基于干细胞的胶质细胞源性神经营养因子和睫状神经营养因子对CLN7型神经元蜡样脂褐质沉积症小鼠模型视网膜退行性病变的治疗研究  

Therapeutic effect of stem cell-based glial cell derived neurotrophic factor and ciliary neurotrophic factor on retinal degeneration of CLN7 neuronal ceroid-lipofuscinosis mouse model

作　　者：魏莹莹 Udo Bartsch Wei Yingying;Udo Bartsch(Department of Ophthalmology,University Medical Center Hamburg-Eppendorf,Hamburg 100731,Germany;Changsha Xiangjiang Aier Eye Hospital,Changsha 410011,China)

机构地区：[1]汉堡-埃彭多夫大学医学中心眼科,德国汉堡100731 [2]长沙湘江爱尔眼科医院,长沙410011

出　　处：《中华眼底病杂志》2024年第7期526-537,共12页Chinese Journal of Ocular Fundus Diseases

基　　金：国家留学基金委项目(201908340079);爱尔眼科集团内部项目(AMF2301D44)。

摘　　要：目的观察CLN7型神经元蜡样脂褐质沉积症小鼠模型视网膜变性的形态学和功能学改变,以及基于神经干细胞(NSC)的胶质细胞源性神经营养因子(GDNF)和(或)睫状神经营养因子(CNTF)对小鼠光感受器细胞的治疗效果。方法鼠龄14 d的CLN7型小鼠100只随机分为实验组、对照组,分别为80、20只。鼠龄14 d的C57BL/6J小鼠20只设定为野生型组(WT组)。对照组、WT组小鼠不作任何处理,于2、4、6月龄时,采用免疫组织化学染色观察视网膜视锥细胞、视杆-双极细胞、视锥-双极细胞分布和数量变化;采用视网膜电图(ERG)检查记录暗视a、b波及明视b波振幅。实验组小鼠随机选取单侧眼为实验眼,玻璃体腔分别注射2μl CNTF-NSC、GDNF-NSC、CNTF-NSC和GDNF-NSC 1:1细胞混合物(GDNF/CNTFNSC),并据此再分为CNTF-NSC组、GDNF-NSC组、GDNF/CNTF-NSC组;对侧眼玻璃体腔注射不含神经营养因子(NTF)的NSC 2μl作为自身对照组。实验组不同治疗亚组于小鼠2、4月龄时,采用免疫组织化学染色观察光感受器细胞的数量;采用ERG检查记录暗视a、b波及明视b波振幅;小鼠4月龄时,观察移植的NSC分化情况和NTF表达。两组间比较采用双因素方差分析。结果与WT组比较,小鼠2、4、6月龄时,对照组小鼠周边区视网膜视锥细胞密度(F=285.10),中央区、周边区视网膜视杆-双极细胞密度(F=823.20、346.20)、视锥-双极细胞密度(F=356.30、210.60)以及暗视a、b波振幅(F=1911.00、387.10)均显著降低,差异有统计学意义(P<0.05);小鼠4、6月龄时,对照组小鼠中央区视网膜视锥细胞密度(F=127.30)及明视b波振幅(F=51.13)均显著降低,差异有统计学意义(P<0.05)。免疫荧光显微镜观察发现,实验组小鼠玻璃体内移植的NSC优先分化为星形胶质细胞,并稳定高水平表达CNTF和GDNF。实验组不同治疗亚组与其自身对照组在不同月龄时视网膜光感受器细胞核行数比较:CNTF-NSC组,2月龄:整个、中央�Objective To observe the morphological and functional changes of retinal degeneration in mice with CLN7 neuronal ceroid-lipofuscinosis,and the therapeutic effects of glial cell derived neurotrophic factor(GDNF)and/or ciliary neurotrophic factor(CNTF)based on neural stem cells(NSC)on mouse photoreceptor cells.Methods A total of 100 CLN7 mice aged 14 days were randomly divided into the experimental group and the control group,with 80 and 20 mice respectively.Twenty C57BL/6J mice aged 14 days were assigned as wild-type group(WT group).Mice in control group and WT group did not receive any interventions.At 2,4,and 6 months of age,immunohistochemical staining was conducted to examine alterations in the distribution and quantity of cones,rod-bipolar cells,and cone-bipolar cells within the retinal of mice while electroretinography(ERG)examination was utilized to record scotopic a and b-waves and photopic b-wave amplitudes.At 14 days of age,the mice in the experimental group were intravitreally injected with 2μl of CNTFNSC,GDNF-NSC,and a 1:1 cell mixture of CNTF-NSC and GDNF-NSC(GDNF/CNTF-NSC).Those mice were then subdivided into the CNTF-NSC group,the GDNF-NSC group,and the GDNF/CNTF-NSC group accordingly.The contralateral eyes of the mice were injected with 2μl of control NSC without neurotrophic factor(NTF)as their own control group.At 2 and 4 months of age,the rows of photoreceptor cells in mice was observed by immunohistochemical staining while ERG was performed to record amplitudes.At 4 months of age,the differentiation of grafted NSC and the expression of NTF were observed.Statistical comparisons between the groups were performed using a two-way ANOVA.Results Compared with WT group,the density of cones in the peripheral region of the control group at 2,4 and 6 months of age(F=285.10),rod-bipolar cell density in central and peripheral retina(F=823.20,346.20),cone-bipolar cell density(F=356.30,210.60)and the scotopic amplitude of a and b waves(F=1911.00,387.10)in central and peripheral retina were significantly de

关 键 词：神经元蜡样质脂褐质沉积症7型 胶质细胞源性神经营养因子 睫状神经营养因子 慢病毒 神经干细胞 眼内干细胞移植 神经保护 

分 类 号：R774.1[医药卫生—眼科] R-332[医药卫生—临床医学]