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软骨组织消化手册

　更新时间：2016-02-16　点击量：1053

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Worthington组织消化指南

引言

组织消化酶主要用于组织培养和细胞生物学研究的原代细胞的分离和收集。尽管这些消化酶已经普遍应用多年，但是对其组织消化和细胞收集的作用机制却没有全面的理解。  由此导致的结果便是选择一种消化方法大多是随机选择或基于过去的经验，而不是在*理解消化原理的基础上选择的消化方案，以及如何根据具体实验目的修正消化方案以获得更好的实验结果。

细胞分离程序的目的是zui大程度的获得功能活性的单个细胞，而影响结果的原因有很多，包括（不限于）以下：

1、组织类型

2、物种来源

3、动物年龄

4、遗传改造情况（如knockouts）

5、所用的消化培养液

6、所用的酶

7、消化酶中的杂质

8、所用消化酶的工作浓度

9、温度

10、孵育时间

通常前四个原因没办法选择，而剩下的原因一般由经验决定以获取zui适实验结果。

通过查阅文献来选择*组织消化酶和zui适消化条件的研究人员通常被互相矛盾的实验数据搞得一头雾水。这些巨大差异来源于组织胞外基质的复杂而动态的特性，以及过去常用的粗制、未提纯的组织分离酶。另外，组织胞外基质由各种蛋白、糖蛋白、脂类和糖脂组成，这些成分的组成比例在不同物种、不同组织、甚至不同发育（年龄）之间存在明显的差异，这些因素都极大的影响了消化方案的制定。目前通常使用的粗制酶，如链霉蛋白酶、NF1:250、以及含有几种其他不同浓度蛋白酶的胶原酶、以及各种多糖酶、核酶和脂酶。

本指南总结了目前常用组织消化酶的zui适消化和细胞收集知识和方法，描述了标准的实验步骤， 以便于制定一个*的细胞分离方案，同时列举了一些相关的参考文献。

下表列出多种组织的适宜消化酶（含参考文献，以组织类型和物种分类，详见链接）

脂肪组织

肾上腺组织

骨组织

脑组织

软骨组织

结肠组织

内皮组织

上皮组织

眼

心脏

肠

肾

肝

肺

淋巴结

乳腺

其它组织

其他组织

肌组织

神经组织

胰腺

腮腺

垂体

前列腺

生殖

动物鳞片

皮肤

脾脏

干细胞

胸腺

甲状腺/甲状旁腺

扁桃体

肿瘤

 

 

 

注：使用Worthington公司消化酶的文献不限于上表链接中所列，一般而言，Worthington公司的组织消化酶可用于大多组织类型的消化。

 

软骨组织消化手册

 

