其他組織消化手冊

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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(yīng)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(dǎo)致的結(jié)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(chǔ)上選擇的消化方案，以及如何根據(jù)具體實驗?zāi)康男拚桨敢垣@得更好的實驗結(jié)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(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適實驗結(jié)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(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(guān)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(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。

其他組織消化手冊

物種	詳細來源	細胞名稱	消化用酶	培養(yǎng)基	參考文獻
馬	馬	Tendon Cells	Collagenase Type 1: 0.1%	DMEM	Nemoto, M., Kizaki, K., Yamamoto, Y., Oonuma, T. and Hashizume, K.: Tenascin-C Expression in Equine Tendon-derived Cells During Proliferation and Migration., J Equine Sci 24, 17-24, 2013
人類	人類, 3-7 歲	Dental pulp stem cells	Collagenase Type 1: 0.3%	DMEM	Salmon, B., Bardet, C, Khaddam, M. Naji, J., Coyac, B., Baroukh, B., Letourneur, F., Lesieur, J., Decup, F., Le Denmat, D., Nicoletti, A., Poliard, A, Rowe, P., Huet, E., Vital, S. and Linglart, A.: MEPE-Derived ASARM Peptide Inhibits Odontogenic Differentiation of Dental Pulp Stem Cells and Impairs Mineralization in Tooth Models of X-linked Hypophosphatemia., PLoS ONE 8, e56749, 2013
			Neutral Protease: 0.4%
人類	人類	Dental pulp stem	Collagenase Type 1: 0.3%	DMEM/F12	Bonnamain, V., Thinard, R., Sergent-Tanguy, S., Huet, P., Bienvenu, G., Naveilhan, P., Farges, J. and Alliot-Licht, B.: Human Dental Pulp Stem Cells Cultured in Serum-Free Supplemented Medium., Front Physiol 4, 357, 2013
			Neutral Protease: 0.4%
人類	人類	Fungiform taste papillae	Collagenase Type 1: 550 u/ml	see reference	Ozdener, H., Spielman, A. and Rawson, N.: Isolation and Culture of Human Fungiform Taste Papillae Cells, J Vis Exp 63, e3730, 2012
			Elastase: 10 u/ml
			Soybean Trypsin Inhibitor: 0.09%
人類	人類	Adipose derived stromal	Collagenase Type 2: 0.075%	DMEM	Panetta, N., Gupta, D., Kwan, M., Wan, D., Commons, G. and Longaker, M.: Tissue Harvest by Means of Suction-Assisted or Third-Generation Ultrasound-Assisted Lipoaspiration Has No Effect on Osteogenic Potential of Human Adipose-Derived Stromal Cells., Plast Reconstr Surg 124, 65, 2009
人類	人類	Carotid artery plaque macrophage	Collagenase Type 4: 450 u/ml	HBSS	Patino WillmarD, Kang Ju-Gyeong, Matoba Satoaki, Mian OmarY, Gochuico BernadetteR, Hwang PaulM: Atherosclerotic plaque macrophage transcriptional regulators are expressed in blood and modulated by tristetraprolin, Circ Res 98, 1282-9, 2006
			Deoxyribonuclease I: 500 u/ml
			Soybean Trypsin Inhibitor: 0.1%
人類	人類	Synoviocytes	Collagenase: 0.2%	DMEM/F12	Chen V, Croft D, Purkis P, Kramer IM: Co-culture of synovial fibroblasts and differentiated U937 cells is sufficient for high interleukin-6 but not interleukin-1beta or tumour necrosis factor-alpha release., Br J Rheumatol 37, 148-56, 1998
人類	人類	Peptic cells	Collagenase Type 4: 0.1%	Ringer solution	Lanas. A., Ner鉚n, J., Esteva, F. and S謾inz, R.: Non-Steroidal Anti-Inflammatory Drugs and Prostaglandin Effects on Pepsinogen Secretion by Dispersed Human Peptic Cells., Gut 36, 657-63, 1995
			Soybean Trypsin Inhibitor: 0.2%
人類	人類	Periapical granuloma	CLSPA: 0.25%	RPMI-1640	Stern MH, Dreizen S, Mackler BF, Levy BM: Isolation and characterization of inflammatory cells from the human periapical granuloma, J Dent Res 61, 1408-12, 1982
昆蟲	昆蟲, 鱗翅類的昆蟲	Lepitopteran	Collagenase Type 3: 0.35%	Dulbecco PBS	Goodwin, R and McCawley, P: Initiating Attached Cell Lines From the Lepidoptera (Insecta), Meth Cell Sci 3, 567, 1977
			Hyaluronidase: 0.01%
小鼠	小鼠, 26-30 克	Myenteric plexus	Collagenase Type 2: 0.13%	Neurobasal A	Smith, T., Grider, J., Dewey, W. and Akbarali, H.: Morphine Decreases Enteric Neuron Excitability via Inhibition of Sodium Channels., PLoS ONE 7, e45251, 2012
