JoVE Logo
发表
联系我们

登录

需要订阅 JoVE 才能查看此. 登录或开始免费试用。

本文内容

  • 摘要
  • 摘要
  • 引言
  • 研究方案
  • 结果
  • 讨论
  • 披露声明
  • 致谢
  • 材料
  • 参考文献
  • 转载和许可

摘要

牛角膜内皮细胞的原代培养物用于研究角膜内皮 - 间充质转换的机制。此外，大鼠角膜内皮冷冻损伤模型被用来证明在体内角膜内皮-间充质转换。

摘要

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet's membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of β-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

引言

Corneal endothelial cells (CECs) play a vital role in maintaining corneal clarity and thus visual acuity by regulating the hydration status of the corneal stroma through active pumping1. Because of the limited proliferative potential of human CECs, the cell number decreases with age, and the repair of corneal endothelial wounds following injury is usually achieved through cell enlargement and migration, rather than cell mitosis2. When the CEC count decreases below a threshold of approximately 500 cells/mm2, the dehydration status of the corneal stroma cannot be maintained, leading to bullous keratopathy and vision impairment3,4.

The limited proliferative potential of human CECs has been attributed to several factors, including reduced expression of the epidermal growth factor and its receptor in aging cells5, antiproliferative TGFβ2 in the aqueous humor6, and contact inhibition2,7. Although some growth factors, such as basic fibroblast growth factor (bFGF), can increase proliferation in a cultured human corneal endothelium, the culture efficiency remains limited8,9. Furthermore, CECs may undergo a phenotypic change during ex vivo expansion, resembling epithelial-mesenchymal transition (EMT)10-13. Endothelial-mesenchymal transition (EnMT) is characterized by cell junction destabilization, apical-basal polarity loss, cytoskeletal rearrangement, alpha smooth muscle actin expression, and type I collagen secretion14. All of these characteristics may abrogate the normal function of CECs, hampering the use of ex vivo cultured CECs in tissue engineering. Moreover, EnMT has been associated with the pathogenesis of several corneal endothelial diseases, including Fuchs endothelial corneal dystrophy and retrocorneal membrane formation15,16. Therefore, understanding the mechanism of EnMT may aid in manipulating the EnMT process and facilitate the regeneration of CECs to enable competent function.

In this study, we described a method for isolating bovine CECs from the corneal button. In the primary culture in vitro, the EnMT process, including a phenotypic change, the nuclear translocation of β-catenin, and EMT regulators snail and slug, was observed. We further described a method for demonstrating EnMT in vivo by using a rat corneal endothelium cryoinjury model. Using these 2 models, we demonstrated that marimastat, a broad-spectrum matrix metalloproteinase (MMP) inhibitor, can suppress the EnMT process. The described protocols facilitate the detailed analysis of the EnMT mechanism and the development of strategies for manipulating the EnMT process for further clinical application.

