摘要
目的探讨磷酸酶-张力蛋白同源物(PTEN)和细胞因子信号转导抑制蛋白3(SOCS3)缺失对视神经损伤大鼠视网膜神经节细胞(RGCs)存活和轴突再生的影响。方法将Thy1-YFP/PTEN^(F/F)SOCS3^(F/F)大鼠作为对照组,玻璃体内注射腺相关病毒(AAV)介导PTEN基因缺失的Thy1-YFP/PTEN^(F/F)大鼠为PTEN^(-/-)组;玻璃体内注射AAV介导SOCS3基因缺失的Thy1-YFP/SOCS3^(F/F)大鼠为SOCS3^(-/-)组;玻璃体内注射AAV介导PTEN基因和SOCS3基因均缺失的Thy1-YFP/PTEN^(F/F) SOCS3^(F/F)大鼠为PTEN^(-/-)SOCS3^(-/-)组,每组各20只。HE染色观察各组大鼠视网膜组织病理学形态,免疫荧光染色鉴定各组大鼠RGCs存活情况,流式细胞仪检测各组大鼠RGCs凋亡情况,并对比轴突再生数量;利用Western blot检测各组大鼠RGCs中GFAP和GAP-43的蛋白相对表达水平。结果对照组大鼠视网膜细胞炎症反应明显,细胞间层次结构混乱、连接差,分散性强;PTEN^(-/-)组和SOCS3^(-/-)组大鼠视网膜整体结构较规则,层次较清晰,炎症反应有所减轻;PTEN^(-/-)SOCS3^(-/-)组大鼠视网膜结构清晰,较规则,细胞之间连接紧密,炎症反应明显减轻。PTEN^(-/-)组和SOCS^(-/-)组大鼠RGCs存活率均显著高于对照组(均为P<0.001)。PTEN^(-/-)SOCS3^(-/-)组大鼠RGCs存活率均明显高于PTEN^(-/-)组和SOCS3^(-/-)组(均为P<0.001)。PTEN^(-/-)组和SOCS3^(-/-)组大鼠RGCs凋亡率显著低于对照组(均为P<0.001),PTEN^(-/-)SOCS3^(-/-)组大鼠RGCs凋亡率均明显低于PTEN^(-/-)组和SOCS3^(-/-)组(均为P<0.001)。PTEN^(-/-)组和SOCS3^(-/-)组大鼠视神经轴突数目均显著多于对照组(均为P<0.001),PTEN^(-/-)SOCS3^(-/-)组大鼠视神经轴突数目均明显多于PTEN^(-/-)组和SOCS3^(-/-)组(均为P<0.001)。PTEN^(-/-)组和SOCS3^(-/-)组大鼠RGCs中GFAP和GAP-43蛋白表达均显著低于对照组(均为P<0.001),PTEN^(-/-)SOCS3^(-/-)组大鼠RGCs中GFAP和GAP-43蛋白表达均明显低于PTEN^(-/-)组和SOCS3^(-/-)组(均为P<0.001)。结论PTEN和SOCS3基因缺失与视神经损伤大鼠RGCs存活和轴突再生具有一定的关系,不仅能够促进RGCs的增殖,抑制其凋亡,还能够促进视神经轴突再生,同时敲减PTEN和SOCS3基因最为显著。
Objective To investigate the effect of phosphatase and tension homolog(PTEN)/suppressor of cytokine signaling 3(SOCS3)deletion on the survival of retinal ganglion cells(RGCs)and axon regeneration in rats with optic nerve injury.Methods Thy1-YFP/PTEN^(F/F)SOCS3^(F/F) rats were classified into the control group;Thy1-YFP/PTEN^(F/F) rats with PTEN deletion mediated by intravitreal injection of adeno-associated virus(AAV)were classified into the PTEN^(-/-)group;Thy1-YFP/SOCS3^(F/F) rats with SOCS3 deletion mediated by intravitreal injection of AAV were classified into the SOCS3^(-/-)group;and Thy1-YFP/PTEN^(F/F)SOCS3^(F/F) rats with PTEN and SOCS3 deletion mediated by intravitreal injection of AAV were classified into the PTEN^(-/-)SOCS3^(-/-)group,with 20 rats in each group.Hematoxylin and eosin(HE)staining was used to observe the pathological morphology of the rat retina,and immunofluorescence staining was used to identify the survival of rat RGCs.Flow cytometry was used to detect the apoptosis of RGCs in rats and compare the number of regenerated axons.Western blot was used to detect the expression of glial fibrillary acidic protein and growth associated protein-43 in RGCs.Results In the control group,retinal cells showed obvious inflammatory response,chaotic hierarchical structure,poor connection,and strong dispersion.In the PTEN^(-/-)and SOCS3^(-/-)groups,the overall structure of the rat retina was relatively regular with a clear hierarchy,and the inflammatory response was slightly reduced.In the PTEN^(-/-)SOCS3^(-/-)group,the rat retina had a clear and regular structure,cells were closely connected,and the inflammatory response was significantly reduced.The survival rate of RGCs in the PTEN^(-/-)and SOCS3^(-/-)groups was significantly higher than that in the control group(both P<0.001),but lower than that in the PTEN^(-/-)SOCS3^(-/-)group(both P<0.001).The apoptosis rate of RGCs in the PTEN^(-/-)and SOCS3^(-/-)groups was significantly lower than that in the control group(both P<0.001),but higher than that in the PTEN^(-/-)SOCS3^(-/-)group(both P<0.001).The number of optic nerve axons in the PTEN^(-/-)and SOCS3^(-/-)groups was significantly higher than that in the control group(both P<0.001),but lower than that in the PTEN^(-/-)SOCS3^(-/-)group(both P<0.001).The expression levels of glial fibrillary acidic protein and growth associated protein-43 in RGCs in the PTEN^(-/-)and SOCS3^(-/-)groups were significantly lower than those in the control group(all P<0.001),but higher than those in the PTEN^(-/-)SOCS3^(-/-)group(all P<0.001).Conclusion The deletion of PTEN and SOCS3 genes has a certain relationship with the survival of RGCs and axon regeneration in rats with optic nerve injury.It can not only promote the proliferation of RGCs to inhibit their apoptosis,but also accelerate the regeneration of optic nerve axons,while significantly knocking down PTEN and SOCS3 genes.
作者
楚妙
熊朝晖
赵丽娜
李康宁
巨朝娟
CHU Miao;XIONG Zhaohui;ZHAO Lina;LI Kangning;JU Chaojuan(Department of Ophthalmology,the First Hospital of Hebei Medical University,Shijiazhuang 050031,Hebei Province,China)
出处
《眼科新进展》
CAS
北大核心
2021年第11期1016-1021,共6页
Recent Advances in Ophthalmology
基金
河北省医学科学研究课题计划项目(编号20190421)。
关键词
PTEN/SOCS3缺失
视神经损伤
视网膜神经节细胞
细胞存活
轴突再生
phosphatase and tension homolog/suppressor of cytokine signaling 3 deletion
optic nerve injury
retinal ganglion cells
survival
axon regeneration
