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ANTISENSE ATTENUATION OF 5 HT2 RECEPTOR EXPRESSION

5 HT2 受体表达的反义衰减

基本信息

批准号：
2251962
负责人：
DANIEL E BENJAMIN
金额：
$ 7.52万
依托单位：
RUTGERS, THE STATE UNIV OF N.J.
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-08-01 至 1998-07-31
项目状态：
已结题

项目摘要

The central serotonergic system is the target of several successful psychotherapeutic drugs, but progress in its study has frequently been impeded because of a lack of selective drugs. The objective of the proposed research is to develop a technique to selectively attenuate the expression of the serotonin 5-HT2A receptor using an antisense oligonucleotide complementary to specific mRNA's, in cultured cells an in vivo. Antisense oligonucleotides act as specific inhibitors of gene expression by inhibiting transcription and/or the translation of mRNA. Experiment 1 will examine the specificity of antisense treatment in affecting the expression of 5-HT2A receptors in C6 cells, the dose response for inhibition of expression, and the time for resynthesis. Oligonucleotides in the sense, antisense and nonsense configurations will be tested for their ability to selectively attenuate the expression of 5- HT2A receptors in cultured C6 glioma cells. Cells will be incubated in the presence of the oligonucleotides and receptor gene transcription will be measured using an RNase protection assay and radioligand binding will be used to estimate receptor expression. Experiment 2 will examine the in vivo specificity of antisense treatment on 5-HT2A receptors in rat brain. Rats will be infused intraventricularly (using Alzet Minipumps) with the three configurations of oligonucleotides. The dose- and time-response for receptor loss and resynthesis will then be determined in rat brain. 5-HT2A receptor binding will be determined autoradiographically and mRNA levels for the 5-HT2A receptors will be determined by in situ hybridization. To verify the specificity of the antisense treatment, the closely related 5- HT2C receptor will also be measured., Experiment 3 will characterize the physiological expression of 5-HT2C receptor will also be measured. Experiment 3 will characterize the physiological expression of 5-HT2A receptor downregulation. The effects of prior antisense treatment ion the corticosterone response to immobilization stress will be assessed. Animals will be immobilized for 60 min and tail blood obtained for the determination of plasma corticosterone (assessed using radioimmunoassay). To better define the respective roles of the 5-HT2A and 5-HT2C receptors, the effect of 5-HT2A receptor attenuation on behavior int he elevated plus- maze and on behavioral responses to the 5-HT2A receptor attenuation on behavior int he elevated plus-maze and on behavioral responses to the 5- HT2A agonist DOI will be determined. The proposed work will help define the physiological role of the 5-HT2A receptor and provide a basis for the development of more efficacious pharmacotherapies for posttraumatic stress disorder, anxiety, and depression.

中枢神经系统是几个成功的目标，精神治疗药物，但其研究的进展往往是由于缺乏选择性药物而受到阻碍。的目的拟议的研究是开发一种技术，选择性地减弱使用反义寡核苷酸表达5-羟色胺5-HT 2A受体与特定mRNA互补的寡核苷酸，在培养的细胞中， vivo. 反义寡核苷酸作为基因的特异性抑制剂通过抑制mRNA的转录和/或翻译来表达。实验1将检查反义处理在小鼠中的特异性。影响C6细胞中5-HT 2A受体的表达，表达抑制和再合成的时间。有义、反义和无义构型的寡核苷酸将测试它们选择性减弱5-羟色胺表达的能力，培养的C6胶质瘤细胞中的HT 2A受体。细胞将在寡核苷酸和受体基因转录的存在将是使用RNA酶保护测定法测量，放射性配体结合将被用于估计受体表达。实验2将检查在反义治疗对大鼠脑中5-HT 2A受体的体内特异性。将向大鼠心室内输注（使用Alzet微型泵）寡核苷酸的三种构型。剂量-时间-反应然后在大鼠脑中测定受体损失和再合成。 5-HT2a 将通过放射自显影和mRNA水平测定受体结合通过原位杂交测定5-HT 2A受体。到验证反义治疗的特异性，与5- 还将测量HT 2C受体。实验3将描述还将测量5-HT 2C受体的生理表达。实验3将表征5-HT 2A的生理表达受体下调预先反义处理对肿瘤细胞增殖的影响将评估皮质酮对固定应激的反应。动物将被固定60分钟，并获得尾血用于测定血浆皮质酮（使用放射免疫测定法评估）。为了更好地定义5-HT 2A和5-HT 2C受体各自的作用， 5-HT 2A受体减弱对正离子升高时行为的影响迷宫和行为反应的5-HT 2A受体衰减行为在他高架十字迷宫和行为反应的5- 将测定HT 2A激动剂DOI。拟议的工作将有助于确定 5-HT 2A受体的生理作用，并提供了依据，开发更有效的创伤后压力药物疗法失调焦虑和抑郁