人掌腱膜挛缩的<em>离体</em>文化为肌成纤维细胞的研究和细胞外基质相互作用
Summary
迪皮特朗病（DD）是手的手掌的纤维增生性疾病。在这里，我们提出了一个协议，从DD文化切除标本的三维（3D）培养体系。这种短期培养系统允许保存的三维结构和纤维化组织的分子性质。
Abstract
Organ fibrosis or “scarring” is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic.
To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells.
As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first “organ” culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.
Introduction
迪皮特朗病（DD），良性纤维增生疾病导致手指的永久弯曲，由于结节和帘线中的手掌的形成。虽然疾病传播是北欧的白种人的特别高，该疾病的潜在的遗传病因仍然是未知的1。 DD的主要特征是过量生产细胞外基质（ECM）蛋白（ 例如，胶原），其形成坚韧的纤维组织占据的肌腱和手和手指的手掌的皮肤之间的空间中的，永久地破坏精细动作手2，3。该疾病的复发表明潜在的遗传改变如纤维化1,4的一个原因。一种有效的治疗方法可能是直接针对在细胞和分子水平上的不可控的纤维化的机制。
我们最近对肝纤维化的工作使我们的发展新颖的三维培养系统，它允许人类纤维化组织的短期培养以药物测试的潜力。该系统有助于克服2D成纤维细胞培养物的限制的方式，为在介导纤维化5定义角色为偏向下调节，通过外显 子跳跃实现，TGFβ途径激活。
我们已经开发出一种方法，以从培养的DD患者体外人体切除标本，研究和肌成纤维细胞之间的相互作用的周围的ECM 5,6。的DD结缔组织纤维化的研究以及其他纤维化疾病依赖于外科切除的组织病理学分析标本，从组织和建立原代培养物或细胞分选程序中的成纤维细胞的分离。这些方法是相当静态的，因为它们不允许外感操纵疾病性质或治疗干预由实验者的。此外，主CELL文化倾向于适应培养条件和它们的基因表达的特性的每个通道在基本上从体内情况不同，即使是在早期传代（通道3之间和6）7，8，我们已成功地保持了废物外科材料在体外培养条件下的时间周期，允许的患者特异性特征的研究和抗纤维化或消炎药的化合物的筛选。
该系统是基于硝化纤维素膜，其允许组织的与介质但不与塑料接触，因此，防止在附接所述组织的改变，如培养的DD成纤维细胞以及其它细胞类型9时以前观察。无胶原凝胶或其他ECM蛋白基质是必需的，因为在DD组织自身产生大量的这些蛋白质。这有利于本土ECM微环境和成交量罪维修CE矩阵基板是组织结构和功能10,11的重要调节器。举例来说，细胞外基质蛋白，如纤连蛋白，层粘连蛋白和胶原蛋白，可能会影响在上皮细胞12同样示于心尖-基底极性的成纤维细胞的前-后极性，13 。极化细胞具有细胞外的分子的非对称分布，它确定细胞迁移和基因表达， 例如，在膜α1β1整联无障碍影响细胞粘附到I型胶原14。由于此3D模型的一个主要目标是保持天然的微环境，无人工的ECM矩阵基板。
简言之：切除标本也同样切在无菌环境中，并放置在nitrocellular膜。如果治疗通过注射给药是必需的组织被注射它们已被放置在该膜后。如果治疗不要求通过注射进行给药挠度则该化合物被添加到培养基（Dulbecco氏改良的Eagle氏培养基（DMEM）中，用1％胎牛血清（FCS），1％青霉素 – 链霉素（P / S））。将培养物维持最多十天，之后将组织固定在4％多聚甲醛（PFA），通过30％蔗糖溶液进行处理，包埋在OCT化合物，并储存在-80℃下，如前所述5。
Protocol
Representative Results
Discussion
培养离体人结缔组织的最关键的步骤是即时使用的手术切除，以确保生存力后的组织的;该组织应保留在任何时候都培养基或盐溶液;保持无菌培养;组织的横截面应该有最大200微米的厚度;建立体外培养体系是最优的时组织是在与介质接触，但尚未完全浸没。介质应当只在Transwell小的外部腔室中的小容积（ 例如，500-600微升每12孔板表面积）加入。
从掌腱膜挛…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors are thankful to the nurses at the AMC that have facilitated the tissue collection. We would also like to acknowledge A.M.A. van der Laan for excellent technical support with the SHG and the two-photon imaging.
