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

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、中央区、周边区差异有统计学意义(F=31.73、75.06、75.06,P<0.05);4月龄:整个、周边区差异有统计学意义(F=12.27、12.27,P<0.05)。GDNF/CNTF-NSC组,2、4月龄:整个(F=27.26、27.26)、周边区(F=16.01、13.55)差异有统计学意义(P<0.05)。GDNF-NSC组,不同月龄整个、中央区、周边区差异均无统计学意义(F=0.00、0.01、0.02,P>0.05)。结论随年龄增长,CLN7型小鼠表现出渐进性加重的退行性变化,这种变化在光感受器细胞和双极细胞的形态结构及功能上是一致的。基于NSC玻璃体内递送的CNTF和GDNF/CNTF均对光感受器细胞有保护作用,但联合使用GDNF/CNTF并未展现出比各自单独使用时更显著的协同保护效果。GDNF对CLN7型小鼠视网膜的形态和功能均没有治疗效果。

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 decreased,with statistical significance(P<0.05).At the age of 4 and 6 months,the density of retinal cone cells(F=127.30)and b-wave photopic amplitude(F=51.13)in the control group were significantly decreased,and the difference was statistically significant(P<0.05).Immunofluorescence microscopy showed that the NSC transplanted in the experimental group preferentially differentiated into astrocytes,and stably expressed CNTF and GDNF at high levels.Comparison of retinal photoreceptor nucleus lines in different treatment subgroups of the experimental group at different ages:CNTF-NSC group,at 2 months of age:the whole,central and peripheral regions were significantly different(F=31.73,75.06,75.06;P<0.05);4 months of age:The difference between the whole area and the peripheral region was statistically significant(F=12.27,12.27;P<0.05).GDNF/CNTF-NSC group,2 and 4 months of age:the whole(F=27.26,27.26)and the peripheral area(F=16.01,13.55)were significantly different(P<0.05).In GDNF-NSC group,there was no statistical significance at all in the whole,central and peripheral areas at different months of age(F=0.00,0.01,0.02;P>0.05).Conclusions CLN7 neuronal ceroidlipofuscinosis mice exhibit progressively increasing degenerative alterations in photoreceptor cells and bipolar cells with age growing,aligning with both morphological and functional observations.Intravitreal administration of stem cell-based CNTF as well as GDNF/CNTF show therapeutic potential in rescuing photoreceptor cells.Nevertheless,the combined application of GDNF/CNTF-NSC do not demonstrate the anticipated synergistic protective effect.GDNF has no therapeutic effect on the retinal morphology and function in CLN7 neuronal ceroid-lipofuscinosis mice.