軟骨組織消化手冊

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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(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(dòng)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(dòng)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(shí)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(dòng)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。

軟骨組織消化手冊

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)
牛	牛, 18-36個(gè)月	Chondrocytes	Collagenase Type 1: 0.1%	DMEM	White, R. and Gibson, J.: The Effect of Oxygen Tension on Calcium Homeostasis in 牛 Articular Chondrocytes., J Orthop Surg Res 5, 27, 2010
牛	牛, 6-8 周	Chondrocytes	Collagenase Type 2: 0.2%	DMEM	Hwang, Y., Sangaj, N. and Varghese, S.: Interconnected Macroporous Poly(ethylene glycol) Cryogels as a Cell Scaffold for Cartilage Tissue Engineering., Tissue Eng Part A 16, 3033-41, 2010
牛	牛, 1-3 個(gè)月	Chondrocytes	Collagenase Type 4: 390 u/ml	hgDMEM	Lima, E., Tan, A., Tai, T., Marra, K., DeFail, A., Ateshian, G. and Hung, C.: Genipin Enhances the Mechanical Properties of Tissue-Engineered Cartilage and Protects Against Inflammatory Degradation When Used as a Medium Supplement., J Biomed Mater Res 91, 692, 2009
牛	控制, 1-2 歲	Chondrocytes	Trypsin: 0.20%	HBSS	Mackintosh, D., and Mason, R.: Pharmacological Actions of 17 Beta-oestradiol on Articular Cartilage Chondrocytes and Chondrosarcoma Chondrocytes in the Absence of Oestrogen Receptors, Biochim Biophys Acta 964, 295, 1988
牛	牛犢, 1-14 天	Chondrocytes	Collagenase Type 2: 0.20%	PBS	Klagsbrun, M.: Large Scale Preparation of Chondrocytes,Methods of Enzymology Vol. 58,, , 560, 1979
犬	犬	Chondrocytes	Collagenase Type 2: 0.3%	Ham's F12	Lee, J., Kim, H., Kim, J., Bae, S., Joo, D., Huh, K., Fang, Y., Jeong, J., Kim, M. and Kim, Y.: Microencapsulation of Pancreatic Islets with Canine Ear Cartilage for Immunoisolation.,Transplant Proc 44, 1091-4, 2012
犬	犬	Chondrocytes	Trypsin: 0.25%	Ham's F12
雞	小雞embryos, HH stage	Mesenchymal	Trypsin: 0.1%	DMEM	Wong, M., and Tuan, R.: Nuserum, A Synthetic Serum Replacement, Supports Chondrogenesis of Embryonic Chick Limb Bud Mesenchymal Cells in Micromass Culture, In Vitro Cell Dev Biol 29A, 917, 1993
雞	小雞embryos, HH stage	Mesenchymal	Trypsin: 0.1%	(see reference)
雞	雞肉,肉雞應(yīng)變, 8-10 周	Matrix vesicles	Trypsin: 0.1%	Tris-buffered saline	Genge, B., Wu, L. and Wuthier, R.: Differential Fractionation of Matrix Vesicle Proteins: Further Characterization of the Acidic Phospholipid-dependent Ca²⁺-Binding Proteins, J Biol Chem 265, 4703, 1990
雞	雞肉,肉雞應(yīng)變, 8-10 周	Epiphyseal growth plate	Trypsin: 0.1%	Tris-buffered saline
雞	雞肉,肉雞應(yīng)變, 8-10 周	Matrix vesicles	Trypsin: 0.1%	(see reference)	Genge, B.R., Wu, L.N.Y., and Wuthier, R.E.: Identification of Phospholipid-dependent Calcium-binding Proteins as Constituents of Matrix Vesicles, J Biol Chem 264, 10917, 1989
雞	小雞胚胎, 白來杭雞, 19 天大的	Fibroblasts	Trypsin: 0.25%	E 199 medium	Gionti, E., Capasso, O., and Cancedda, R.: The Culture of Chick Embryo Chondrocytes and the Control of Their Differentiated Functions in Vitro, J Biol Chem 258 (11), 7190, 1983
