Visualization and quantification of gene expression involved in pain transmission and regulation with molecular probes

使用分子探针对参与疼痛传递和调节的基因表达进行可视化和定量

• 批准号: 07557010
• 负责人: SATOH Masamichi
• 金额: $ 8.7万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07557010/
• 关键词: in situ hybridization; mRNA; double staining; opioid receptor; pain; digoxigenin; image analysis

For the purpose to increase the sensitivity of an in situ hybridization technique with nonradioactive probes, we examined the several methods for nonradioactive labeling of RNA probes. Simiar sensitivities were obtained between digoxigenin- and fluorescein-labeled antisense RNA probes to detect the target mRNA,but the background signals were lower in the results with digoxigenin-labeled probe than with fluorescein-labeled one. For an analysis of the results of double in situ hybridization histochemistry, we developed an apparatus to simultaneously obtain both bright- and dark-field images, which were transferred to a personal computer and analyzed using a software for image analysis, NIH image. Coexistence of the mu-opioid receptor mRNA with the mRNA for preprotachykinin A (PPTA), a precursor protein of substance P,in the rat dorsal root ganglia was examined by this system, and we could quantitatively analyze the expression of mu-opioid receptor mRNA,which was detected by a ^<35>S-labeled probe, specifically in the cells expressing PPTA mRNA,which was visualized by a digoxigenin-labeled probe. Furthermore, we developed a method to analyze the of film-autoradiogram, and showed that expressions of mu-and kappa-, but not delta-, opioid receptor mRNAs were enhanced in the superfacial layrs of spinal dorsal horn of the arthritic rats.

为了提高非放射性探针原位杂交技术的灵敏度，我们研究了几种非放射性标记RNA探针的方法。地高辛标记的反义RNA探针和荧光素标记的反义RNA探针检测靶mRNA的灵敏度相似，但地高辛标记的探针检测结果的背景信号低于荧光素标记的探针。为了分析双重原位杂交组织化学的结果，我们开发了一种装置，可以同时获得亮场和暗场图像，将其传输到个人计算机上，并使用图像分析软件NIH image进行分析。利用该系统检测了大鼠背根神经节中μ阿片受体mRNA与P物质前体蛋白前原速激肽A（PPTA）mRNA的共存，并可定量分析μ阿片受体mRNA的表达，该表达可通过<35>地高辛标记的探针检测，特别是在表达PPTA mRNA的细胞中。此外，我们开发了一种分析胶片放射自显影的方法，并显示在关节炎大鼠脊髓背角的表层中，μ-和κ-阿片受体mRNA的表达增强，而不是δ-阿片受体mRNA的表达。

项目成果

Yabuuchi,K.: "Induction of interleukin-1β mRNA in the hypothalmus following subcuxtaneous inuectxions of formalin into the rat hind paws." Neuroxsci.Lett.207. 109-112 (1996)

Yabuuchi, K.：“将福尔马林注射到大鼠后爪后，下丘脑中白细胞介素 1β mRNA 的诱导” Neuroxsci.Lett.207 (1996)。

Maekawa, K.: "Expression of μ-and κ-,but not δ-,opioid receptor mRNAs are enhanced in the spinal dorsal horn of the arthritic rats." Pain. 64. 365-371 (1996)

Maekawa, K.：“关节炎大鼠脊髓背角中 μ- 和 κ- 而不是 δ- 的阿片受体 mRNA 表达增强。”64. 365-371 (1996)。

Yabuuchi, K.: "Induction of interleukin-1β mRNA in the hypothalamus following subcutaneous injections of fromalin into the rat hind paws." Neurosci. Lett.207. 109-112 (1996)

Yabuuchi, K.：“向大鼠后爪皮下注射白细胞介素 1β mRNA。Neurosci。109-112。”

Minami, M.: "Double in situ hybridization study on co-existence of μ-,δ-and κ-opioid receptor mRNAs with preprotachykinin A mRNA in the rat dorsal root ganglia." Mol. Brain Res.30. 203-210 (1995)

Minami, M.：“大鼠背根神经节中 μ-、δ- 和 κ-阿片受体 mRNA 与前速激肽 A mRNA 共存的双重原位杂交研究。” 1995)

Minami,M.: "Double in situ hybridization study on co-exsitence of μ-,δ-and κ-opioid receptor mRNAs with preprotachykinin A mRNA in the rat dorsal rootx gxanglia." Mol.Brain Res.30. 203-210 (1995)

Minami, M.：“大鼠背根 gxanglia 中 μ-、δ- 和 κ-阿片受体 mRNA 与前原速激肽 A mRNA 共存的双重原位杂交研究。” 1995)