Expression Of CD44s In Cultured Human Trabecular Meshwork Cells

Purpose:To study the culture of human trabecular meshwork cells(HTCs) in vitroand to observe their biological character. To investigate the effect of tumornecrosis factor-α(TNF-α) on CD44s expression in cultured human trabecularmeshwork cells.Methods:(1).In the first part,The trabecular tissues from human eyeswere primarily cultured.The morphologic features and growingcharacteristics of the cultured cells were observed by invertedmicroscope,electron microscope and immunohistochemistry.The proliferativecurve of the cultured cells was obtained by colorimetric assay with MTT.(2).CD44s expressed in cultured human trabecular meshwork cells was measured byquantitative flow cytometry and semi-quantitative RT-PCR after treated with0.0ng/ml(control),5ng/ml,10ng/ml,20ng/ml TNF-αfor 24h.(3).Effect of TNF-αon expression of CD44s was confirmed by MTT assay.Results: (1).The primary cells in culture were observed from the edgeof the cultured tissues after 10-15 days.The cultured cells wereround,oval,fusiform and multiangular.The trabecular cells showed apicalvillons projections and had a high density of secondary lysosome.Thejunctions between the trabecular cells observed most frequently were gapjunction.Laminin(LN),fibronectin(FN) and neuron specific enolase(NSE) ofthe cultured cells expressed were identified with immunohistochemicalmethod.(2).The mean fluorescence intensity of CD44s expressed by the cellstreated with 0.0ng/ml(control),5ng/ml,10ng/ml,20ng/ml TNF-αwas 73.93±12.39,76.79±9.00,89.89±14.64,91.65±14.96,respectively. Thedifference between these 10ng/ml,20ng/ml TNF-αtreated groups and that ofthe control group was statistically significant.(3).The relative gray valueof CD44smRNA/G3PDH of the cells treated with 0.0ng/ml(control),5ng/ml,10ng/ml,20ng/ml TNF-αwas 0.58±0.10,0.63±0.09,0.79±0.12,0.91±0.13,respectively. The difference between these 10ng/ml,20ng/ml TNF-αtreated groups and that of the control group was statistically significant.The difference between the 10ng/ml and that of 20ng/ml TNF-αtreated groupswas also statistically significant.(4).Adhesion of trabecular meshworkcells to hyaluronan was increased by TNF-α.OD value of the four groups were0.86±0.13,0.97±0.13,1.14±0.14,1.23±0.12,respectively.The differencebetween 10ng/ml,20ng/ml TNF-αtreated groups and that of the control groupwas statistically significant. Conclusion:(1).Human trabecular meshwork cells(HTCs) can besuccessfully cultured in vitro as long as the technology of cell culture andthe characteristic anatomy of trabecular meshwork are known-well.Theidentification of cultured HTCs must be combined with three aspects:thegrowing characteristics and morphological features of cultured cells underlight and electron microscopes,and immunohistochemistry.(2).TNF-αcouldincrease CD44s expression in cultured HTCs. (3).Adhesion assay alsoconfirmed that TNF-αcould increase CD44s expression in cultured HTCs.