Expression of Hepatocyte Growth Factor in Frozen Human RPE Cells and Effects of Low Molecular Weight Heparin (Fragmin) on Hepatocyte Growth Factor

Abstract

Abstract: : Purpose: To study hepatocyte growth factor (HGF) level in frozenhuman retinal pigment epithelium cells (hRPE) and evaluate theeffect of HGF induced hRPE proliferation. Furthermore, to investigatethe influence of Fragmin, a low molecular weight heparin, onHGF expression and cell proliferation both in vitro and in vivo.Method: Cultured hRPE cells (3rd-5th passages) were frozenat –80°C for 15 seconds and 60 seconds. The followingsubstances were tested: (1) The frozen cells were incubatedfor further. (2) Intravitreous injection of frozen cells inRabbits. The cell supernate and vitreous samples were collectedat 3 days and 6 days. The level of HGF was monitored by ELISA.(3) Some vitreous samples of test (2) were added to non-freezinghRPE cells culture media and the cell proliferation was examinedby MTT assay. (4) The frozen cells were incubated in media withFragmin (25ul/ml) for 3 days and 6 days and collected theirsupernate and measured by ELISA and (5) Adding Fragmin to theculture media of non-freezing hRPE cells cultured with the rabbitvitreous and the cell proliferation was examined by MTT assay.Results: HGF concentration in frozen hRPE cells (mg/ml, Table1). HGF level in rabbit vitreous injected frozen hRPE cells(pg/ml, Table 2). Enhanced proliferation was observed in non-freezinghRPE cells after cultured with the vitreous, especially, in60-second –frozen group.Conclusion: Freezing can increaseexpression of HGF in hRPE cells. The supernate (samples) afterfrozen hRPE cells can induce the proliferation of hRPE cells.The HGF level decrease significantly in frozen hRPE cells byFragmin Table 1 

Table 2 

Keywords: proliferative vitreoretinopathy • growth factors/growth factor receptors • drug toxicity/drug effects