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型变态反应和中枢神经系统的组胺能神经传递。蛋白激酶C(PKC)的激活导致组胺H1受体失敏，导致受体信号的抑制。PKC激活还通过基因表达诱导组胺H1受体表达上调，增强受体信号转导。不同的蛋白激酶C同工酶被认为参与了组胺H1受体信号的两种调节机制。为了鉴定这两种机制中的PKC同工酶，利用含有核酶基因的载体pcDNA3设计了敲除每一种PKC同工酶的特异性核酶。1Zeo+CMV(-)。首先，从5‘端PKC-α序列中筛选出5个核酶插入片段，分别为Ribo-PKC-α1~Ribo-PKCα5。核酶产生载体，pcDNA3。1Zeo+Cmv(-)-PKC-α1~pcDNA3.1ZEO+CMV(-)-PKC-α5。1Zeo+CMV(-)。将组胺H1受体基因导入U373星形细胞瘤细胞。为了达到这一目的，高转染率是必不可少的。然而，对U373细胞的转染率并不高。获得稳定表达核酶的PKCα敲除细胞系。然而，表达核酶的U373细胞未能敲除PKCα。低毒的高效转染剂PolyFect转染剂对HeLa细胞有很高的转染率(70-80%)。在HeLa细胞中不能将pKCα基因敲除。然后用PKC激活的佛波酯将其导入到PKC水平很低的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淘汰赛的建立并不成功。

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.