Materials
| Dulbecco’s Modified Eagle’s Medium | Invitrogen | 11965-084 | |
| fetal calf serum (FCS) | Gibco | high glucose, heat inactivated | |
| penicillin-streptomycin | Invitrogen | 15070-063 | |
| Cell culture inserts | Millicell | PIHA01250 | 0.45um pore size, 12mm diameter |
| anti-collagen type I | Southern Biotech | 1310-08 | |
| anti- α smooth muscle actin | Sigma | A2547 | |
| anti-collagen type III | Southern Biotech | 1330-01 | |
| Alexa Fluor555 Donkey Anti-Mouse IgG (H+L) | Invitrogen | A-31570 | |
| Alexa Fluor 488 Donkey Anti-Goat IgG (H+L) Antibody | Invitrogen | A-11055 | |
| TOPRO-3 | Invitrogen | T3605 | |
| methylsalicylate | Sigma | M6752 | |
| paraformaldehyde | Sigma | P6148 | |
| Tissue Tek OCT | Sakura | 25608-930 | |
| Microsccope glass coverslips | Menzel-Glaser | BB024060A1 | 24x60mm |
| Microscope SuperFrost slides | Menzel-Glaser | AA00000102E | 76x26mm |
| VECTASHIELD HardSet Mounting Medium | Vector laboratories | H-1400 | |
| Leica TCS SP5 II confocal microscope | Leica Microsystems | Argon-488, 514 nm and HeNe-633 nm laser lines | |
| Zeiss 710 NLO upright confocal microscope | Jena, Germany | equipped with femtosecond Spectra – Physics Deep See MP laser (Santa Clara, United States) using a Plan -Apochromat 20x/1.0 NA water-immersion objective. |
References
- Shih, B., Bayat, A. Scientific understanding and clinical management of Dupuytren disease. Nat Rev Rheumatol. 6 (12), 715-726 (2010).
- Bisson, M. A., McGrouther, D. A., Mudera, V., Grobbelaar, A. O. The different characteristics of Dupuytren’s disease fibroblasts derived from either nodule or cord: expression of α-smooth muscle actin and the response to stimulation by TGF-β1. The Journal of Hand Surgery. 28 (4), 351-356 (2003).
- Brickley-Parsons, D., Glimcher, M. J., Smith, R. J., Albin, R., Adams, J. P. Biochemical changes in the collagen of the palmar fascia in patients with Dupuytren’s disease. The Journal of Bone & Joint Surgery. 63 (5), 787-797 (1981).
- Berndt, A., Kosmehl, H., Katenkamp, D., Tauchmann, V. Appearance of the myofibroblastic phenotype in Dupuytren’s disease is associated with a fibronectin, laminin, collagen type IV and tenascin extracellular matrix. Pathobiology. 62 (2), 55-58 (1994).
- Karkampouna, S., et al. Novel ex vivo culture method for the study of Dupuytren’s disease: effects of TGFβ type 1 receptor modulation by antisense oligonucleotides. Mol Ther Nucleic Acids. 3, e142 (2014).
- Kruithof-de Julio, M., et al. Canonical Wnt signaling regulates Nkx3.1 expression and luminal epithelial differentiation during prostate organogenesis. Developmental Dynamics. 242 (10), 1160-1171 (2013).
- Krause, C., Kloen, P., ten Dijke, P. Elevated transforming growth factor β and mitogen-activated protein kinase pathways mediate fibrotic traits of Dupuytren’s disease fibroblasts. Fibrogenesis & Tissue Repair. 4 (1), 14 (2011).
- Gorman, D. B., Wu, Y., Seney, S., Zhu, R. D., Gan, B. S. Wnt expression is not correlated with beta-catenin dysregulation in Dupuytren’s Disease. J Negat Results Biomed. 5, 13 (2006).