研究方案

所有的程序遵循本研究中，在视觉与眼科声明协会研究给予了动物在眼科和视觉研究的用途和台大医院的机构动物护理和使用委员会批准。

1.隔离，原代培养制备与牛的CEC的免疫染色

  1. 从本地屠宰场获得新鲜牛的眼睛。
  2. 消毒在3分钟10％w / v的聚乙烯吡咯烷酮 - 碘溶液中的眼睛。用磷酸盐缓冲盐水（PBS）溶液洗涤。
  3. 收获在无菌条件下解剖刀和剪刀角膜按钮。去皮后弹力与在解剖显微镜下用镊子膜（请注意，在这项研究中，牛眼睛被一个本地屠场去核，因此，在第一研究过程是preenucleated眼睛的在实验室消毒）。
  4. 在1毫升试试孵育后弹力膜PSIN在37℃下30分钟。收集通过离心牛的CEC在112×g离心5分钟。重悬细胞于1ml补充激素上皮培养基（SHEM）含有HEPES缓冲的Dulbecco改进的Eagle培养基和哈姆的F12培养基，补充有5％胎牛血清，0.5％二甲亚砜，2纳克/毫升人表皮的等体积的生长因子，5毫克/毫升的胰岛素，5毫克/毫升转铁蛋白，5纳克/毫升硒，1纳摩尔/升霍乱毒素，50毫克/毫升庆大霉素，和1.25毫克/毫升两性霉素B的
  5. 种子的细胞（每眼约1×10 5个细胞）到一个直径6cm培养皿。培养他们在SHEM。孵育在37℃下的菜在5％CO 2在空气气氛中。改变培养基每3天。
  6. 当细胞达到汇合时，把它们洗干净的PBS，并在37°C孵育他们在1ml胰蛋白酶的5分钟。离心112×g离心5分钟收集它们。重悬在1ml的闪的细胞沉淀。
    1. 算在血球细胞。种子在盖玻片的细胞在24孔板一个1×10 4每孔的密度，并培养细胞的SHEM。调查马立马司他的EnMT抑制效果，孵育细胞SHEM 10马立马司他的μM加入到培养基中，并改变它的培养基，每3天。
  7. 固定细胞在含有250微升4％多聚甲醛的，pH值7.4所指示的时间点上，在室温下30分钟。用250微升0.5％的Triton X-100的5分钟通透。用10％的牛血清白蛋白阻断30分钟。
  8. 孵育针对活性β-连环蛋白（ABC），蜗牛和蛞蝓的初级抗体的细胞在4℃下过夜。使用的初级抗体的稀释度为1:200。抗体稀释在10mM PBS中，1％w / v的牛血清白蛋白，和0.09％w / v的叠氮化钠组成的抗体稀释液。
  9. 用PBS洗涤细胞两次15分钟，并孵育米，所述的Alexa Fluor缀合的二级抗体:1小时在室温下（1 100在抗体稀释液）。
  10. 染液通过覆盖细胞用2微克/毫升4'​​，6-二脒基-2-苯基的细胞核，洗涤细胞两次，用PBS进行15分钟，并用抗褪色安装溶液装入它们。
  11. 通过使用激光扫描共聚焦显微镜用20X物镜放大倍率获得在405和488纳米的激发波长免疫荧光图像。

2.鼠角膜内皮细胞冷冻损伤模型和房内注射

  1. 麻醉12周大的雄性SD大鼠用2％甲苯噻嗪（5.6毫克/千克）和替来他明以及唑拉西泮（18毫克/千克）的肌内注射。轻轻捏住动物的皮肤，以确认在​​不存在皮肤抽搐适当麻醉。
  2. 灌输0.5％丙美卡因盐酸一滴对每只大鼠的右眼，以尽量减少眼痛和眨眼反射。灌输四环素软膏到左眼防止角膜干燥。
  3. 冷却在液氮中的不锈钢探针（直径= 3毫米）。不锈钢探针应用于右眼的角膜中央，持续30秒。在操作过程中频繁地灌输的PBS于右眼防止角膜干燥。
  4. 灌输0.1％阿托品和0.3％硫酸庆大霉素冷冻损伤后立即和每日一次，以减轻来自睫状肌痉挛造成眼部疼痛和预防感染。
    1. 手术后，继续使用热灯老鼠温暖，麻醉后观察其恢复每隔15分钟，直到他们重新获得电机控制。此外，应用四环素软膏右眼以防止在恢复期间角膜干。
  5. 重复角膜冷冻损伤连续3天。
  6. 用于递送马立马司他或bFGF的到大鼠眼睛的前房，如前所述麻醉大鼠。灌输一滴0.5％丙美卡因盐酸成右眼的，以尽量减少眼痛和眨眼反射。
  7. 灌溉用无菌PBS眼表面。通过上述和平行的平面中插入在paralimbal透明角膜附着到1ml注射器一个地下30针虹膜的手术显微镜下进行前房穿刺术。
  8. 打开针头斜面向上，并稍微踩下角膜伤口排出一些房水，降低眼压。注入0.02毫升药物的前房内。轻轻压缩与针撤出期间棉尖端的针道。
  9. 拍摄外眼在手术显微镜下所指示的时间点。

3.收获鼠角膜按钮和免疫

  1. 安乐死的大鼠，将它们放置在一个安乐死腔和以每分钟的腔室容积的10-30％的填充速率注入100％的CO 2。保持二氧化碳注入一个缺乏呼吸和褪色的眼睛的颜色后附加分。
  2. 穿透眼鼠在用锋利的刀片将角膜缘。切沿角膜缘剪刀眼角膜。拼合幻灯片上的眼角膜。作出额外的放射状切口，如果角膜蜷缩起身体。
  3. 修复角膜用250微升4％多聚甲醛的，pH为7.4，在室温下30分钟。用250微升0.5％的Triton X-100的5分钟通透，并用10％的牛血清白蛋白阻断30分钟。
  4. 在抗体稀释液200:为1的稀释孵育针对ABC初级抗体的角膜在4℃下过夜。用PBS洗角膜两次15分钟，并与二级抗体孵育它们:在室温下1小时（1 100在抗体稀释液）。染液通过覆盖细胞用2微克/毫升的4'，6-二脒基-2-苯基细胞核，并用PBS洗涤细胞两次15分钟。
  5. 安装在防褪色安装解决方案的眼角膜。 OB在20X物镜放大倍率覃在405激发波长的免疫荧光图像和561 nm的激光扫描共聚焦显微镜。

