论文标题:单核细胞趋化蛋白-1在增生性玻璃体视网膜病变中的作用研究
Experimental Study of Monocyte Chemotactic Protein-1 in the Development of Proliferative Vitreoretinopathy
论文作者
论文导师 惠延年,论文学位 博士,论文专业 眼科学
论文单位 第四军医大学,点击次数 101,论文页数 119页File Size5150K
2000-05-01论文网 http://www.lw23.com/lunwen_546609027/
proliferative vitreoretinopathy; monocyte chemotactic protein-1; retinal pigment epithelial cell; macrophage; T-lymphocyte;immunohistochemistry; in situ hybridization; migration;dexamethasone; daunorubicin; Calphostin C; Verapamil
目的：增生性玻璃体视网膜病变(proliferative vitreoretinopathy，PVR)是有多种炎性因子、炎症细胞和组织细胞参与的损伤修复过程，巨噬细胞在PVR的发病过程中有重要的作用，单核细胞趋化蛋白—1(monocyte chemotactic protein-1，MCP—1)可以特异性地趋化巨噬细胞和淋巴细胞向炎症的部位聚集，而视网膜色素上皮细胞(retinal pigment epithelium，RPE)是其中主要的增殖细胞。本实验研究的目的是(1)评价巨噬细胞调理液(MCM)对培养人RPE合成MCP-1的作用；(2)观察MCP—1对RPE移行的作用和地塞米松和道诺霉素对这种作用的影响，以及细胞信号转导通道阻滞剂对这个过程的作用；(3)观察视网膜脱离后不同时期的临床标本组织中MCP—1的表达与巨噬细胞和淋巴细胞的关系。 方法：(1)在培养人RPE的培养液中加入20％培养人巨噬细胞调理液(MCM)、白细胞介素—1β(IL—1β)或肿瘤坏死因子—α(TNF—α)(0.2，2，20ng／ml)培养2、4、8、16、24和48h后，对培养细胞爬片进行免疫组化和原位杂交染色，并对培养RPE的上清液作Western blot检测MCP—1的含量。同时在培养液中加入地塞米松(10~(-8)，10~(-7)，10~(-6)M)或道诺霉素(2μg，20μg，200μg／L)重复上述实验，观察在此条件下MCM对MCP—1合成的作用；(2)处于生长融合状态的单层RPE用剃须刀片刮出一片无细胞的区域，在无血清培养液中加入MCP—1、IL—1β和TNF—α，培养20h后计数进入刮痕区的细胞，观察这些因素对RPE移行的作用。同时另设实验组在培养液中分别加入MCP—1中和抗体、地塞米松、道诺霉素、蛋白激酶C抑制剂(Calphostin C，CC)和钙离子通道阻滞剂维拉帕米(Verapamil，Ver)，观察这些条件下MCP—1对RPE移行作用的变化。最后用MTT法检测了MCP—1对RPE的促增殖作用；(3)选用视网膜手术取材标本，包括裂孔源性视网膜脱离视网膜下液中的细胞成份，B级PVR玻璃体手术集取物以及PVR C／D级的视网膜增殖膜，免疫组化法检测其中MCP—1、CD68和CD3的表达状态。 结果：(1)Western blot检测表明培养的人RPE在无药物作用的条件下，MCP—1的分泌为阴性，20％MCM、IL—1β和TNF—α培养条件下，2h时可以检测到分泌的MCP—1，而且随着时间和药物浓度的增加而上升，并在16h达到较高水平，但是MCM培养过程中MCP—1的分泌浓度较低，约为IL—1β条件下的1／5。原位杂交和免疫组化结果验证了Western blot的结果。加入地塞米松
Aim: Proliferative vitreoretinopathy (PVR) is an excessively wound healing response that involved many kinds of inflammatory cytokines, and migration and proliferation of cells. Macrophages have been proved a role in the development PVR. Monocyte Chemotactic Protein-1 (MCP-1) can specifically recruit macrophages and lymphocytes into the inflammatory area. Retinal pigment epithelial cells (RPE) are predominant proliferative cells in PVR. This experiment aim to (1) examine the effects of cultured macrophage conditioned media (MCM) on RPE production and gene expression of MCP-1, (2) investigate the effects of MCP-1 on modulation of the migration and proliferation of RPE. Additional experiments were conducted to test the changes of this modulation effect under the action of several agents, and (3) investigate the relationship of the presence of MCP-1, macrophages and T-lymphocytes in specimens obtained from eyes with retinal detachment and PVR.Methods: (1) Human RPE were cultured with 20% macrophage-conditioned mediua (MCM), IL-1β (0.2ng to 20ng/ml) or TNF-α (0.2ng to 20ng/ml),and with additional dexamethasone (10~(-8), 10~(-7), 10~(-6)M) or daunorubicin (2μg,20μg,200μg/L) for 2, 4, 8, 24 or 48 h. Secreted MCP-1 in the supernatant of cultured RPE was measured with Western blot assay, and intracellular MCP-1 was detected with in situ hybridization and immunohistochemistry. (2) We used an in vitro wound healing model in which a small area of a confluent monolayer of RPE was denuded with a razor blade. The cultures were subsequently incubated with MCP-1 (0.2ng/ml to 20ng/ml),
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