雞	小雞胚胎, 白來杭雞, 19 天大的	Epithelial-like	Trypsin: 0.25%	E 199 medium
雞	小雞胚胎, 19 天大的	Chondrocytes	Trypsin: 0.75%	Coon's modified F-12	Capasso, O., Gionti, E., Pontarelli, G., Ambesi-Impiobato, F., Nitsch, L., Tajana, G., and Cancedda, R.: The Culture of Chick Embryo Chondrocytes and the Control of Their Differentiated Functions In Vitro, Exp Cell Res 142, 197, 1982
雞	小雞胚胎, 白來杭雞	Wing buds	Trypsin: 0.1%	Saline G	Ahrens, P., Solursh, M., and Reiter, R.: Stage-Related Capacity for Limb Chondrogenesis in Cell Culture, Dev Biol 60, 69, 1977
馬	馬	Chondrocytes	Collagenase Type 2: 0.08%	DMEM/F12	Ley, C., Svala, E., Nilton, A., Lindahl, A., Eloranta, M., Ekman, S. and Skioldebrand, E.:Effects of High Mobility Group Box Protein-1, Interleukin-1B, and Interleukin-6 on Cartilage Matrix Metabolism in Three-Dimensional Equine Chondrocyte Cultures., Conn Tissue Res 52, 290-300, 2011
山羊	山羊	Chondrocytes	Collagenase Type 2: 2%	DMEM	Bekkers,J, Creemers, L, Tsuchida, A, van Rijen, M, Custers, R, Dhert, W and Saris, D.:One-Stage Focal Cartilage Defect Treatment with Bone Marrow Mononuclear Cells and Chondrocytes Leads to Better Macroscopic Cartilage Regeneration Compared to Microfracture in Goats., Osteoarthritis Cartilage 21, 950-6, 2013
人類	人類	Chondrocytes	Collagenase Type 2: 0.15%	DMEM	Capsoni, F., Ongari, A., Lonati, C., Accetta, R., Gatti, S. and Catania, A.: Alpha-Melanocyte-Stimulating-Hormone (a-MSH) Modulates Human Chondrocyte Activation Induced by Proinflammatory Cytokines., BMC Musculoskelet Disord 16, 154, 2015
人類	人類	Chondrocytes	Collagenase: 0.3%	DMEM	Tamai, M., Nagasao, T., Yanaga, H., Hamamoto, Y., Kogure, T. and Tanaka, Y.:Correction of Secondary Deformity after Nuss Procedure for Pectus Excavatum by Means of Cultured autologous Cartilage Cell Injection.,Int J Surg Case Rep Vol. 15,, , 70-3, 2015
人類	人類, 52-82 歲	Chondrocytes	Collagenase Type 2: 0.2%	DMEM/F12	Pallu, S., Francin, P., Guillaume, C., Gegout-Pottie, P., Netter, P., Mainard, D., Terlain, B. and Presle, N.: Obesity Affects the Chondrocyte Responsiveness to Leptin in Patients with Osteoarthritis., Arthritis Res Ther 12, R112, 2010
人類	人類, 52-82 歲	Chondrocytes	Pronase: 0.15%	DMEM/F12
人類	人類	Synoviocytes	Collagenase Type 1: 0.4%	DMEM	Kim Wan-Uk, Kwok Seung-Ki, Hong Kyung-Hee, Yoo Seung-Ah, Kong Jin-Sun, Choe Jongseon, Cho Chul-Soo: Soluble Fas ligand inhibits angiogenesis in rheumatoid arthritis,Arthritis Res Ther 9, R42, 2007
人類	人類, 26-68 歲	Meniscus and cartilage	Collagenase Type 2: 0.15%	DMEM	Marsano A, Millward-Sadler SJ, Salter DM, Adesida A, Hardingham T, Tognana E, Kon E, Chiari-Grisar C, Nehrer S, Jakob M, Martin I: Differential cartilaginous tissue formation by human synovial membrane, fat pad, meniscus cells and articular chondrocytes,Osteoarthritis Cartilage 15, 48-58, 2007
人類	人類, 64-83 歲	Chondrocytes	Collagenase Type 2: 0.08%	DMEM/F12	Tallheden T, Bengtsson C, Brantsing C, Sjogren-Jansson E, Carlsson L, Peterson L, Brittberg M, and Lindahl A.: Proliferation and Differentiation Potential of Chondrocytes from Osteoarthritic Patients, Arthritis Res Ther 7(3), R560, 2005
