Exposure to an antisense oligonucleotide decreases corticotropin-releasing factor receptor binding in rat pituitary cultures.
Exposure to an antisense oligonucleotide decreases corticotropin-releasing factor receptor binding in rat pituitary cultures.
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暴露于反义寡核苷酸会降低大鼠垂体培养物中促肾上腺皮质激素释放因子受体的结合。
DOI：
10.1046/j.1471-4159.1995.64052358.x
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发表时间：
1995
影响因子：
4.7
通讯作者：
Nemeroff,CB
中科院分区：
文献类型：
--
作者：
Owens,MJ;Mulchahey,JJ;Kasckow,JW;Plotsky,PM;Nemeroff,CB
促肾上腺皮质激素释放因子(CRF)似乎整合了哺乳动物对应激的内分泌、自主神经、免疫和行为反应。为了进一步研究CRF在中枢神经系统中的作用，我们利用原代培养的大鼠垂体前叶细胞，研究了针对CRF受体的“反义基因敲除”策略的有效性。根据大鼠CRF受体起始密码子下游的序列，合成了15聚体反义寡核苷酸(5‘CTG-CGG-GCG-CCG-TCC 3’)和“杂乱”对照(5‘CGT-CCG-CGC-GCT-GCG 3’)。在四个不同的实验中，暴露于10微克分子/L的反义寡核苷酸40-67小时后，125I-绵羊促肾上腺皮质激素释放因子与垂体细胞的结合比对照(无寡核苷酸)或10微克分子/L的“杂乱”寡核苷酸显著减少(17-36%)。此外，与杂乱寡核苷酸相比，10µm ol/L的反义寡核苷酸使CRF受体结合量减少22%，在100pmol/L刺激30分钟后，促肾上腺皮质激素的反应也显著减弱。因此，CRF受体反义寡核苷酸明显降低CRF受体的功能表达。该技术可用于研究CRF受体在中枢神经系统内的产生动力学和生理功能。
Corticotropin‐releasing factor (CRF) appears to integrate the endocrine, autonomic, immunologic, and behavioral responses of mammals to stress. To investigate further the role of CRF in the CNS, we have begun investigating the usefulness of “antisense knockdown” strategies directed against the CRF receptor using rat anterior pituitary gland primary cell cultures. The 15‐mer antisense (5′ CTG‐CGG‐GCG‐CCG‐TCC 3′) and “scrambled” control (5′ CGT‐CCG‐CGC‐GCT‐GCG 3′) oligonucleotides were synthesized based on the rat CRF receptor sequence just downstream of the initiation codon. In each of four separate experiments, exposure to 10 µmol/L of antisense oligonucleotide for 40–67 h resulted in significant (17–36%) decreases in125I‐ovine CRF binding to pituitary cells as compared with either control (no oligonucleotide) or 10 µmol/L of “scrambled” oligonucleotide. Moreover, compared with scrambled oligonucleotide, exposure to 10 µmol/L of antisense oligonucleotide, which produced a 22% decrease in CRF receptor binding, also resulted in a significant attenuation of the adrenocorticotrophic hormone response following a 30‐min challenge with 100 pmol/L of CRF. Thus, CRF receptor antisense oligonucleotides apparently reduce functional expression of CRF receptors. This technique may be useful in studying the kinetics of CRF receptor production and the physiological functions of CRF receptors within the CNS.