睫状上皮营养因子对氧化损伤RGC-5的保护作用

Ciliary Neurotrophic Factor Mediated RGC-5 Survival from Oxidative Damage

目的:探究睫状上皮营养因子(CNTF)对过氧化氢所致视网膜神经节细胞(RGC)-5损伤模型的作用。方法:对处于生长对数期的RGC-5予以不同浓度过氧化氢处理不同时间后,根据细胞存活率选择适当的浓度及时间进行造模。造模成功后,予以CNTF进行干预。成功构建携带CNTF基因的慢病毒载体后,将重组慢病毒载体转染的骨髓间充质干细胞(BMSC)与RGC-5共培养。并且由于BMSC可分泌多种神经营养因子,如脑源性神经营养因子(BDNF)、神经胶质细胞源性神经营养因子(GDNF)。RT-PCR检测共培养3d和7d后CNTF和BDNF、GDNF的含量。最后在两种细胞共培养7d后予以过氧化氢损伤,检测细胞存活率。结果:随着过氧化氢浓度及作用时间的增加,细胞存活率随之降低。当过氧化氢浓度为125μmol/L时,达到凋亡高峰,选择该浓度造模。RGC-5与重组慢病毒载体转染的BMSC共培养后,RT-PCR检测发现CNTF和BDNF在培养7d后表达量增多,有统计学意义,而GDNF培养7d后表达量与对照组无统计学差异。当共培养7d后,过氧化氢损伤后检测发现RGC-5存活率增加。结论:CNTF对过氧化氢损伤的RGC-5有保护作用,并且推测在该过程中BDNF同样也发挥了保护作用。

Objective:To invenstigate whether cilary neurotrophic factor（CNTF）has neuroprotective effect on RGC-5 against oxidative damage induced by H2O2.Methods:RGC-5 on logarithmic phase identified by Brn3 awas culturing with additional H2O2 from 50 to 150μmol/L at four time points respectively to determine an optimal concentration and time point.Then,intervention of CNTF was conducted by RGC-5 co-cultured with BMSCs transfected by recombinant lentiviral plasmid carrying CNTF gene in H2O2-induced model.As BMSCs could secrete several neurotrophic factors such as BDNF and GDNF,mRNA level of BDNF,GDNF and CNTF was detected by RT-PCR after 3 days and 7 days.Finally,RGC-5 was co-cultured with BMSCs for seven days and survival rate was detected after H2O2 damage.Results:Along with prolonged time and increased concentration,H2O2 aggravated RGC-5 injury.125μmol/L of H2O2,as an apoptotic peaky concentration,was chosen to construct cell model.The mRNA level of CNTF and BDNF had statistical significance after seven days compared with control group while GDNF hadn’t.After recombinant lentiviral plasmid was implemented,the data showed CNTF increased cell survival rate a-gainst H2O2.Conclusion:Taken together,the results suggest CNTF plays a positive role against oxidative damage.Meanwhile,BDNF,as a neurotrophic factors may be also have an protective effect on injured cells.