人類	人類, 成年的	Synovial	Collagenase: 0.15%	DMEM	Liagre B, Vergne-Salle P, Corbiere C, Charissoux JL, and Beneytout JL.: Diosgenin, a Plant Steroid, Induces Apoptosis in Human Rheumatoid Arthritis Synoviocytes with Cyclooxygenase-2 Overexpression, Arthritis Res Ther 6(4), R373, 2004
			Hyaluronidase: 0.1%
			Deoxyribonuclease I: 0.015%
人類	人類, 成年的	Chondrocytes	Collagenase Type 2: 0.15%	DMEM	Jakob M, Demarteau O, Schafer D, Stumm M, Heberer M, and Martin I.: Enzymatic Digestion of 成年的 Human Articular Cartilage Yields a Small Fraction of the Total Available Cells, Conn Tissue Res 44, 173, 2003
人類	人類, 成年的	Synoviocytes	Collagenase Type 1: 0.1%	RPMI	McEvoy A., Murphy E., Ponnio T., Conneely O., Bresnihan B., FitzGerald O., and Murphy E.: Activation of Nuclear Orphan Receptor NURR1 Transcription by NF-kappa B and Cyclic Adenosine 5'-Monophosphate Response Element-binding Protein in Rheumatoid Arthritis Synovial Tissue, J Immunol 168(6), 2979, 2002
人類	人類, 15-60 歲	Chondrocytes	Collagenase Type 2: 0.2%	DMEM	Rotter N, Bonassar LJ, Tobias G, Lebl M, Roy AK, Vacanti CA: Age dependence of cellular properties of human septal cartilage: implications for tissue engineering, Arch Otolaryngol Head Neck Surg 127, 1248-52, 2001
人類	人類	Synovial fibroblasts	Collagenase: 0.4%	DMEM	Sarkissian M, Lafyatis R: Integrin engagement regulates proliferation and collagenase expression of rheumatoid synovial fibroblasts, J Immunol 162, 1772-9, 1999
人類	人類	Septal chondrocytes	Collagenase Type 2: 0.2%	DMEM/F12	Dunham B P, Koch R J: Basic fibroblast growth factor and insulinlike growth factor I support the growth of human septal chondrocytes in a serum-free environment, Arch Otolaryngol Head Neck Surg 124, 1325-30, 1998
			Hyaluronidase: 0.01%
			Deoxyribonuclease I: 0.015%
人類	人類 ,風(fēng)濕性關(guān)節(jié)炎	Synovial tissue	Trypsin: 0.05%	DMEM	天er, J., Krane, S., Russell, R., and Robinson, D.: Production of Collagenase and Prostaglandins by Isolated Adherent Rheumatoid Synovial Cells, Proc Natl Acad Sci U S A 73 (3), 945, 1976
人類	人類 ,風(fēng)濕性關(guān)節(jié)炎	Synovial tissue	Trypsin: 0.05%	(see reference)
人類	人類, 13-62 歲	Articular chondrocytes	Trypsin: 0.2%	BSS	Srivastava, V.M.L., Malemud, C.J., Hough, A.J., Bland, J.H., and Sokoloff, L.: Preliminary Experience with Cell Culture of Human Articular Chondrocytes, Arthritis Rheum 17, 165, 1974
人類	人類, 26-84 歲	Chondrocytes	Collagenase:	GBSS	Manning, W.K., and Bonner, W.M.: Isolation and Culture of Chondrocytes From Human 成年的 Articular Cartilage, Arthritis Rheum 10, 235, 1967
小鼠	小鼠, 2-5 天	Chondrocytes	Trypsin: 0.25%	DMEM	Otsuru, S., Hofmann, T., Raman, P., Olson, T., Guess, A., Dominici, M. and Horwitz, E.:Genomic and Functional Comparison of Mesenchymal Stromal Cells Prepared Using Two Isolation Methods., Cytotherapy 17, 262-70, 2015
小鼠	小鼠, 2-5 天	Chondrocytes	Collagenase Type 1: 86.5 u/ml	DMEM
小鼠	小鼠, 1 天	Chondrocytes	Collagenase: 0.2%	DMEM	Terpstra, L, Prud'homme, J, Arabian, A, Takeda, S, Karsenty, G, Dedhar, S, and St-Arnaud, R.: Reduced Chondrocyte Proliferation and Chondrodysplasia in Mice Lacking the Integrin-linked Kinase in Chondrocytes, J Cell Biol 162, 139, 2003
