光基因技术选择性调控星形胶质细胞对其活化增殖的影响  

The effect of selective optogenetic regulation on astrocyte activation and proliferation

作　　者：魏文洁 苏醒 谢敏杰[1] 王伟[1] 骆翔 喻志源[1] Wei Wenjie;Su Xing;Xie Minjie(Department of Neurology,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030)

机构地区：[1]华中科技大学同济医学院附属同济医院神经内科,武汉430030 [2]同济医院肿瘤科

出　　处：《卒中与神经疾病》2020年第4期425-429,共5页Stroke and Nervous Diseases

基　　金：国家自然科学基因面上项目(NO.81771341);国家自然科学基金青年项目(NO.81600990);湖北省自然科学基金(2017CFB705)。

摘　　要：目的探讨光敏感通道蛋白Channelrhodopsin2 (ChR2)选择性调控星形胶质细胞对其活化增殖的影响。方法离体培养SD大鼠乳鼠皮层星形胶质细胞,采用慢病毒载体Lenti-GFAP-ChR2-YFP感染星形胶质细胞后以470 nm波长的蓝光特定模式进行光刺激激活ChR2通道,观察在光刺激0、3、6、12、24 h后星形胶质细胞细胞周期的变化。结果成功将光敏感通道蛋白ChR2表达在星形胶质细胞中;经光刺激3、6、12、24 h后星形胶质细胞均表现出S期细胞比例增加,且以12及24 h较为显著。结论激活光敏感通道ChR2可促进星形胶质细胞增殖;光基因技术可运用于星形胶质细胞活化增殖的研究,它时空精准的特性使星形胶质细胞作为神经系统疾病治疗靶点具有更高的应用前景。Objective To study the effect of selective optogenetic regulation by Channelrhodopsin2(ChR2)on astrocyte activation and proliferation. Methods Cortical astrocytes were cultured from SD neonate rats and the purity was assessed by immunofluorescence staining of glial fibrillary acidicprotein(GFAP). A lentiviral vector, Lenti-GFAP-ChR2-YFP, was used to transduce culturedastrocytes. ChR2 was expressed selectively under the control of GFAP promoter into astrocytes. By photostimulation using blue light with a wavelength of 470 nm, ChR2 was activated. Cell cycledistribution was assessed by cytometry after 0, 3, 6, 12 and 24 h of stimulation. Results Channelrhodopsin-2 was expressed in astrocytes successfully. Photostimulation of ChR2 inastrocytes led to an increase of percentage of S phase after 0, 3, 6, 12 and 24 h of stimulation, with a very robust increase after 12 and 24 h of stimulation particularly. Conclusion The activation of ChR2 promoted astrocyte proliferation. Optogenetic technology could be applied to the study of astrogliosis. Characterized by precise regulation both temporally and spatially, optogenetics made astrocytes being atherapeutic target in numerous neurological diseases much more promising.

关 键 词：光基因技术 ChR2 星形胶质细胞 细胞周期 增殖 

分 类 号：Q253[生物学—细胞生物学]