原发性开角型青光眼小梁细胞的体外培养及其生物学特性

Biological characteristics of in vitro trabecular cells from primary open-angle glaucomatous eye

背景原发性开角型青光眼（POAG）是一种常见致盲性眼病，其特点是房水外流阻力增加导致眼压增高。位于房水外流通道的小梁网调节房水的外流，因此研究小梁网细胞的生物学特性有着重要的意义。目的探讨POAG小梁细胞体外培养的方法及其生物学特性。方法经小梁切除术收集8例开角型青光眼患者患眼的带小梁网的深层巩膜组织块进行体外原代和传代培养，用鼠抗人层黏连蛋白（LM）单克隆抗体、兔抗人纤维连结蛋白（FN）单克隆抗体、鼠抗人神经元特异性烯醇化酶（NSE）单克隆抗体进行免疫组织化学检测以对传代细胞进行鉴定，在透射电子显微镜下对传代细胞的超微结构进行观察，并将传代小梁细胞的生物学特性与本研究组前期培养的正常小梁细胞进行比较。结果组织块培养10d左右，可见细胞从其边缘向外生长。传代细胞在4d内处于对数生长期，其后进入平台期，第7天细胞基本融合。第3代POAG小梁细胞及正常人眼小梁细胞中可见FN、LM和NSE均呈阳性表达，证实传代细胞为小梁细胞，而空白对照组细胞未见FN、LM和NSE表达。第3代POAG小梁细胞和正常小梁细胞中FN的A450值分别为0．354±0．06和0．26±0．01，LM的A450值分别为0．34±0．03和0．25±0．02，差异均有统计学意义（FN：t=14．446，P=0．001；LM：t=9．346，P=0．001）。与正常小梁细胞比较，第3代POAG小梁细胞表面的微绒毛、细胞质的溶酶体及吞噬小泡含量减少。结论采用组织块培养法可成功在体外培养POAG小梁细胞，该研究结果为研究青光眼的发病机制提供了细胞学基础。

Background Primary open angle glaucoma （POAG） is a major blindness-causing disease, characterized by elevated intraocular pressure due to an insufficient outflow of aqueous humor. The trabecular meshwork lining the aqueous outflow pathway modulates the aqueous outflow facility. To study the biological characteristics of the trabecular meshwork ceils has important significance. Objective This study was to culture the trabecular cells from primary open-angle glaucomatous eye （POAG） and study the biologic characteristics of passaged cells. Methods The deep scleral tissue with trabecular meshwork was obtained during the trabeculectomy from 8 eyes with POAG. The trabecular cells were primarily cultured and passaged in vitro. The generation 3 cells were identified by immunoehemistry with the laminin （LM）, fibronectin （FN） and neuron specific endolase （NSE） monoelonal antibodies. The uhrastructure was examined to observe the biological characteristics of the ceils under the transmission electronic microscope. The experimental results were compared among POAG group,normal control group and blank control group. Results The primarily cultured POAG trabecular cells migrated from the edge of tissue mass about 10 days. The cells of generation 3 presented the logarithmic phase in the first 4 days and fused in the 7th day. FN,LM and NSE were positively expressed in the generated cells in POAG group and normal control group rather than blank control group. The MOD values of the generation 3 cells for FN in POAG group and normal control group were0.35±0.06 and 0.26±0.01, and those for LM were 0.34：t=0.03 and 0.25 ±0.02 respectively, showingstatistically significant difference between these two groups （ FN ： t = 14. 446, P〈0. 001 ; LM ： t = 9. 346, P〈0. 001 ）. The microvilli, cytolysosome and phagocytic vesicle were obviously decreased in the trabcular cells of POAG group compared with normal control group under the transmission electron microscope. Conclusion The trabecular meshwork ceils from POAG can be successfully cultured and passaged in vitro. It provides a cytology basis for further glaucoma research.