Effects And Mechanisms Of Ciliary Neurotrophic Factor On Rat Retinal Ganglion Cells In Vitro

Purpose: Recently, it was reported in many studies that apoptosis is the mainly death way of retinal ganglion cells (RGCs) of glaucoma. Following the deep researches of glaucoma pathological mechanism, the protective effects of neurotrophin were more and more important for therapy of glaucoma. Ciliary neurotrophic factor (CNTF) can induce differentiation of nerve cells, decrease apoptosis of nerve cells and accelerate regeneration of the cells. Some experiments showed that CNTF enhance the survival of RGCs. But the effect that CNTF prevent RGCs from apoptosis was not reported and the mechanisms that CNTF protect RGCs are little understood. The purpose of this study was to observe the effects of CNTF and explore the mechanisms of CNTF on rat retinal ganglion cells in vitro.Method: At this research, the retina of Wistar rats within postnatal 2 or 3 days were dissociated into cell suspension with 0.05% trypsin digestion. After cultured 1 and 4 days RGCs were identified with immunohistochemistry method using anti-rat Thy-1.1 monoclonal antibody. TUNEL method was used to observe apoptotic RGCs. In order to study the effects of CNTF on cultured cells, the expriments was divided into 10ng.ml-1,20ng.ml-1,40ng.ml-1 CNTF groups and control group. The time of survival RGCs was recorded. The indirect numbers of living cells after cultured 1, 3 and 5 days were tested by MTT colorimetric microassay. The rate of apoptotic RGCs that cultured 3 days was tested by flow cytometer. The expression of bcl-2 protein in RGCs that cultured 3 days with 20ng.ml-1 CNTF was determined by Western blot.Result: The cell membrane and neurite of cultured 1 and 4 days RGCs weredyed buffy by Thy-1.1 antibody with immunohistochemistry method and the positive rate of retinal ganglion cells was over 90 percent. There are positive TUNEL cell in cultured RGCs. The time of survival RGCs was prolonged 2 to 4 days in CNTF groups, the numbers of surviving RGCs added significantly at cultured 3 and 5days in all concentrations of CNTF (P<0.01~0.05) by MTT measured, and 20ng.ml-1 CNTF was higher than others. Flow cytometer results show that CNTF significantly reduced the rate of apoptotic RGCs for cultured 3 days (P<0.01~0.05), and 20ng.ml-1 CNTF were more significantly effective than it in 10ng.ml-1 and 40ng.ml-1(P<0.01). Meanwhile, Western blot analysis is shown that the expression of bcl-2 protein was obviously increased in retinal ganglion cells for cultured 3 days with 20ng.ml-1 CNTF.Conclusion: The methods that we used at this study can successfully obtained RGCs for research aim. CNTF furthermore promote RGCs survival by preventing RGCs from apoptosis. The effect of CNTF decrease RGCs apoptosis may relate to upregulation of the expression of bcl-2 gene.