Nerve Growth Factor Prevents Apoptosis Induced By Light In Cultured Human Retinal Pigment Epithelium

Background and ObjectiveEnvironment and artificial light have the potential damage to retinal.For many years, it is an important discussion for retinal light damage. Forrecent 30 years, with the more and more optics apparatus are using in theclinic, people begin to research retinal light damage, not only to nature light,but also to the optics apparatus and visual light. It is a difficult problem toophthalmology field to find the mechanism, pathogens and diagnosis of thedegeneration of retina. Some researches have indicated that some opticsapparatus such as slit-lamp, indirect-ophthalmoscope and operativemicroscope have damage to retina. Investigators also have found thatrepeated irradiation can make accumulative effect. Long-time, low intension,intermittent sun radiation might have relation to AMD. It has been improvedthat retinal light damage can simulate the animal retina degenerationmodel,therefore it is very important to discuss the relationship betweendamage factors and retinal light damage, how to reduce and protect theretinal light damage. We design the experiment to investigate the protectiveeffect of nerve growth factor (NGF) on apoptosis of cultured human retinalpigment epithelium (hRPE).Methods1,The eye balls were from two young men died for accident, refer to Hu's. Follow the edge of cornea make a cut and derelict the cornea, lens, vitreous body and retina. Put these two eye balls in D-hank's in 4Co. All the process was completed in 4 hours. hRPE cells were isolated andcultured in primary manner in vitro and identified by monoclonal antibody of keratin. The primary cells were observed by phase contrast microscope every day. The classical cells were round with many melanin drops. The cells became to be fusiform and few melanin drops with transferring of culture.2,Subcultured hRPE cells were treated by (2000+500)Lx visible light to be established cell apototic model. The protective effect of NGF on apoptosis of cultured hRPE cells were assessed by an acridine orange (AO) staining method, transmission electron microscopy (TEM) and flow cytometry. hRPE cells were isolated and cultured in primary manner in vitro and identified by monoclonal antibody of keratin. The primary cells were observed by phase contrast microscope every day. The classical cells were round with many melanin drops. The cells became to be fusiform and few melanin drops with transferring of culture. We select the 3rd to 6th generation cells to do the experiment.3,AO (acridine orange ) staining a. 1% acetum differentiated for 30'; b. 0.01%AO stained for 10min; c. PH 4.8 PBS (phposphate balanced solution) washed for 1min; d. 0.1%M CaCl2 differentiated for 30'then washed; e. PBS washed for three times in three different staining glasses, every time for 10'; f. Use the PBS to seal the cells for keeping.4,Observe and detect the cellsa. Observe the configuration of the cells: using the microscope, transmission electron microscopy(TEM) .b.Detect the cells: we using the flow cytometry to divided the cells to Q1, Q2,Q3 and Q4. they indicate: necrotic and mechanical damaged cells, secondary necrotic cells, the viable cells and the apoptic cells.5,Statistical analysis: We use the SPSS to analyze the data. α=0.05. we use F test to compare the relationships between several groups; we use the t test to cmompare the relationships between two groups.Results1,Cell culture and identify After the irradiation of the visible light, we can see the hRPE cellschanged, we can observe the apoptosic cells.2,Flow cytometry It has significant difference between the 6h group with the none lightgroup (p﹤0.01). There has no significant difference(p﹥0.01).There hassignificant difference between the 12h group and 24h group not only onapoptosic cells or necrotic cells (p﹤0.01).There has significant differencein apoptosic cells between the 6h group and 12h group (p﹤0.01), not innecrotic cells (p﹥0.01). There has significant difference between the 24hgroup and 6h group, not only in apoptosic cells and necrotic cells (p﹤0.01).There has significant difference between 12h group and 24h group inapoptosic cells (p﹥0.01), but there has significant difference in necroticcells (p﹤0.01).