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。两个主要的地点 青光眼的损害是小梁网络(TM)，导致眼压升高，以及视网膜 在青光眼中逐渐死亡的神经节细胞(RGC)，导致与此相关的视力丧失 疾病。通过对青光眼的研究，人们对青光眼的细胞和分子生物学有了很大的了解 培养的TM细胞和RGC。然而，目前的TM细胞和RGC培养有很大的局限性。 TM细胞生长相对较慢，衰老较快，将研究局限于早期细胞中相对较少的细胞数量 段落。视网膜节细胞是终末分化的，因此原代视网膜节细胞必须从新生儿制备。 每进行一次实验就用啮齿动物的眼睛。虽然已经产生了永生化的细胞系，但这些细胞 在培养中不断增殖，并失去了原代TM细胞的其他表型特征 RGC。确实需要新的TM细胞和RGC系，它们可以很容易地繁殖，并且仍然 保留原代细胞培养的表型特征。ImmortoMouse是作为一个 独特的资源，可以从各种组织中产生条件永生化的细胞，这将 当细胞在允许的温度下培养时，细胞持续增殖，但恢复正常 在不允许的温度下生长时的表型。我们的假设是有条件地永生 可以从ImmortoMouse中分离出小鼠RGC和TM细胞系，具有以下特点 在不允许的条件下生长的原代TM细胞和RGC。以下具体目标将 应对这一假设。(SA#1)我们将开发和表征条件永生化的小鼠TM 在允许的条件下(在33oC与干扰素一起培养)快速增殖的细胞株，并将具有 在不允许的条件下(39℃培养，不含干扰素)培养的原代TM细胞的特征。 (SA#2)我们将开发和表征条件永生化的小鼠RGC系，这些系将迅速 在允许的条件下增殖，在非培养条件下生长将具有原代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)我们将分离和鉴定 第一个有条件永生化的TM和RGC细胞系。(B)我们还开发了一种新的方法，使用 吞噬磁性微球纯化小鼠TM细胞。这些线路将向更多的人开放 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，以及(C)测试潜在的新治疗药物。 好了！

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. !