物种详细来源细胞名称消化用酶培养基参考文献
牛牛, 18-36个月ChondrocytesCollagenase Type 1: 0.1%DMEMWhite, 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ChondrocytesCollagenase Type 2: 0.2%DMEMHwang, 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 个月ChondrocytesCollagenase Type 4: 390 u/mlhgDMEMLima, 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 岁ChondrocytesTrypsin: 0.20%HBSSMackintosh, 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
牛牛犊, 1-14 天ChondrocytesCollagenase Type 2: 0.20%PBSKlagsbrun, M.: Large Scale Preparation of Chondrocytes,Methods of Enzymology Vol. 58,, , 560, 1979
犬犬ChondrocytesCollagenase Type 2: 0.3%Ham's F12Lee, 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
Trypsin: 0.25%
鸡小鸡embryos, HH stageMesenchymalTrypsin: 0.1%DMEMWong, 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
(see reference)
鸡鸡肉,肉鸡应变, 8-10 周Matrix vesiclesTrypsin: 0.1%Tris-buffered salineGenge, 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
Epiphyseal growth plate
鸡鸡肉,肉鸡应变, 8-10 周Matrix vesiclesTrypsin: 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
鸡小鸡胚胎, 白来杭鸡, 19 天大的FibroblastsTrypsin: 0.25%E 199 mediumGionti, 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
Epithelial-like
鸡小鸡胚胎, 19 天大的ChondrocytesTrypsin: 0.75%Coon's modified F-12Capasso, 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
鸡小鸡胚胎,  白来杭鸡Wing budsTrypsin: 0.1%Saline GAhrens, P., Solursh, M., and Reiter, R.: Stage-Related Capacity for Limb Chondrogenesis in Cell Culture, Dev Biol 60, 69, 1977
马马ChondrocytesCollagenase Type 2: 0.08%DMEM/F12Ley, 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ChondrocytesCollagenase Type 2: 2%DMEMBekkers,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
人类人类ChondrocytesCollagenase Type 2: 0.15%DMEMCapsoni, 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
人类人类ChondrocytesCollagenase: 0.3%DMEMTamai, 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
人类人类, 52-82 岁ChondrocytesCollagenase Type 2: 0.2%DMEM/F12Pallu, 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
Pronase: 0.15%
人类人类SynoviocytesCollagenase Type 1: 0.4%DMEMKim 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
人类人类, 26-68 岁Meniscus and cartilageCollagenase Type 2: 0.15%DMEMMarsano 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
人类人类, 64-83 岁ChondrocytesCollagenase Type 2: 0.08%DMEM/F12Tallheden 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
人类人类, 成年的SynovialCollagenase: 0.15%DMEMLiagre 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%
人类人类, 成年的ChondrocytesCollagenase Type 2: 0.15%DMEMJakob 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
人类人类, 成年的SynoviocytesCollagenase Type 1: 0.1%RPMIMcEvoy 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
人类人类, 15-60 岁ChondrocytesCollagenase Type 2: 0.2%DMEMRotter 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
人类人类Synovial fibroblastsCollagenase: 0.4%DMEMSarkissian M, Lafyatis R: Integrin engagement regulates proliferation and collagenase expression of rheumatoid synovial fibroblasts, J Immunol 162, 1772-9, 1999
人类人类Septal chondrocytesCollagenase Type 2: 0.2%DMEM/F12Dunham 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%
人类人类 ,风湿性关节炎Synovial tissueTrypsin: 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
(see reference)
人类人类, 13-62 岁Articular chondrocytesTrypsin: 0.2%BSSSrivastava, 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
人类人类, 26-84 岁ChondrocytesCollagenase: GBSSManning, 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ChondrocytesTrypsin: 0.25%DMEMOtsuru, 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
Collagenase Type 1: 86.5 u/ml
小鼠小鼠, 1 天ChondrocytesCollagenase: 0.2%DMEMTerpstra, 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
绵羊绵羊, 2 个月ChondrocytesCollagenase Type 2: 0.3%Ham's F-12Kojima 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
猪猪, 1岁ChondrocytesCollagenase Type 1: 0.2%DEMEMChowdhury, 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
猪猪, 2-4 个月ChondronsNeutral Protease: 0.3%PBSGraff RD, Lazarowski ER, Banes AJ, Lee GM: ATP release by mechanically loaded porcine chondrons in pellet culture, Arthritis Rheum 43, 1571-9, 2000
Collagenase: 0.2%
兔兔, 新西兰, 1.2-1.4公斤ChondrocytesCollagenase Type 2: 0.025%DMEMJu, 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
Pronase: 0.2%
兔兔, 新西兰, 1.8-2.3公斤ChondrocytesHyaluronidase: .05%Gey's solutionMehraban 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, 新西兰, 白色, 4-6 周 & 22-25 周ChondrocytesProtease XIV: 5 mg/g of tissueHam's F-12Plaas, 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ChondrocytesTrypsin: 0.2%Gey's BSSBenya, 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, 新西兰白或 荷兰的, 1 周ChondrocytesTrypsin: 0.1%Saline G, CMFSchindler, 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
(还有 人类, 初生)
兔兔, 新西兰白, 幼小的, 2.25 - 3.3 公斤Articular chondrocytesTrypsin: 0.2%Gey's BSSGreen, J.R., and William, T.: Articular Cartilage Repair. Behavior of Rabbit Chondrocytes During Tissue Culture and Subsequent Allografting, Clin Orthop Relat Res , 237, 1976
Hyaline
兔兔, 新西兰白, 雄性, 250-350 克EpiphysealTrypsin: 0.25%Eagle's basal mediumBentley, G., and Greer , R.: Homotransplantation of Isolated Epiphyseal and Articular Cartilage Chondrocytes into Joint Surfaces of Rabbits, Nature 230, 385, 1971
Articular cartilage
大鼠大鼠, SD, 年幼的, 100-120 克ChondrocytesTrypsin: 0.2%Ham's F-12 mediumShimomura, 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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