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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(jīng)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(jīng)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(jù)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(yǎng)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(jīng)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(shù)據(jù)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(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(nèi)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(jīng)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。
腸組織消化手冊
| 物種 | 詳細來源 | 細胞名稱 | 消化用酶 | 培養(yǎng)基 | 參考文獻 |
| 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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