- Streuli, C. H., Schmidhauser, C., Kobrin, M., Bissell, M. J., Derynck, R. Extracellular matrix regulates expression of the TGF-beta 1 gene. The Journal of Cell Biology. 120 (1), 253-260 (1993).
- Vogel, V., Sheetz, M. Local force and geometry sensing regulate cell functions. Nat Rev Mol Cell Biol. 7 (4), 265-275 (2006).
- Gottrup, F., Ågren, M. S., Karlsmark, T. Models for use in wound healing research: A survey focusing on in vitro and in vivo adult soft tissue. Wound Repair and Regeneration. 8 (2), 83-96 (2000).
- Gudjonsson, T., et al. Normal and tumor-derived myoepithelial cells differ in their ability to interact with luminal breast epithelial cells for polarity and basement membrane deposition. Journal of Cell Science. 115 (1), 39-50 (2002).
- Xu, R., Boudreau, A., Bissell, M. Tissue architecture and function: dynamic reciprocity via extra- and intra-cellular matrices. Cancer and Metastasis Reviews. 28 (1-2), 167-176 (2009).
- Jokinen, J., et al. Integrin-mediated cell adhesion to type I collagen fibrils. Journal of Biological Chemistry. 279 (30), 31956-31963 (2004).
- Au-Yong, I. T. H., Wildin, C. J., Dias, J. J., Page, R. E. A review of common practice in Dupuytren surgery. Techniques in Hand & Upper Extremity Surgery. 9 (4), 178-187 (2005).
- Deries, M., Collins, J. J. P., Duxson, M. J. The mammalian myotome: a muscle with no innervation. Evolution & Development. 10 (6), 746-755 (2008).
- Deries, M., et al. Extracellular matrix remodeling accompanies axial muscle development and morphogenesis in the mouse. Developmental Dynamics. 241 (2), 350-364 (2012).
- Martins, G. G., et al. Dynamic 3D Cell Rearrangements Guided by a Fibronectin Matrix Underlie Somitogenesis. PLoS ONE. 4 (10), e7429 (2009).
- Yokomizo, T., et al. Whole-mount three-dimensional imaging of internally localized immunostained cells within mouse embryos. Nat. Protocols. 7 (3), 421-431 (2012).
- Karkampouna, S., de Julio, M. K. r. u. i. t. h. o. f. -. Fibrosis: a novel approach for an old problem. Receptors & Clinical Investigation. 1 (5), (2014).
- Wolf, K., et al. Collagen-based cell migration models in vitro and in vivo. Seminars in Cell & Developmental Biology. 20 (8), 931-941 (2009).
- Freund, I., Deutsch, M., Sprecher, A. Connective tissue polarity. Optical second-harmonic microscopy, crossed-beam summation, and small-angle scattering in rat-tail tendon. Biophysical Journal. 50 (4), 693-712 (1986).
- Mohler, W., Millard, A. C., Campagnola, P. J. Second harmonic generation imaging of endogenous structural proteins. Methods. 29 (1), 97-109 (2003).
- Boulesteix, T., et al. Micrometer scale ex vivo multiphoton imaging of unstained arterial wall structure. Cytometry Part A. 69A (1), 20-26 (2006).
- Tomasek, J. J., Gabbiani, G., Hinz, B., Chaponnier, C., Brown, R. A. Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat Rev Mol Cell Biol. 3 (5), 349-363 (2002).
- Hinz, B., Mastrangelo, D., Iselin, C. E., Chaponnier, C., Gabbiani, G. Mechanical tension controls granulation tissue contractile activity and myofibroblast differentiation. The American Journal of Pathology. 159 (3), 1009-1020 (2001).
- Eastwood, M., McGrouther, D. A., Brown, R. A. A culture force monitor for measurement of contraction forces generated in human dermal fibroblast cultures: evidence for cell-matrix mechanical signalling. Biochimica et Biophysica Acta (BBA) – General Subjects. 1201 (2), 186-192 (1994).
- Verjee, L. S., Midwood, K., Davidson, D., Eastwood, M., Nanchahal, J. Post-transcriptional regulation of α-smooth muscle actin determines the contractile phenotype of Dupuytren’s nodular cells. Journal of Cellular Physiology. 224 (3), 681-690 (2010).
