睫状神经营养因子对培养大鼠视网膜神经节细胞凋亡的影响

EFFECT OF CILIARY NEUROTROPHIC FACTOR ON APOPTOSIS IN CULTURED RAT RETINALGANGLION CELL

目的 :观察睫状神经营养因子 (CNTF)对培养大鼠视网膜神经节细胞 (RGCs)凋亡的影响。方法 :15只生后 2～ 3dWistar大鼠 ,视网膜采用胰酶消化法制成细胞悬液后接种于 2 4孔培养板 (每孔 1× 10 5个细胞 ) ,取培养 72h的细胞行免疫细胞化学鉴定。将培养的细胞随机分为对照组、三种浓度CNTF组 (10ng/ml、2 0ng/ml、4 0ng/ml) ,培养 72h后采用TUNEL法和流式细胞仪检测培养大鼠视网膜神经节细胞凋亡的变化。结果 :培养 72h的细胞 90 %以上为视网膜神经节细胞 ,TUNEL法检测显示培养细胞有凋亡细胞存在 ,流式细胞仪检测结果显示对照组凋亡率为 (11.95± 4 .4 0 0 ) % ,10ng/ml、2 0ng/ml、4 0ng/mlCNTF组凋亡率分别为(7.883± 2 .14 6 ) % ,(6 .4 17± 3.317) % ,(8.0 33± 2 .0 2 3) % ,CNTF组凋亡率显著降低 (P <0 .0 1～ 0 .0 5 ) ,其中2 0ng/mlCNTF组差异非常显著 (P <0 .0 1)。结论 :CNTF能降低培养大鼠视网膜神经节细胞凋亡率 ,其促视网膜神经节细胞存活可能是通过减少视网膜神经节细胞凋亡来起作用 ,而高浓度CNTF对视网膜神经节细胞有毒副作用。

Objective:To observe the effect of ciliary neurotrophic factor(CNTF) with different concentration on apoptosis in cultured rat retinal ganglion cell. Methods:The retina of Wistar rats within postnatal 2 or 3 days were dissociated into cell suspension with 0.05% trypsin digestion. After 72 hours in culture RGC were identified with immtmohist ochemistry method using anti-rat Thy- 1.1 monoclonal antibody. The expriments was divided into 10ng/ml,20ng/ml, 40ng/ml CNTF groups ( Ⅰ, Ⅱ,Ⅲgroups) and control group. After 3 days in culture ,TUNEL method and flow cytometer was used to observe the change of apoptosis in cultured rat retinal ganglion cell. Results:The positive rate of retinal ganglion cells was over 90 %. There are positive TUNEL cell in cultured RGCs. 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 CNTF were more significantly effective than it m 10ng/ml and 40ng/ml( P < 0.01 ).Conclusion:CNTF could reduced the rate of apoptotic of cultured RGCs, and CNTF promote RGCs survival by preventing RGCs from apoptosis. The high level of CNTF perhaps has toxicity.