上海交通大学学报（医学版）

上海交通大学学报(医学版) ›› 2020, Vol. 40 ›› Issue (08): 1036-1040.doi: 10.3969/j.issn.1674-8115.2020.08.006

• 论著·基础研究 • 上一篇    下一篇

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

王晓丹1，杨 钊1，沈帆霞1, 2   

  1. 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，旧金山94110，美国
  • 出版日期:2020-08-28 发布日期:2020-08-28
  • 通讯作者: 沈帆霞，电子信箱：sfx20140602@outlook.com。
  • 作者简介:王晓丹（1983—），女，主治医师，硕士；电子信箱：13405066606@163.com。
  • 基金资助:
    国家自然科学基金（81771281，81471177）。

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

WANG Xiao-dan1, YANG Zhao1, SHEN Fan-xia1, 2   

  1. 1. Ruijin Hospital North, Shanghai Jiao Tong University School of Medicine, Shanghai 201801, China; 2. Center for Cerebrovascular Research, Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco 94110, California, USA
  • Online:2020-08-28 Published:2020-08-28
  • Supported by:
    National Natural Science Foundation of China (81771281, 81471177).

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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。

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

Abstract:

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 of β-galactosidase (β-gal) encoded by the LacZ gene in the ischemic area and liver tissue. Immunofluorescence double staining was used to detect the expression of β-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 of β-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 of β-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.

Key words: adeno-associated virus serotype 9 (AAV9), cerebral ischemia, hypoxia-responsive element (HRE), astrocyte

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