小鼠	小鼠	Spleen, bone marrow endothelial	Collagenase Type 4: 0.3-1.0%	PBS	Shi, C., Jia, T., Mendez-Ferrer, S., Hohl, T., Serbina, N., Lipuma, L., Leiner, I., Li, M., Frenette, P. and Pamer, E.: Bone Marrow Mesenchymal Stem and Progenitor Cells Induce Monocyte Emigration in Response to Circulating Toll-Like Receptor Ligands., Immunity 34, 590, 2011
			Deoxyribonuclease I: 20 u/ml
小鼠	小鼠	Cochleaer	Trypsin: 0.125%	DMEM/F-12	Jan, T., Chai, R., Sayyid, Z. and Cheng, A.: Isolating LacZ-Expressing Cells from Mouse Inner Ear Tissues Using Flow Cytometry., J Vis Exp 58, e3432, 2011
小鼠	小鼠, 6-8 周	Bone marrow mesenchymal stem	Collagenase Type 1: 0.25%	RPMI 1640	Xu, S., De Becker, A., Van Camp, B., Vanderkerken, K. and Van Riet, I.: An Improved Harvest and In Vitro Expansion Protocol for Murine Bone Marrow-Derived Mesenchymal Stem Cells., J Biomed Biotechnol 2010, 105940, 2010
小鼠	小鼠, 3-6 周	Ear mesenchymal stem	Collagenase Type 1: 0.2%	DMEM/F12	Staszkiewicz, J., Gimble, J., Manuel, J. and Gawronska-Kozak, B.: IFATS Collection: Stem Cell Antigen-1-Positive Ear Mesenchymal Stem Cells Display Enhanced Adipogenic Potential.,Stem Cells 26, 2666, 2008
小鼠	小鼠, 6-10 周	Salivary Gland and Stomach	Collagenase Type 4: 0.8%	RPMI-1640	Ji, H., Rinen, F., Waltzinger, C., Meier, D, Bilancio, A., Pearce, W., Hirsch, E., Wymann, M., Ruckle, T., Camps, M., Vanhaesebroeck, B., Okkenhaug, K. and Rommel, C.:Inactivation of PI3Kgamma and PI3Kdelta Distorts T-cell Development and Causes Multiple Organ Inflammation., Blood 110, 2940, 2007
			Deoxyribonuclease I: 1.0%
小鼠	小鼠	Bone marrow	Collagenase Type 1: 0.15%	PBS	Bertoncello, I. and Williams, B.: Hematopoietic Stem Cell Characterization by Hoechst 33342 and Rhodamine 123 Staining., Methods Mol Biol 263, 181, 2004
			Neutral Protease: 0.15%
小鼠	小鼠, 25-30 g	Tracheal inflammatory cells	Collagenase Type 4: 0.1%	RPMI 1640	Minamoto Kanji, Pinsky DavidJ: Recipient iNOS but not eNOS deficiency reduces luminal narrowing in tracheal allografts, J Exp Med 196, 1321-33, 2002
			Deoxyribonuclease I: 50 u/ml
			Soybean Trypsin Inhibitor: 0.1%
豬	豬, 6 月	Synoviocytes	Hyaluronidase: 660 u/ml	DMEM	Kean, T and Dennis, J.: Synoviocyte Derived-Extracellular Matrix Enhances Human Articular Chondrocyte Proliferation and Maintains Re-Differentiation Capacity at Both Low and Atmospheric Oxygen Tensions., PLoS ONE 10, e0129961, 2015
			Trypsin: 0.25%
			Collagenase Type 2: 583 u/ml
兔	兔, 新西蘭, 8-10 周	Tenocytes and tendon stem cells	Collagenase Type 1: 0.3%	DMEM	Zhang, J. and Wang, J.: Characterization of Differential Properties of Rabbit Tendon Stem Cells and Tenocytes., BMC Musculoskelet Disord 11, 10, 2010
			Neutral Protease: 0.4%
大鼠	Lewis大鼠, M	Synovial macrophages	Collagenase Type 1: 250 u/ml	DMEM	Jain, S., Tran, T. and Amiji, M.: Macrophage Repolarization with Targeted Alginate Nanoparticles Containing IL-10 Plasmid DNA for the Treatment of Experimental Arthritis.,Biomaterials 61, 162-77, 2015
大鼠	Wistar大鼠, 7-9 周	Gingival mitochondria	Collagenase Type 1: 0.115-0.130 %	HBSS	Kaneko, N., Rikimaru, T., Fujimura, T., Mori, S., Hidaka, S. and Kaya, H.: Preparation of Rat Gingival Mitochondria with an Improved Isolation Method., Int J Dent 2010, 275103, 2010
大鼠	大鼠, 雄性, 250-350克	Submandibular acinar	Collagenase: 0.05%	PBS	Silver, N., Proctor, GB, Arno, M. and Carpenter GH: Activation of mTOR Coincides with Autophagy During Ligation-induced Atrophy in the Rat Submandibular Gland., Cell Death Dis1, e14, 2010
大鼠	大鼠, 8-10 周	Synovial cells	Collagenase Type 1: 250 u/ml	RPMI 1640	Moghaddami, M., Cleland, L. and Mayrhofer, G.: MHC II+ CD45+ Cells from Synovium-Rich Tissues of Normal Rats: Phenotype, Comparison with Macrophage and Dendritic Cell Lineages and Differentiation into Mature Dendritic Cells In Vitro., Int Immunol 17, 1103, 2005

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