眼組織消化手冊

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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(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(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。

眼組織消化手冊

物種	詳細來源	細胞名稱	消化用酶	培養(yǎng)基	參考文獻
牛	牛	Pericyte	Collagenase: 0.2%	DMEM	Bryan, B. and D'Amore, P.: Pericyte Isolation and USe is Endothelial/Pericyte Coculture Models, Meth Enzymol 443, 315, 2008
牛	牛	Microvascular endothelial	Collagenase/Dispase: 0.1%	MEM	Bowman, P., Betz, A., and Goldstein, G.: Primary Culture of Microvascular Endothelial Cells From Bovine Retina, In Vitro 18 (7), 626, 1982
雞	小雞, 胚胎, 6 天	Retinal cells	Trypsin: 0.005%	DMEM/F12	Jacob Vanessa, Rothermel Andrae, Wolf Peter, Layer Paul G: Rhodopsin, violet and blue opsin expressions in the chick are highly dependent on tissue and serum conditions, Cell Death Differ 180, 159-68, 2005
雞	小雞, 胚胎, 6 天	Retinal cells	Deoxyribonuclease I: 0.005%	DMEM/F12
雞	小雞胚胎	Corneal epithelia	Collagenase: 0.08%	HBSS	Reenstra Wende R, Orlow Daniel L, Svoboda Kathy K H: ECM-stimulated signaling and actin reorganization in embryonic corneal epithelia are Rho dependent, Inv Ophthal Visual Sci 43, 3181-9, 2002
雞	小雞胚胎	Corneal epithelia	Trypsin: 0.08%	HBSS
雞	小雞胚胎	Retinal	Trypsin: 0.6%	DMEM	Seigel, G.: The Golden Age of Retinal Cell Culture, Mol Vis 5, 4, 1999
雞	(包括大鼠)	Retinal	Trypsin: 0.6%	DMEM
雞	小雞, 胚胎, 10-14 天	Flat, retina	Trypsin: 0.1%	Tyrode's solution, CMF	Moyer, M., Bullrich, F., and Sheffield, J.: Emergence of Flat Cells From Glia in Stationary Cultures of Embryonic Chick Neural Retina, In Vitro Cell Dev Biol 26, 1073, 1990
魚類	魚, 斑馬魚	Retinal	Papain: 10 u/ml	L-15	Diekmann, H., Kalbhen, P. and Fischer, D.: Characterization of Optic Nerve Regeneration using Transgenic Zebrafish., Front Cell Neurosci 9, 118, 2015
人類	人類	Corneal stromal stem	Neutral Protease: 1.2 u/ml	DMEM	Du, Y., Roh, D., Funderburgh, M., Mann, M., Marra, K., Rubin, J., Li, X. and Funderburgh, J.: Adipose-Derived Stem Cells Differentiate to Keratocytes In Vitro.,Mol Vis 16, 2680, 2010
人類	人類	Corneal stromal stem	Collagenase: 0.1%	DMEM
人類	人類, 18-68 歲	Corneal endothelial	Collagenase: 0.2%	DMEM/F12	Li Wei, Sabater Alfonso L, Chen Ying-Ting, Hayashida Yasutaka, Chen Szu-Yu, He Hua, Tseng Scheffer C G: A novel method of isolation, preservation, and expansion of human corneal endothelial cells, Inv Ophthal Visual Sci 48, 614-20, 2007
人類	人類, 18-68 歲	Corneal endothelial	Neutral Protease: 1.0%	DMEM/F12
人類	人類	Corneal limbal epithelial sheet	Neutral Protease: 5%	see reference	Espana EM, Romano AC, Kawakita T, Di Pascuale M, Smiddy R, and Tseng SC:Novel enzymatic isolation of an entire viable human limbal epithelial sheet, Inv Ophthal Visual Sci 44(10), 4275, 2003
人類	人類, 5-65 歲	Retinal pigment epithelial (RPE)	Trypsin: 0.25%	HBSS	Von Recum, H., Okano, T., Kim, S, and Bernstein, P.: Maintenance of Retinoid Metabolism in Human Retinal Pigment Epithelium Cell Culture, Exp Eye Res 69, 97, 1999
猴	獼猴和狒狒	Retinal	Papain: 20-40 u/ml	Ames' solution	Han ,Y., Jacoby, R. and Wu, S.: Morphological and Electrophysiological Properties of Dissociated Primate Retinal Cells., Brain Res 875, 175, 2000
