经静脉注射的单链9型腺相关病毒介导基因在星形胶质细胞表达  

Single-stranded adeno-associated virus serotype 9 mediated gene expression in astrocyte via intravenous delivery

作　　者：王晓丹 杨钊 沈帆霞 WANG Xiao-dan;YANG Zhao;SHEN Fan-xia(Ruijin Hospital North,Shanghai Jiao Tong University School of Medicine,Shanghai 201801,China;Center for Cerebrovascular Research,Department of Anesthesia and Perioperative Care,University of California,San Francisco,San Francisco 94110,California,USA)

机构地区：[1]上海交通大学医学院附属瑞金医院北院,上海201801 [2]美国加州大学旧金山分校麻醉与围术期医学系脑血管研究中心,美国旧金山94110

出　　处：《上海交通大学学报（医学版）》2020年第8期1036-1040,共5页Journal of Shanghai Jiao tong University:Medical Science

基　　金：国家自然科学基金(81771281,81471177)。

摘　　要：目的·探讨静脉注射含低氧反应元件(hypoxia-responsive element,HRE)启动子的单链9型腺相关病毒(single-stranded adeno-associated virus serotype 9,ssAAV9)能否介导LacZ基因在脑缺血区表达,及其可转染的脑细胞类型。方法·建立永久性左侧远端大脑中动脉闭塞(distal middle cerebral artery occlusion,dMCAO)小鼠模型,模型建立1 d和5 d后检测脑缺血区中低氧诱导因子-1(hypoxia-inducible factor-1,HIF-1)的表达。dMCAO模型建立1 h经颈静脉注射AAV9-H9LacZ载体,注射5 d后X-gal染色检测脑缺血区和肝组织中LacZ基因编码蛋白β-半乳糖苷酶(β-galactosidase,β-gal)的表达,免疫荧光双染色检测β-gal在星形胶质细胞、神经元及血管内皮细胞内的表达。结果·dMCAO模型建立1 d和5 d后小鼠缺血病灶局部HIF-1表达增高。β-gal主要在AAV9-H9LacZ注射小鼠的缺血半暗带区表达,其肝脏组织无β-gal阳性表达。脑缺血区β-gal主要与胶质细胞原纤维酸性蛋白共表达,少量β-gal与神经元特异核蛋白共表达。结论·经静脉注射的ssAAV9主要介导基因在脑缺血区星形胶质细胞表达。HRE能有效控制LacZ基因主要在脑缺血区表达。Objective·To investigate the expression of LacZ gene mediated by intravenous injection of the single-stranded adeno-associated virus serotype 9(ssAAV9)containing hypoxia-responsive element(HRE)promoter in the cerebral ischemic area,and further identify the types of brain cells that can be transfected by the vector.Methods·A mouse model of permanent left distal middle cerebral artery occlusion(dMCAO)was established.The expression of hypoxia-inducible factor-1(HIF-1)in cerebral ischemic area was detected at 1 and 5 days after ischemia.The ssAAV vector containing HRE-regulated LacZ gene was packaged into the capsid of AAV9 virus(AAV9-H9LacZ),and AAV9-H9LacZ was injected into mice through jugular vein 1 h after the establishment of dMCAO model.Five days after injection of AAV9-H9LacZ,X-gal staining was used to detect the expression ofβ-galactosidase(β-gal)encoded by the LacZ gene in the ischemic area and liver tissue.Immunofluorescence double staining was used to detect the expression ofβ-gal in astrocyte,neurons and vascular endothelial cells.Results·The expression of HIF-1 was increased 1 and 5 days after ischemia.β-gal was mainly expressed in ischemic penumbra of mice injected with AAV9-H9LacZ.There was no positive expression ofβ-gal in the liver tissue of mice.β-gal was mainly co-expressed with glial fibrillary acidic protein as an astrocyte-specific marker,and a little ofβ-gal was co-expressed with neuron-specific nuclear protein.Conclusion·After cerebral ischemia,intravenous injection of AAV9-H9LacZ can effectively mediate gene expression in astrocyte in the cerebral ischemia area.HRE can effectively control the expression of the LacZ gene in cerebral ischemia.

关 键 词：9型腺相关病毒 脑缺血 低氧反应元件 星形胶质细胞 

分 类 号：R741[医药卫生—神经病学与精神病学]