Retrovirus-mediated transfer of an angiotensin type I receptor (AT1-R) antisense sequence decreases AT1-Rs and angiotensin II action in astroglial and neuronal cells in primary cultures from the brain.
Retrovirus-mediated transfer of an angiotensin type I receptor (AT1-R) antisense sequence decreases AT1-Rs and angiotensin II action in astroglial and neuronal cells in primary cultures from the brain.
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DOI：
10.1073/pnas.92.4.1162
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发表时间：
1995-02
影响因子：
11.1
通讯作者：
D. Lu;K. Yu;M. Raizada
中科院分区：
文献类型：
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作者：
D. Lu;K. Yu;M. Raizada
AT 1-R参与了血管紧张素II（AII）在脑中的许多细胞和生理作用。构建了含有AT 1B-R反义序列（AT 1B-AS）的逆转录病毒载体（LNSV）（简称LNSV-AT 1B-AS），并用于确定利用病毒介导的基因转移来控制大鼠脑星形胶质细胞和神经元细胞中AT 1-R和AII作用的可行性。简言之，将对应于AT 1-R cDNA的nt-132至+1128的1.26-kb反义序列克隆到LNSV载体中，将该载体转染到PA 317细胞中，并在G418中选择转染的细胞。将脑细胞与含有LNSV-AT 1B-AS病毒颗粒的培养基一起孵育，表明AT 1B-AS整合到基因组中并在脑细胞中转录。这与AT 1-R的显著减少和AII刺激的c-fos mRNA的增加有关，这是AT 1-R功能的一个指标。这些观察结果表明，AT 1B-AS基因可以转移到培养的星形胶质细胞中的LNSV，这种转移抑制AT 1-Rs和AII刺激的细胞活性。此外，这种方法的有用性，以研究AII依赖的病理生理学在原代神经元培养，特别是从大脑，是建立。
The AT1-R has been implicated in many cellular and physiological actions of angiotensin II (AII) in the brain. A retrovirus vector (LNSV) containing an AT1B-R antisense sequence (AT1B-AS) (termed LNSV-AT1B-AS) was constructed and used to determine the feasibility of using viral-mediated gene transfer to control AT1-Rs and AII actions in astroglial and neuronal cells in primary cultures from rat brain. Briefly, a 1.26-kb antisense sequence corresponding to nt -132 to +1128 of AT1-R cDNA was cloned into the LNSV vector, the vector was transfected into PA317 cells, and transfected cells were selected in G418. Incubation of brain cells with culture medium containing LNSV-AT1B-AS viral particles showed that AT1B-AS was integrated into the genome and transcribed in brain cells. This was associated with a significant decrease in AT1-Rs and in the AII-stimulated increase of c-fos mRNA, a measure of AT1-R function. These observations show that the AT1B-AS gene can be transferred into astroglial cells in culture by LNSV and that such a transfer inhibits AT1-Rs and the AII stimulation of cellular activities. In addition, the usefulness of this approach to study AII-dependent pathophysiology in primary neuronal cultures from brain, in particular, is established.