结果

牛的CEC的分离后，将细胞在体外培养， 图1给出了牛的CEC的相位对比图像。细胞在汇合的六边形表示细胞没有通过细胞分离过程中角膜基质成纤维细胞的污染。 图2描绘了使用针对ABC，蜗牛和蛞蝓的抗体在一个指定的时间点进行的免疫染色。除了 ​​在体外培养的表型变化，观察到的ABC和EMT调节的相应核易位。 图3示出马...

讨论

的CEC是众所周知的倾向中的细胞增殖经受EnMT。制定战略用于抑制EnMT过程用于治疗目的时，EnMT机构的透彻理解是必要的。我们描述了2模型，研究EnMT，即在体 ​​外培养模型大鼠角膜内皮冷冻损伤模型牛CEC。我们的研究结果证明了这两款车型的EnMT过程。此外，马立马司他的EnMT抑制效应再现这两种模式，这表明这些2种型号共享相同的机制。

体外培养模型牛CEC提?...

披露声明

The authors have no competing financial interests to declare.

致谢

We thank the staff of the Second Core Lab, Department of Medical Research, National Taiwan University Hospital for their technical support.

材料

NameCompanyCatalog NumberComments
trypsinThermoFisher Scientific12604-013
Dulbecco’s modified Eagle medium and Ham's F12 mediumThermoFisher Scientific11330
fetal bovine serumThermoFisher Scientific26140-079
dimethyl sulfoxideSigmaD2650
human epidermal growth factorThermoFisher ScientificPHG0311
insulin, transferrin, selenium ThermoFisher Scientific41400-045
cholera toxinSigmaC8052-1MG
gentamicinThermoFisher Scientific15750-060
amphotericin BThermoFisher Scientific15290-026
paraformaldehydeElectron Microscopy Sciences111219
Triton X-100SigmaT8787 
bovine serum albuminSigmaA7906
marimastatSigmaM2699-25MG
anti-active beta-catenin antibodyMillpore05-665
anti-snail antibodySanta cruzsc28199
anti-slug antibodySanta cruzsc15391
goat anti-mouse IgG (H+L) secondary antibodyThermoFisher ScientificA-11001for staining of ABC of bovine CECs
goat anti-mouse IgG (H+L) secondary antibodyThermoFisher ScientificA-11003for staining of ABC of rat corneal endothelium
goat anti-rabbit IgG (H+L) secondary antibodyThermoFisher ScientificA-11008for staining of snail and slug of bovine CECs
antibody diluentGenemed Biotechnologies10-0001
4',6-diamidino-2-phenylindoleThermoFisher ScientificD1306
mounting mediumVector LaboratoriesH-1000
laser scanning confocal microscopeZEISSLSM510
xylazine BayerN/A
tiletamine plus zolazepamVirbacN/Aveterinary drug
proparacaine hydrochloride ophthalmic solutionAlconN/Aveterinary drug
0.1% atropineWu-Fu Laboratories Co., LtdN/Aclinical drug 
0.3% gentamicin sulfateSinphar GroupN/Aclinical drug 
basic fibroblast growth factorThermoFisher ScientificPHG0024clinical drug 

