the nonselective cation channel activated by muscarinic stimulation in murine stomach, Am J Physiol/Gastro 284, G604-16, 2003 |
| 小鼠 | 小鼠 | Lamina propria lymphocytes | Collagenase Type 4: 300 u/ml | PBS | Wu Y, Wang X, Csencsits KL, Haddad A, Walters N, Pascual DW: M cell-targeted DNA vaccination, Proc Natl Acad Sci U S A 98, 9318-23, 2001 |
| 小鼠 | 小鼠, F14 | Intestinal mesenchymal | Collagenase Type 2: 0.03% | HBSS | Sakagami Y, Inaguma Y, Sakakura T, Nishizuka Y: Intestine-like remodeling of adult mouse glandular stomach by implanting of fetal intestinal mesenchyme, Cancer Res 44, 5845-9, 1984 |
| 大鼠 | 大鼠, 出生后的, 成年的 | Gut Neural Crest Stem Cells | Collagenase Type 4: 0.1% | HBSS | Kruger, G., Mosher, J., Bixby, S., Joseph, N., Iwashita, T. and Morrison, S.: Neural Crest Stem Cells Persist in the Adult Gut but Undergo Changes in Self-Renewal, Neuronal Subtype Potential, and Factor Responsiveness., Neuron 35, 657, 2002 |
| Trypsin: 0.025% | |||||
| 大鼠 | SD大鼠,胚胎 | Sciatic Nerve and Gut Neural Crest Stem Cells | Collagenase Type 4: 0.025% | HBSS | Bixby, S., Kruger, G., Mosher, J., Joseph, N. and Morrison, S.: Cell-Intrinsic Differences Between Stem Cells from Different Regions of the Peripheral Nervous System Regulate the Generation of Neural Diversity., Neuron 35, 643, 2002 |
| Trypsin: 0.005% | |||||
| Deoxyribonuclease I: 0.05% | |||||
| 大鼠 | 大鼠,雄性, 18-24 天 | Intestinal epithelial | Collagenase Type 1: 0.1% | DMEM | Quaroni A, Wands J, Trelstad RL, Isselbacher KJ: Epithelioid cell cultures from rat small intestine. Characterization by morphologic and immunologic criteria, J Cell Biol 80, 248-65, 1979 |
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