綿羊	綿羊, 2 個(gè)月	Chondrocytes	Collagenase Type 2: 0.3%	Ham's F-12	Kojima Koji, Bonassar Lawrence J, Roy Amit K, Mizuno Hirokazu, Cortiella Joaquin, Vacanti Charles A: A Composite Tissue-Engineered Trachea Using Sheep Nasal Chondrocyte and Epithelial Cells, FASEB J 17, 823-8, 2003
豬	豬, 1歲	Chondrocytes	Collagenase Type 1: 0.2%	DEMEM	Chowdhury, T., Schulz, R., Rai, S., Thuemmler, C., Wuestneck, N., Bader, A and Homandberg, G: Biomechanical Modulation of Collagen Fragment-Induced Anabolic and Catabolic Activities in Chondrocyte/Agarose Constructs., Arthritis Res Ther 12, R82, 2010
豬	豬, 2-4 個(gè)月	Chondrons	Neutral Protease: 0.3%	PBS	Graff RD, Lazarowski ER, Banes AJ, Lee GM: ATP release by mechanically loaded porcine chondrons in pellet culture, Arthritis Rheum 43, 1571-9, 2000
豬	豬, 2-4 個(gè)月	Chondrons	Collagenase: 0.2%	PBS
兔	兔, 新西蘭, 1.2-1.4公斤	Chondrocytes	Collagenase Type 2: 0.025%	DMEM	Ju, X., Deng, M., Ao, Y., Yu, C., Wang, J., Yu, J., Cui, G. and Hu, Y.: Protective Effect of Sinomenine on Cartilage Degradation and Chondrocytes Apoptosis., Yakugaku Zasshi 130, 1053-60, 2010
兔	兔, 新西蘭, 1.2-1.4公斤	Chondrocytes	Pronase: 0.2%	DMEM
兔	兔, 新西蘭, 1.8-2.3公斤	Chondrocytes	Hyaluronidase: .05%	Gey's solution	Mehraban F, Tindal MH, Proffitt MM, Moskowitz RW.: Temporal pattern of cysteine endopeptidase (cathepsin B) expression in cartilage and synovium from rabbit knees with experimental osteoarthritis: gene expression in chondrocytes in response to interleukin-1 and matrix depletion, Ann Rheum Dis 56, 108, 1997
			Collagenase Type 2: 0.2%
			Trypsin: 0.2%
兔	兔, 新西蘭, 白色, 4-6 周 & 22-25 周	Chondrocytes	Protease XIV: 5 mg/g of tissue	Ham's F-12	Plaas, A., Sandy, J., and Kimura, J.: Biosynthesis of Cartilage Proteoglycan & Link Protein Articular Chondrocytes, J Biol Chem 263, 7560, 1988
兔	兔, 白色, 雄性, 8 周	Chondrocytes	Trypsin: 0.2%	Gey's BSS	Benya, P.D., Padilla, S.R., and Nimni, M.E.: The Progeny of Rabbit Articular Chondrocytes Synthesize Collagen Types I and III and Type I Trimer, but Not Type II, Biochemistry 16, 865, 1977
兔	兔, 新西蘭白或荷蘭的, 1 周	Chondrocytes	Trypsin: 0.1%	Saline G, CMF	Schindler, F.H., Ose, M.A., and Solursh, M.: The Synthesis of Cartilage Collagen by Rabbit and Human Chondrocytes in Primary Cell Culture, In Vitro 12, 44, 1976
兔	(還有人類, 初生)	Chondrocytes	Trypsin: 0.1%	Saline G, CMF
兔	兔, 新西蘭白, 幼小的, 2.25 - 3.3 公斤	Articular chondrocytes	Trypsin: 0.2%	Gey's BSS	Green, J.R., and William, T.: Articular Cartilage Repair. Behavior of Rabbit Chondrocytes During Tissue Culture and Subsequent Allografting, Clin Orthop Relat Res , 237, 1976
兔	兔, 新西蘭白, 幼小的, 2.25 - 3.3 公斤	Hyaline	Trypsin: 0.2%	Gey's BSS
兔	兔, 新西蘭白, 雄性, 250-350 克	Epiphyseal	Trypsin: 0.25%	Eagle's basal medium	Bentley, G., and Greer , R.: Homotransplantation of Isolated Epiphyseal and Articular Cartilage Chondrocytes into Joint Surfaces of Rabbits, Nature 230, 385, 1971
兔	兔, 新西蘭白, 雄性, 250-350 克	Articular cartilage	Trypsin: 0.25%	Eagle's basal medium
大鼠	大鼠, SD, 年幼的, 100-120 克	Chondrocytes	Trypsin: 0.2%	Ham's F-12 medium	Shimomura, Y., Yoneda, T., and Suzuki, F.: Osteogenesis by Chondrocytes from Growth Cartilage of Rat Rib, Calcif Tissue Res 19, 179, 1975

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