参考文献

  1. Maurice, D. M. The location of the fluid pump in the cornea. J Physiol. 221 (1), 43-54 (1972).
  2. Joyce, N. Proliferative capacity of the corneal endothelium. Progress in Retinal and Eye Research. 22 (3), 359-389 (2003).
  3. Morishige, N., Sonoda, K. H. Bullous keratopathy as a progressive disease: evidence from clinical and laboratory imaging studies. Cornea. 32, 77-83 (2013).
  4. Bates, A. K., Cheng, H., Hiorns, R. W. Pseudophakic bullous keratopathy: relationship with endothelial cell density and use of a predictive cell loss model. A preliminary report. Curr Eye Res. 5 (5), 363-366 (1986).
  5. Wilson, S. E., Lloyd, S. A. Epidermal growth factor and its receptor, basic fibroblast growth factor, transforming growth factor beta-1, and interleukin-1 alpha messenger RNA production in human corneal endothelial cells. Invest Ophthalmol Vis Sci. 32 (10), 2747-2756 (1991).
  6. Chen, K. H., Harris, D. L., Joyce, N. C. TGF-beta2 in aqueous humor suppresses S-phase entry in cultured corneal endothelial cells. Invest Ophthalmol Vis Sci. 40 (11), 2513-2519 (1999).
  7. Senoo, T., Obara, Y., Joyce, N. C. EDTA: a promoter of proliferation in human corneal endothelium. Invest Ophthalmol Vis Sci. 41 (10), 2930-2935 (2000).
  8. Yue, B. Y., Sugar, J., Gilboy, J. E., Elvart, J. L. Growth of human corneal endothelial cells in culture. Invest Ophthalmol Vis Sci. 30 (2), 248-253 (1989).
  9. Peh, G. S., Beuerman, R. W., Colman, A., Tan, D. T., Mehta, J. S. Human corneal endothelial cell expansion for corneal endothelium transplantation: an overview. Transplantation. 91 (8), 811-819 (2011).
  10. Zhu, C., Rawe, I., Joyce, N. C. Differential protein expression in human corneal endothelial cells cultured from young and older donors. Mol Vis. 14, 1805-1814 (2008).
  11. Ko, M. K., Kay, E. P. Regulatory role of FGF-2 on type I collagen expression during endothelial mesenchymal transformation. Invest Ophthalmol Vis Sci. 46 (12), 4495-4503 (2005).
  12. Lee, H. T., Kay, E. P. FGF-2 induced reorganization and disruption of actin cytoskeleton through PI 3-kinase, Rho, and Cdc42 in corneal endothelial cells. Mol Vis. 9, 624-634 (2003).
  13. Pipparelli, A., et al. ROCK Inhibitor Enhances Adhesion and Wound Healing of Human Corneal Endothelial Cells. PloS one. 8 (4), e62095 (2013).
  14. Roy, O., Leclerc, V. B., Bourget, J. M., Theriault, M., Proulx, S. Understanding the process of corneal endothelial morphological change in vitro. Invest Ophthalmol Vis Sci. 56 (2), 1228-1237 (2015).
  15. Jakobiec, F. A., Bhat, P. Retrocorneal membranes: a comparative immunohistochemical analysis of keratocytic, endothelial, and epithelial origins. Am J Ophthalmol. 150 (2), 230-242 (2010).
  16. Okumura, N., et al. Involvement of ZEB1 and Snail1 in excessive production of extracellular matrix in Fuchs endothelial corneal dystrophy. Lab Invest. 95 (11), 1291-1304 (2015).
  17. Okumura, N., et al. Enhancement on primate corneal endothelial cell survival in vitro by a ROCK inhibitor. Invest Ophthalmol Vis Sci. 50 (8), 3680-3687 (2009).
  18. Zhu, Y. T., Chen, H. C., Chen, S. Y., Tseng, S. C. G. Nuclear p120 catenin unlocks mitotic block of contact-inhibited human corneal endothelial monolayers without disrupting adherent junctions. J Cell Sci. 125 (15), 3636-3648 (2012).
  19. Ho, W. T., et al. Inhibition of Matrix Metalloproteinase Activity Reverses Corneal Endothelial-Mesenchymal Transition. Am J Pathol. 185 (8), 2158-2167 (2015).
  20. Kay, E. D., Cheung, C. C., Jester, J. V., Nimni, M. E., Smith, R. E. Type I collagen and fibronectin synthesis by retrocorneal fibrous membrane. Invest Ophthalmol Vis Sci. 22 (2), 200-212 (1982).
  21. Li, C., et al. Notch Signal Regulates Corneal Endothelial-to-Mesenchymal Transition. Am J Pathol. 183 (3), 786-795 (2013).
  22. Okumura, N., et al. The ROCK Inhibitor Eye Drop Accelerates Corneal Endothelium Wound Healing. Invest Ophthalmol Vis Sci. 54 (4), 2493-2502 (2013).
  23. Mannermaa, E., Vellonen, K. S., Urtti, A. Drug transport in corneal epithelium and blood-retina barrier: emerging role of transporters in ocular pharmacokinetics. Adv Drug Deliv Rev. 58 (11), 1136-1163 (2006).

转载和许可

请求许可使用此 JoVE 文章的文本或图形

请求许可

探索更多文章

114 catenin N cadherin

This article has been published

Video Coming Soon

JoVE Logo

政策

使用条款

隐私

联系我们

向图书馆推荐

JoVE 新闻简报

科研

教育

发表

图书馆员

关于 JoVE

版权所属 © 2025 MyJoVE 公司版权所有，本公司不涉及任何医疗业务和医疗服务。