猴	獼猴和狒狒	Retinal	Deoxyribonuclease I: 400 u/ml	Ames' solution
猴	猴, 獼猴, 青少年	Conjunctival lymphocytes	Collagenase Type 1: 0.02%	RPMI 1640	Whittum-Hudson JA, Taylor HR: Antichlamydial specificity of conjunctival lymphocytes during experimental ocular infection, Infect Immun 57, 2977, 1989
小鼠	小鼠, 成年的	Retinal photoreceptor	PDS kit: with modifications	EBSS	Feodorova, Y., Koch, M., Bultman, S., Michalakis, S. and Solovei, I.: Quick and Reliable Method for Retina Dissociation and Separation of Rod Photoreceptor Perikarya from Adult Mice., MethodsX 2, 39, 2015
小鼠	小鼠, 4-8 天	Photoreceptors	PDS kit: per instructions	Neurobasal	Balmer, J., Zulliger, R., Roberti, S. and Enzmann, V.: Retinal Cell Death Caused by Sodium Iodate Involves Multiple Caspase-Dependent and Caspase-Independent Cell-Death Pathways., Int J Mol Sci 16, 15086-103, 2015
小鼠	小鼠, 胚胎和 P0蛋白	Retinal	Papain: 8 u/ml	DPBS	Dvoriantchikova, G., Perea-Martinez, I., Pappas, S., Barry, A., Danek, D., Dvoriantchikova, X., Pelaez, D. and Ivanov, D.: Molecular Characterization of Notch1 Positive Progenitor Cells in the Developing Retina., PLoS ONE 10, e0131054, 2015
小鼠	小鼠, 胚胎和 P0蛋白	Retinal	Deoxyribonuclease I: 124 u/ml	DPBS
小鼠	小鼠	Vitreous cells	Collagenase: 0.1%	DMEM	Iqbal, N., Xu, L., Devitt, C. and Skapek, S.: Isolation and Characterization of Mammalian Cells Expressing the Arf Promoter During Eye Development.,Biotechniques 56, 239-49, 2014
小鼠	小鼠	Vitreous cells	Hyaluronidase: 0.03%	DMEM
小鼠	小鼠, 6 周	Retinal	PDS kit: per instructions	DMEM	Singh, M., Issa, P, Butler, R., Martin, C., Lipinski, D., Sekaran, S., Barnard, A. and Maclaren, R.: Reversal of End-Stage Retinal Degeneration and Restoration of Visual Bunction by Photoreceptor Transplantation., Proc Natl Acad Sci U S A , , 2013
小鼠	小鼠	Retinal ganglion	CLSPA: 240 u/ml	Ames	Schmidt, T. and Kofuji, P.: An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells., J Vis Exp 47, 2367, 2011
小鼠	小鼠	Retinal ganglion	Hyaluronidase: 0.2%	Ames
小鼠	小鼠, 成年的	Cilary epithelial	PDS kit: per instructions	EBSS	Gualdoni, S., Baron, M., Lakowski, J., Decembrini, S., Pearson, R., Ali, R. and Sowden, J.: Isolation and Culture of Adult Ciliary Epithelial Cells, Previously Identified as Retinal Stem Cells, and Retinal Progenitor Cells., Curr Protoc Stem Cell Biol Chapter 1, Unit 1H.4, 2011
小鼠	小鼠, 出生后一天的	Retinal progenitor	Collagenase: 0.1%	HBSS	Jiang, C. Klassen, H., Zhang, X. and Young, M.: Laser Injury Promotes Migration and Integration of Retinal Progenitor Cells into Host Retina., Mol Vis 16, 983, 2010
小鼠	小鼠	Retinal	Collagenase: 0.1%	RPMI	Amadi-Obi, A., Yu, C., Liu, X., Mahdi, R., Clarke, G., Nussenblatt, R, Gery, I., Lee, Y. and Egwuagu, C.: TH17 Cells Contribute to Uveitis and Scleritis and are Expanded by IL-2 and Inhibited by IL-27/STAT1., Nat Med 13, 711, 2007
小鼠	小鼠	Retinal	Deoxyribonuclease I: 0.001%	RPMI
小鼠	小鼠, 成年的	Retinal	Papain: 50 u/ml	DMEM/F-12	Jadhav AP, Cho SH and Cepko CL: Notch activity permits retinal cells to progress through multiple progenitor states and acquire a stem cell property, Proc Natl Acad Sci U S A 103, 18998, 2006
