Conditionally immortalized TM cell and RGC lines from Animal Model

摘要 青光眼是世界上不可逆转的视力丧失和失明的主要原因。两大网站 青光眼损害是小梁网（TM），导致眼内压升高，以及视网膜损害 神经节细胞（RGC）在青光眼中逐渐死亡，导致与此相关的视力丧失。 疾病通过对青光眼的细胞和分子生物学的研究， 培养的TM细胞和RGCs。然而，目前的TM细胞和RGC培养物存在显著的局限性。 TM细胞生长相对缓慢，衰老迅速，限制了对早期细胞相对较小细胞数量的研究。 段落。RGC是终末分化的，因此，原代RGC必须从新生儿中制备。 啮齿动物眼睛进行的每个实验。虽然已经产生了永生化细胞系，但这些细胞 在培养物中连续增殖，并已丧失原代TM细胞的其他表型特征， RGC。对于新的TM细胞和RGC系存在明确的需求，其可以容易地繁殖并且仍然是 保留原代细胞培养物的表型特征。ImmortoMouse是作为一种 从各种组织中产生条件永生化细胞的独特资源， 当细胞在允许的温度下培养时， 在非允许温度下生长时的表型。我们的假设是条件永生化 可以分离来自ImmortoMouse的小鼠RGC和TM细胞系，其将具有以下特征： 原代TM细胞和RGC在非允许条件下生长时。以下具体目标将 解决这个假设。(SA#1）我们将开发和表征条件永生化小鼠TM 在允许的条件下（在33 ℃下与干扰素一起培养）快速增殖的细胞系， 当在非允许条件下生长时（在39 ° C无IFN下培养），原代TM细胞的特征。 (SA#2）我们将开发和表征条件永生化小鼠RGC系，其将快速地 在允许的条件下增殖，并且当在非限制性条件下生长时将具有初级RGC的特征。 允许的条件。这个项目在几个方面都很有创新性。(A)我们将分离并表征 第一个条件永生化的TM和RGC细胞系。(B)我们还开发了一种新的方法， 吞噬磁性微球以纯化小鼠TM细胞。这些线路将提供给广泛的 各种视觉研究人员：（a）更好地了解TM和RGC生物学，（B）发现和表征 新的致病途径，和（c）测试潜在的新治疗剂。 !

Abstract Glaucoma is a leading cause of irreversible vision loss and blindness in the world. Two major sites of glaucoma damage are the trabecular meshwork (TM), which leads to elevated intraocular pressure, and retinal ganglion cells (RGCs) that progressively die in glaucoma, leading to the loss of vision associated with this disease. A great deal has been learned about the cell and molecular biology of glaucoma through research on cultured TM cells and RGCs. However, there are significant limitations with current TM cell and RGC cultures. TM cells grow relatively slowly and rapidly senesce, limiting studies to relatively small cell numbers of early cell passages. RGCs are terminally differentiated, and therefore, primary RGCs must be prepared from neonatal rodent eyes for each experiment conducted. Although immortalized cell lines have been generated, these cells continuously proliferate in culture and have lost other phenotypic characteristics of primary TM cells and RGCs. There is a definite need for new TM cell and RGC lines that can be easily propagated and still retain the phenotypic characteristics of primary cell cultures. The ImmortoMouse was developed as a unique resource to generate conditionally immortalized cells from a wide variety of tissues that will continuously proliferate when the cells are cultured at permissive temperatures, but regain their normal phenotypes when grown at non-permissive temperatures. Our hypothesis is that conditionally immortalized mouse RGC and TM cell lines from the ImmortoMouse can be isolated, which will have the characteristics of primary TM cells and RGCs when grown under non-permissive conditions. The following Specific Aims will address this hypothesis. (SA#1) We will develop and characterize conditionally immortalized mouse TM cell lines that will rapidly proliferate under permissive conditions (culture at 33oC with ¿IFN) and will have characteristics of primary TM cells when grown under non-permissive conditions (culture at 39oC without ¿IFN). (SA#2) We will develop and characterize conditionally immortalized mouse RGC lines that will rapidly proliferate under permissive conditions and will have characteristics of primary RGCs when grown under non- permissive conditions. This project is very innovative in several ways. (A) We will isolate and characterize the first conditionally immortalized TM and RGC cell lines. (B) We also have developed a novel method using phagocytized magnetic microspheres to purify mouse TM cells. These lines will be made available to a wide variety of vision researchers to: (a) better understand TM and RGC biology, (b) discover and characterize new pathogenic pathways, and (c) test potential new therapeutic agents. !