Zhong L, Li M
Department of Ophthalmology, First School of Clinical Medicine, Beijing Medical University, Beijing 100034.
Zhonghua Yan Ke Za Zhi. 1999 May;35(3):186-9, 9.
To understand transforming growth factor-beta(1) (TFG-beta(1)) influencing microfilament actins in cultured human trabecular cells (HTCs) and approach to the effect of resistance of aqueous outflow in the pathogenesis and mechanism of primary open angle glaucoma (POAG).
Trabecular meshwork was collected from 68 eyeballs of 34 human donors under 6 years old within 24 hours after death. HTCs were primarily cultured and subcultured. Cultured cells were observed by light and electron microscopes. Laminin (LN) and IVcollagen (IVC) in extracellular matrix (ECM) were immunohistochemically stained by S-P method. Various doses (0, 1, 5 ng/ml) of TFG-beta(1) and 30 microg/ml neutralizing antibody of TFG-beta(1) were respectively added to the third passage cells before confluence and acted for 6, 12, 24 hours. Stress fibers stained with fluorescein isocyanate (FITC)-phalloidin were observed under light and electron microscopes. Various doses (0, 0.5, 1, 5 ng/ml) of TFG-beta(1) and 30 microg/ml neutralizing antibody of TFG-beta(1) were respectively added to the third passage cells after confluence for one week and acted for 6, 12, 24, 48 hours. The positive numbers of labeled F-actins were quantitatively evaluated by flow cytometry. Various doses (0, 0.5, 1, 5 ng/ml) of TFG-beta(1) and 30 microg/ml neutralizing antibody of TFG-beta(1) were respectively added to the third passage cells after confluence for one week and acted for 6, 12, 24, 36, 48 hours. The amount of beta-actin mRNA expression was semi-quantitatively evaluated by densitometry with reverse transcription-polymerase chain reaction (RT-PCR) using specific oligonucleotide primers for beta-actin and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) as an internal standard.
According to the growing characteristics and morphological features of the cultured cells, they were identified as HTCs. In comparison with the control group, TGF-beta(1) could increase the stress fibrous bundle, the contents of F-actins and the expression of beta-actin mRNA; the effects of TFG-beta(1) appeared time dependent and were antagonized by neutralizing antibody of TFG-beta(1). When TFG-beta(1) was at higher concentration and longer acting time, cell proliferation was inhibited, cell membrane ruptured and cells peeled off in cultured HTCs before confluence.
Application of reverse transcription-semiquantitative PCR can promote the pathogenic research of POAG at genetic level, in a certain extent, TFG-beta(1) can obviously upregulate microfilament actins in the confluent HTCs, enhance the contractility of trabecular cells and decrease the resistance of aqueous outflow in trabecular meshwork.
了解转化生长因子-β1（TFG-β1）对培养的人小梁细胞（HTCs）中微丝肌动蛋白的影响，探讨其在原发性开角型青光眼（POAG）发病机制及房水流出阻力中的作用。
取34例6岁以下人类供体死亡后24小时内的68只眼球的小梁网，进行HTCs的原代培养和传代培养。用光镜和电镜观察培养的细胞。采用S-P法对细胞外基质（ECM）中的层粘连蛋白（LN）和IV型胶原（IVC）进行免疫组织化学染色。在汇合前分别向第三代细胞中加入不同剂量（0、1、5 ng/ml）的TFG-β1和30 μg/ml的TFG-β1中和抗体，作用6、12、24小时。用异硫氰酸荧光素（FITC）-鬼笔环肽染色观察应力纤维，在光镜和电镜下观察。汇合一周后分别向第三代细胞中加入不同剂量（0、0.5、1、5 ng/ml）的TFG-β1和30 μg/ml的TFG-β1中和抗体，作用6、12、24、48小时。采用流式细胞术定量评估标记F-肌动蛋白的阳性细胞数。汇合一周后分别向第三代细胞中加入不同剂量（0、0.5、1、5 ng/ml）的TFG-β1和30 μg/ml的TFG-β1中和抗体，作用6、12、24、36、48小时。以β-肌动蛋白和甘油醛-3-磷酸脱氢酶（G3PDH）特异性寡核苷酸引物，用逆转录-聚合酶链反应（RT-PCR）和光密度法半定量评估β-肌动蛋白mRNA表达量，以G3PDH作为内参。
根据培养细胞的生长特性和形态特征，鉴定为HTCs。与对照组相比，TGF-β1可增加应力纤维束、F-肌动蛋白含量及β-肌动蛋白mRNA表达；TGF-β1的作用呈时间依赖性，并被TFG-β1中和抗体拮抗。当TFG-β1浓度较高、作用时间较长时，可抑制汇合前培养的HTCs细胞增殖，导致细胞膜破裂、细胞脱落。
逆转录-半定量PCR的应用可在一定程度上促进POAG在基因水平的发病机制研究，TFG-β1可明显上调汇合后HTCs中的微丝肌动蛋白，增强小梁细胞的收缩性，降低小梁网房水流出阻力。