小鼠	小鼠, 2-5 個月	Mouse retinal and bipolar	Papain: 20 u/ml	HBSS	Maxeiner Stephan, Dedek Karin, Janssen-Bienhold Ulrike, Ammermuller Josef, Brune Hendrik, Kirsch Taryn, Pieper Mario, Degen Joachim, Kruger Olaf, Willecke Klaus, Weiler Reto: Deletion of connexin45 in mouse retinal neurons disrupts the rod/cone signaling pathway between AII amacrine and ON cone bipolar cells and leads to impaired visual transmission, J Neurosci 25, 566-76, 2005
小鼠	小鼠, 2-5 個月	Mouse retinal and bipolar	Deoxyribonuclease I: 200 u/ml	HBSS
小鼠	小鼠, 4 周	Retinal endothelial cells	Collagenase Type 1: 0.1%	DMEM	Su X, Sorenson CM, and Sheibani N.: Isolation and characterization of murine retinal endothelial cells, Mol Vis 9, 171, 2003
豬	豬, 5-60 公斤	Retinal pigment epithelial cells	Collagenase: 2%	DMEM	Wiencke, A., Kiilgaard, J., Nicolini, J., Bundgaard, M., Ropke, C., and La Cour, M.: Growth of Cultured Porcine Retinal Pigment Epithelial Cells, Acta Opthalmol Scand 81(2), 170, 2003
兔	兔, New Zealand, Adult	Corneal Endothelial	Collagenase Type 2: 0.2%	HBSS	Lai, J., Cheng, H. and Ma, D.: Investigation of Overrun-Processed Porous Hyaluronic Acid Carriers in Corneal Endothelial Tissue Engineering, PLoS ONE 10, 1371, 2015
兔	兔	Corneal keratocytes	Hyaluronidase: 0.05%	DMEM	Hao, M., Flynn, K., Nien-Shy, C., Jester, B., Winkler, M., Brown, D., La Schiazza, O., Bille, J., Jester, J.: In Vivo Non Linear Optical (NLO) Imaging in Live Rabbit Eyes Using the Heidelbert Two-Photon Laser Opthalmoscope, Exp Eye Res 91, 308, 2010
兔	兔	Corneal keratocytes	Collagenase: 0.2%	DMEM
兔	兔, 新西蘭	Corneal keratocytes	Trypsin: 0.25%	PBS	Stramer Brian M, Kwok Michael G K, Farthing-Nayak Pamela J, Jung Jae-Chang, Fini M Elizabeth, Nayak Ramesh C: Monoclonal antibody (3G5)-defined ganglioside: cell surface marker of corneal keratocytes, Inv Ophthal Visual Sci 45, 807-12, 2004
兔	兔, 新西蘭	Corneal keratocytes	Collagenase: 0.5%	PBS
兔	兔, 成年的	Retinal neurons	Papain: 26 u/ml	DMEM	Brockway LM, Zhou Z-H, Bubien JK, Jovov B, Benos DJ, Keyser KT: Rabbit retinal neurons and glia express a variety of ENaC/DEG subunits, Am J Physiol Cell Physiol283, C126-34, 2002
兔	兔, 新西蘭, 成年的, 雄性, 白化變種,	Epithelial	Trypsin: 0.25%	HBSS/DMEM	Johnson-Muller, B., and Gross, J.: Regulation of Corneal Collagenase Production: Epithelial-Stromal Cell Interactions, Proc Natl Acad Sci U S A 75 (9), 4417, 1978
兔	2 公斤	Epithelial	Trypsin: 0.25%	HBSS/DMEM
大鼠	SD大鼠,6-8 周	Retinal	PDS kit: per instructions	EBSS	Vigneswara, V., Esmaeili, M., Deer, L., Berry, M., Logan, A. and Ahmed, Z.: Eye Drop Delivery of Pigment Epithelium-Derived Factor-34 Promotes Retinal Ganglion Cell Neuroprotection and Axon Regeneration., Mol Cell Neurosci 68, 212-21, 2015
大鼠	大鼠, SD	Retinal ganglion	PDS kit: with modifications	Neurobasal	Tan, C., Andrews, M., Kwok, J., Heintz, T., Gumy, L., Fassler, R. and Fawcett, J.:Kindlin-1 Enhances Axon Growth on Inhibitory Chondroitin Sulfate Proteoglycans and Promotes Sensory Axon Regeneration., J Neurosci 32, 7325, 2012
