新型光敏蛋白PsCatCh2.0治疗视网膜退行性疾病的长期有效性和安全性研究

Long-term effectiveness and safety of new channelrhodopsin PsCatCh2.0 in the treatment of retinal degenerative diseases

目的观察新型光敏蛋白PsCatCh2.0转染至视网膜色素变性模型rd1小鼠视网膜1年后视网膜神经节细胞(RGC)对光反应、视网膜炎症及细胞凋亡情况。方法雄性rd1小鼠24只随机均分为rd1实验组、rd1对照组,各12只。rd1实验组小鼠鼻侧角巩膜缘下1 mm处玻璃体腔注射重组腺相关病毒(rAAV)2/2-巨细胞病毒启动子(CMV)-PsCatCh2.0-增强型绿色荧光蛋白(EGFP)1.5μl,2周后颞侧角巩膜缘下1 mm处再次注射相同剂量的重组病毒。注射后1年,用膜片钳技术记录表达PsCatCh2.0的RGC对光反应;行免疫荧光染色评估PsCatCh2.0在视网膜中的表达情况;行视网膜铺片染色评估重组病毒的转染效率,并计数RGC;使用苏木精-伊红染色评估内层视网膜厚度;采用蛋白免疫印迹法检测视网膜中核因子(NF)-κB p65的蛋白表达;采用实时荧光定量聚合酶链反应检测各组小鼠视网膜中肿瘤坏死因子(TNF)-α、白细胞介素(IL)-6、B淋巴细胞瘤-2相关X蛋白(Bax)mRNA相对表达量;采用原位末端标记法观察各组小鼠视网膜细胞凋亡情况。组间比较采用单因素方差分析。结果注射重组病毒后1年,表达PsCatCh2.0的RGC产生的电流约为30 pA。PsCatCh2.0-EGFP与RGC呈共定位表达;少量与无长突细胞共定位,几乎不与水平细胞、双极细胞共定位。rd1实验组、rd1对照组小鼠RGC密度、内层视网膜厚度比较,差异均无统计学意义(F=14.35、0.05,P>0.05)。rd1实验组小鼠视网膜NF-κB p65蛋白表达量以及TNF-α、IL-6 mRNA表达均低于rd1对照组,差异均有统计学意义(F=4.61、5.91、5.78,P<0.05)。rd1实验组小鼠视网膜中呈红色荧光的凋亡细胞数量少于rd1对照组,Bax mRNA表达低于rd1对照组,差异有统计学意义(F=7.52,P<0.01)。结论玻璃体腔注射rAAV2/2-CMV-PsCatCh2.0-EGFP后1年,表达PsCatCh2.0的RGC仍可产生光电流;长期转染表达PsCatCh2.0对RGC无明显细胞毒性作用,也不增加视网膜的炎性反应。

Objective To explore the light response,retinal inflammation and apoptosis of the retinal ganglion cells(RGCs)1 year after the new type of channelrhodopsin PsCatCh2.0 was transfected into the retina of rd1 mice.Methods Twenty-four male rd1 mice were randomly divided into rd1 experimental group and rd1 control group,12 mice in each group.1.5μl of recombinant adeno-associated virus(rAAV)2/2-cytomegalovirus(CMV)-PsCatCh2.0-enhanced green fluorescent protein(EGFP)was injected into the vitreous cavity 1 mm below the corneoscleral limbus of mice in the rd1 experimental group,and the same dose of recombinant virus was injected 2 weeks later at temporal side 1 mm below the corneoscleral limbus.One year after virus injection,the light response of RGCs expressing PsCatCh2.0 was recorded by patch clamp technique;the expression of PsCatCh2.0 in the retina was evaluated by immunofluorescence staining;the transfection efficiency of recombinant virus was evaluated by the transfection efficiency of virus and the number of RGCs.Hematoxylineosin staining was performed to measure the inner retinal thickness.Western blotting was used to detect the protein expression of nuclear factor(NF)-κB p65 in retina;real-time quantitative polymerase chain reaction was used to detect the relative expression of tumor necrosis factor(TNF)-α,interleukin(IL)-6 and Bax mRNA.Terminal deoxynucleotidyl transferase kit was used to observe the apoptosis of retinal cells in each group of mice.Results One year after the intravitreal injection of recombinant virus,PsCatCh2.0-expressing RGCs can still generate 30 pA photocurrent.The virus PsCatCh2.0-EGFP was mainly transfected into RGCs,and partly transfected into amacrine cells,almost no transfection was seen in bipolar and horizontal cells.There were no significant differences in the number of RGCs and thickness of the inner retina between the rd1 experimental group and the rd1 control group(F=14.35,0.05;P>0.05),while the rd1 experimental group NF-κB p65 protein expression,TNF-αand IL-6 mRNA quantification were significantly lower than those of rd1 control group(F=4.61,5.91,5.78;P<0.05).The number of red fluorescent apoptotic cells in the retina of mice in the rd1 experimental group was less than that in the rd1 control group,and the Bax mRNA expression was lower than that in the rd1 control group,and the difference was statistically significant(F=7.52,P<0.01).Conclusion One year after intravitreal injection of recombinant virus,the PsCatCh2.0 expressing RGCs can still generate photocurrent.Long term transfection and expression of PsCatCh2.0 has no obvious cytotoxic effect on RGCs,nor it increases the inflammatory effect of the retina of rd1 mice with retinal degeneration.