[Influence of transforming growth factor-beta1 on actin in cultured human trabecular cells]

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.