Single-cell characterization of endothelin system gene expression in the cerebellum in situ.
Single-cell characterization of endothelin system gene expression in the cerebellum in situ.
复制标题
小脑原位内皮素系统基因表达的单细胞特征。
DOI：
10.1097/00005344-199800001-00102
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发表时间：
1998
影响因子：
3
通讯作者：
Martin Paul
中科院分区：
文献类型：
--
作者：
Kai M. Schmidt;S. Tuschick;F. Kirchhoff;A. Verkhratsky;Lutz Liefeldt;H. Kettenmann;Martin Paul
为了评价内皮素（ET）系统的成分在单个浦肯野神经元和Bergmann胶质细胞原位表达，膜片钳记录结合多重RT-PCR方法。从20至28日龄小鼠中快速分离小脑切片。对细胞进行形态学和电生理学表征，吸出细胞内容物并立即进行逆转录。将cDNA用作多重PCR反应中的模板，所述多重PCR反应含有对ET-1、ET-2和ET-3、ET-转化酶1（ECE-1）和ECE-2以及ETA和ETB受体特异性的引物。将所得PCR产物用作仅含有一对嵌套引物的第二PCR反应中的模板。从阳性细胞中获得了特异性单带，并通过PCR产物的DNA测序证实了这一点。在25个浦肯野神经元中，ECE-1 mRNA和ECE-2 mRNA的阳性率分别为84%和68%。未检测到ET和ETA受体mRNA，仅1个细胞ETB受体mRNA阳性。在Bergmann胶质细胞中，ETB受体mRNA占主导地位。25个细胞中68%为阳性。ETA受体mRNA阳性率为16%，ECE-1 mRNA阳性率为8%，ECE-2 mRNA阳性率为12%。同样，没有检测到ET mRNA。这些结果证实了ETB受体在Bergmann神经胶质细胞中的作用，并为ECE-1和ECE-2在浦肯野神经元中的表达提供了证据。
To evaluate the expression of components of the endothelin (ET) system in single Purkinje neurons and Bergmann glial cells in situ, patch-clamp recording was combined with a multiplex RT-PCR approach. Cerebellar slices were rapidly isolated from 20- to 28-day-old mice. Cells were characterized morphologically and electrophysiologically and cell contents were aspirated and immediately reverse-transcribed. The cDNA was used as a template in a multiplex PCR reaction containing primers specific for ET-1, ET-2, and ET-3, ET-converting enzyme 1 (ECE-1) and ECE-2, and ETA and ETB receptors. The resulting PCR products were used as templates in a second PCR reaction containing only one pair of nested primers. Specific single bands were obtained from positive cells, which was confirmed by DNA sequencing of the PCR products. Of the 25 Purkinje neurons assayed, 84% were positive for ECE-1 mRNA and 68% for ECE-2 mRNA. No ET and ETA receptor mRNAs were detected, and only one cell was positive for ETB receptor mRNA. In Bergmann glial cells, ETB receptor mRNA was predominant. A total of 68% of the 25 cells assayed were positive. Sixteen percent were positive for ETA receptor mRNA, 8% for ECE-1 mRNA, and 12% for ECE-2 mRNA. Again, no ET mRNAs were detected. These results confirm the role of the ETB receptor in Bergmann glial cells and provide evidence for expression of ECE-1 and ECE-2 in Purkinje neurons.