新型光遗传学工具Channelrhodopsin-XXM2.0和PsCatCh2.0恢复视觉功能的可行性分析  

Feasibility analysis of new optogenetics tools Channelrhodopsin-XXM2.0 and Channelrhodopsin-PsCatCh2.0 to restore visual function

作　　者：陈飞[1] 沈吟[1] Chen Fei;Shen Yin(Eye Center,Renmin Hospital of Wuhan University,Wuhan 430060,China)

机构地区：[1]武汉大学人民医院眼科中心,430060

出　　处：《中华眼底病杂志》2020年第11期838-845,共8页Chinese Journal of Ocular Fundus Diseases

基　　金：国家重点研发计划(2017YFE0103400)。

摘　　要：目的探讨新型光遗传学工具Channelrhodopsin-XXM2.0(XXM2.0)、Channelrhodopsin-PsCatCh2.0(PsCatCh2.0)的光敏感性及动力学特征,分析其是否可用于光遗传学视觉功能的恢复。方法通过分子生物学技术将XXM2.0和PsCatCh2.0的基因片段连接到包含抗氨苄青霉素筛选基因和报告基因的载体pCIG(c)-msFoxn3上,形成新质粒pCIG(c)-msFoxn3-XXM2.0、pCIG(c)-msFoxn3-PsCatCh2.0。将构建的新质粒转染到HEK 293T细胞上,用HEKA膜片钳系统行全细胞模式记录对光反应。在2.7×10^16、4.7×10^15、6.4×10^14 photons/(cm2·s)的光强下记录电流大小;在2.7×10^16 photons/(cm2·s)光强下刺激XXM2.0、PsCatCh2.0并让其充分恢复,在Clampfit 10.6软件中分析开放和关闭时间常数;在相同光强下,设置2~32 Hz的光脉冲刺激,记录XXM2.0、PsCatCh2.0的响应情况,并在重复光刺激后间隔4000 ms和200 ms来分析电流衰减情况。组间比较均采用独立样本t检验。结果在XXM2.0、PsCatCh2.0序列两端引入限制性内切酶位点EcoRⅠ和EcoRⅤ,酶切后通过T4 DNA连接酶成功构建新质粒pCIG(c)-msFoxn3-XXM2.0和pCIG(c)-msFoxn3-PsCatCh2.0,并转染表达在HEK 293T细胞上。XXM2.0和PsCatCh2.0均表现出光强依赖性,光强越大电流越大。在视网膜安全阈值内的光强6.4×10^14 photons/(cm2·s)刺激下,XXM2.0和PsCatCh2.0仍产生较大电流,分别为(92.8±142.0)、(13.9±5.6)pA;两者比较,差异无统计学意义(t=1.24、1.24,P=0.28、0.29)。XXM2.0、PsCatCh2.0的开放时间常数分别为(23.9±6.7)、(2.4±0.8)ms,关闭时间常数分别为(5803.0±568.2)、(219.9±25.6)ms;两者比较,XXM2.0开放时间常数、关闭时间常数均较PsCatCh2.0大,差异均有统计学意义(t=7.10、31.60,P=0.00、0.00)。在响应频率方面,XXM2.0和PsCatCh2.0均能很好地响应32 Hz的高频率脉冲光刺激,并在较长时间(4000 ms)和较短时间(200 ms)间隔的重复光刺激后均保持较小的电流衰减率。结论XXM2.0和PsCatCh2.0均可在对视网膜安全的光Objective To explore the light sensitivity and kinetic of the new optogenetics tools Channelrhodopsin-XXM2.0 (XXM2.0) and Channelrhodopsin-PsCatCh2.0 (PsCatCh2.0), and analyze whether they could be used to restore the visual function by optogenetics.Methods Molecular biology techniques were used to link the gene fragments of XXM2.0 and PsCatCh2.0 to the vector pCIG(c)-msFoxn3 containing ampicillin resistant screening gene and reporter gene to form new plasmid pCIG(c)-msFoxn3-XXM2.0 and pCIG(c)-msFoxn3-PsCatCh2.0. The constructed plasmids were transfected into HEK 293T cells, and light responses were recorded in the whole cell mode with the HEKA patch clamp system. The photocurrent was recorded under three light intensity included 2.7×10^16, 4.7×10^15, and 6.4×10^14 photons/(cm2·s). And then, XXM2.0 and PsCatCh2.0 were stimulated with 2.7×10^16 photons/(cm2·s) and fully recovered. The opening and closing time constants were analyzed with Clampfit 10.6 software. At the same light intensity, photocurrents of XXM2.0 and PsCatCh2.0 were recorded by the light pulse stimulating of 2-32 Hz. The current attenuation was analyzed at long intervals of 4000 ms and short intervals of 200 ms after repeated stimulation. Comparisons between groups were performed by independent samples t test.Results Restriction endonuclease sites of EcoRⅠ and EcoRⅤ were successfully introduced at XXM2.0 and PsCatCh2.0 sequences. When the digestion was completed, they were ligated by T4 DNA ligase to construct new plasmids pCIG(c)-msFoxn3-XXM2.0 and pCIG (c)-msFoxn3-PsCatCh2.0, and then transfected on HEK 293T cells. The light intensity dependence was showed in XXM2.0 and PsCatCh2.0. The greater light intensity was accompanied by the greater photocurrent. Under the light intensity 6.4×10^14 photons/(cm2·s) below the retinal safety threshold, large photocurrent was still generated in XXM2.0 and PsCatCh2.0 with 92.8±142.0 and 13.9±5.6 pA (t=1.24, 1.24;P=0.28, 0.29). The opening time constants of XXM2.0 and PsCatCh2.0 were 23.9±6.7 and

关 键 词：视觉 眼 光遗传学 Channelrhodopsin-XXM2.0 Channelrhodopsin-PsCatCh2.0 

分 类 号：R774[医药卫生—眼科]