A Magnetic Bead-Based Method for Mouse Trabecular Meshwork Cell Isolation
A Magnetic Bead-Based Method for Mouse Trabecular Meshwork Cell Isolation
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DOI：
10.1167/iovs.13-12033
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发表时间：
2013-05-01
影响因子：
4.4
通讯作者：
Clark, Abbot F.
中科院分区：
文献类型：
--
作者：
Mao, Weiming;Liu, Yang;Clark, Abbot F.
目的。老鼠已被广泛用于青光眼研究。然而，由于小鼠眼睛的尺寸较小，很难解剖小鼠小梁网络(MTM)组织并建立MTM细胞株。为了解决这一问题，我们利用小梁细胞的吞噬特性，开发了一种新的基于磁珠的方法，使我们能够分离出纯的MTM细胞。方法麻醉后，将多达2亩的L荧光微球或磁性微球注入小鼠眼内。分别于注射后1~7d处死小鼠，摘除眼球进行荧光和透射电子显微镜观察，观察微珠的分布和定位。为分离MTM细胞，在注射磁珠后7天解剖10~15只无菌小鼠眼前节。用胶原酶A消化组织，用磁场和反复洗涤的方法进行纯化。结果透射电子显微镜研究表明，磁珠定位于小鼠的TM，而不存在于角膜和巩膜成纤维细胞中。培养的MTM细胞形态与人TM细胞相似。MTM细胞表达TM标志物，包括IV型胶原、层粘连蛋白和α-平滑肌肌动蛋白。此外，100 nM地塞米松处理的MTM细胞表现出更多的交联化肌动蛋白网络的形成和myocin表达的诱导。结论：磁珠法是一种有效的分离MTM细胞的方法，所需的显微切割技术最少。这将是一种从小动物中分离TM细胞用于青光眼研究的有用方法。
PURPOSE. Mice have been used widely for glaucoma research. However, due to the small size of the mouse eye, it is difficult to dissect mouse trabecular meshwork (MTM) tissues and establish MTM cell strains. To circumvent this problem, we took advantage of the phagocytic property of trabecular meshwork (TM) cells, and developed a novel magnetic bead-based method that enables us to isolate pure MTM cells.METHODS. After anesthesia, up to 2 mu L of fluorescent or magnetic microbeads were injected intracamerally into the mouse eyes. To study the distribution and localization of the beads, mice were sacrificed 1 to 7 days after injection, and eyes were enucleated for fluorescent or transmission electron microscopy (TEM) study, respectively. To isolate MTM cells, anterior segments injected with magnetic beads were dissected from 10 to 15 sterilized mouse eyes 7 days after injection. The tissues were digested with collagenase A and purified by using a magnetic field as well as repeated washing.RESULTS. TEM studies showed that the magnetic beads were located in the mouse TM, but not in corneal or scleral fibroblast cells. Cultured MTM cells were similar morphologically to human TM cells. MTM cells expressed TM markers, including collagen IV, laminin, and alpha-smooth muscle actin. Also, MTM cells treated with 100 nM dexamethasone showed increased formation of cross-linked actin networks and induction of myocilin expression.CONCLUSIONS. The magnetic bead-based method is efficient for isolating MTM cells with minimal microdissection techniques required. It will be a useful approach for isolating TM cells from small animals for glaucoma research.