大鼠	SD大鼠, 180-220克	Retinal ganglion	Papain: 0.2%	Neurobasal	Ma, J., Yu, W., Wang, Y., Cao, G., Cai, S., Chen, X., Yan, N., Yuan, Y., Zeng, H., Fleenor, D., Liu, X. and Pang, I.: Neuroprotective Effects of C-type Natriuretic Peptide on Rat Retinal Ganglion Cells., Inv Ophthal Visual Sci 51, 3544-53, 2010
大鼠	Fishe大鼠, 成年的	Retinal	PDS kit: per instructions	MEM	Suzuki, T., Mandai, M., Akimoto, M., Yoshimura, N. and Takahashi, M.: The Simultaneous Treatment of MMP-2 Stimulants in Retinal Transplantation Enhances Grafted Cell Migration into the Host Retina., Stem Cells 24, 2406, 2006
大鼠	大鼠, 暗條紋, 3-4 周	Ciliary-derived eye	PDS kit: with modifications	DMEM/F12	Akagi T, Mandai M, Ooto S, Hirami Y, Osakada F, Kageyama R, Yoshimura N, Takahashi M.: Otx2 homeobox gene induces photoreceptor-specific phenotypes in cells derived from adult iris and ciliary tissue, Inv Ophthal Visual Sci 45, 4570, 2004
大鼠	大鼠, 胚胎 17 天	Retinal	Trypsin: 0.6%	DMEM	Seigel, G.: The Golden Age of Retinal Cell Culture, Mol Vis 5, 4, 1999
大鼠	大鼠, SD	Retina	Papain: 120 U	HBSS, PBS	Jing, S., Wen, D., Yu, Y., Holst, P., Luo, Y., Fang, M., Tamir, R., Antonio, L., Hu, Z., et al.: GDNF-Induced Activation of the Ret Protein Tyrosine Kinase is Mediated by GDNFR-a, a Novel Receptor for GDNF, Cell 85, 1113, 1996
大鼠	大鼠, 4-14 week	Retinal pigment epithelial cells	Collagenase Type 1: 65 u/ml	CF Hanks with EDTA	Wang N, Koutz CA, and Anderson RE.: A method for the isolation of retinal pigment epithelial cells from adult rats, Inv Ophthal Visual Sci 34(1), 101, 1993
大鼠	大鼠, 4-14 week	Retinal pigment epithelial cells	Hyaluronidase: 220 u/ml	CF Hanks with EDTA
大鼠	大鼠, 6-8 天	Retinal pigment epithelial	Neutral Protease: 2%	DMEM	Chang CW, Roque RS, Defoe DM, and Caldwell RB.: An improved method for isolation and culture of pigment epithelial cells from rat retina, Curr Eye Res 10(11), 1081, 1991
大鼠	大鼠, 長伊萬斯, 1-15 天	Neurons, visual cortex	Papain: 20 u/ml	BSS	Huettner, J., and Baughman, R.: Primary Culture of Identified Neurons From the Visual Cortex of Postnatal Rats, J Neurosci 6, 3044, 1986
大鼠	大鼠, 長伊萬斯, 1-15 天	Neurons, visual cortex	Papain: 20 u/ml	(see reference)
大鼠	大鼠, SD, 小狗	Retina	Trypsin: 0.25%	Ham's F-12	Sarthy PV, Curtis BM, and Catterall WA.: Retrograde Labeling, Enrichment, and Characerization of Retinal Ganglion Cells from the Neonatal Rat, J Neurosci 3 (12), 2532, 1983
蠑螈	蠑螈	Neurons	Papain: 14 u/ml	Ringers	Clarke, R, Wang, J. and Townes-Anderson, E.: Using Laser Tweezers For Manipulating Isolated Neurons In Vitro., J Vis Exp 19, 911, 2008
蠑螈	蠑螈, 18-25 厘米	Retina	Papain: 14 u/ml	Saline	Townes-Anderson, E., MacLeish, P., and Raviola, E.: Rod Cells Dissociated from Mature Salamander Retina: Ultrastruture and Uptake of Horseradish Peroxidase, J Cell Biol 100, 175, 1985
蠑螈	蠑螈 (A. tigrinum)	Photoreceptors, retina	Papain: 0.05%	(see reference)	Bader, C., MacLeish, P., and Schwartz, E.: Responses to Light of Solitary Rod Photoreceptors Isolated From Tiger Salamander Retina, Proc Natl Acad Sci U S A 75, 3507, 1978
龜	龜(紅耳龜)	Retinal	Papain: 0.1% (13.5 u/mg)	Kreb's Ringer	Lam, D.: Biosynthesis of Acetylcholine in Turtle Photoreceptors, Proc Natl Acad Sci U S A 69, 1987, 1972

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