Regulation of histamine H1 receptor functions by ribozyme-induced isozyme-specific suppression

组胺H1受体介导外周组织的1型过敏和中枢神经系统的组胺能神经传递。蛋白激酶C （PKC）的激活诱导组胺H1受体脱敏，导致受体信号的抑制。PKC激活还通过基因表达诱导组胺H1受体上调，增强受体信号传导。不同的蛋白激酶C同工酶被认为参与组胺H1受体信号的两种调节机制。为了鉴定PKC同工酶在两种机制中的作用，利用含有核酶cDNA的载体pcDNA3设计了敲除PKC同工酶的特异性核酶。1 Zeo +巨细胞病毒(-)。首先，从PKC-α序列的5′位点（Ribo-PKC-α1 ~ Ribo-PKC-α 5）中选择5个核酶插入物。核酶生成质粒，pcDNA3。1 Zeo+CMV(-)- pkc -α1 ~ pcDNA3。1 Zeo+CMV(-)- pkc -α5，用pcDNA3构建。1 Zeo +巨细胞病毒(-)。用表达U373星形细胞瘤细胞的组胺H1受体转染质粒。高转染率是实现这一目的的必要条件。U373细胞转染率不高。稳定转染获得表达核酶的PKCα敲除细胞系。然而，表达核酶的U373细胞不能敲除PKCα。有效的低毒性转染试剂PolyFect转染试剂对HeLa细胞具有很高的转染率（70-80%）。转染质粒和表达核酶均不能敲除HeLa细胞中的PKCα。然后将质粒通过激活PKC的磷酸酯处理转染到PKC水平极低的HeLa细胞中。在pma处理的HeLa细胞中，核酶对PKCα生成的影响非常有限。总之，由于条件设置的困难和一些未知的原因，PKC敲除的建立没有成功。

Histamine H1 receptors mediate type-1 allergy in peripheral tissues and histaminergic neurotransmission in CNS. Activation of the protein kinase C (PKC) induced histamine H1 receptor desensitization, leading to the suppression of the receptor signaling. PKC activation also induced histamine H1 receptor up-regulation through gene expression, enhancing the receptor signaling. Different protein kinase C isozymes were considered to be involved in the two regulatory mechanisms of histamine H1 receptor signaling. In order to identify PKC isozymes in the two mechanisms, specific ribozyme to knock out each PKC isozyme was designed using a vector containing cDNA for ribozyme, pcDNA3. 1 Zeo+CMV(-). Initially, five ribozyme inserts selected from PKC-α sequences at 5'-site, Ribo-PKC-α1 〜 Ribo-PKCα5 were made. Ribozyme generating plasmids, pcDNA3. 1 Zeo+CMV(-)-PKC-α1 〜 pcDNA3. 1 Zeo+CMV(-)-PKC-α5, were constructed using pcDNA3. 1 Zeo+CMV(-). The plasmid was transfected with histamine H1 receptor expressing U373 astrocytoma cells. High transfection rate was essential for the purpose. The transfection rate to U373 cells, however, was not high. Stable transfection was achieved to obtain the PKCα knock out cell line expressing the ribozyme. U373 cells expressing ribozyme, however, failed in knocking out PKCα. Effective transfection reagent with low toxicity, PolyFect Transfection Reagent, showed very high transfection rate (70-80%) to HeLa cells. Transfection of the plasmid and expression of the ribozyme could not knock out PKCα in HeLa cells. Then the plasmid was transfected to HeLa cells with very low level of PKC by the treatment of PKC-activating phorbol ester. The effect of the ribozyme on PKCα generation was very limited in the PMA-treated HeLa cells. In conclusion, establishment of the PKC knock out was not successful due to the difficulties of condition settings and several unknown reasons.