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Molecular cloning and characterization of a novel sequences with enhanced expression in permanent ischemic rat orain

在永久性缺血大鼠口腔中表达增强的新序列的分子克隆和表征

基本信息

批准号：
12470194
负责人：
TOHDA Michihisa
金额：
$ 2.75万
依托单位：
TOYAMA MEDICAL AND PHARMACEUTICAL UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

To clarify the molecular mechanisms of generation of dementia caused by chronic cerebral hypoperfusion, some kinds of intrinsic factors which changed the mRNA expression were cloned by differential display from the rat brain treated by permanent occlusion of bilateral common carotide arteries (2VO). Among them, further detail studies were done for two novel factors which expressions were enhanced by 2VO. One was isolated as a factor that the expression was enhanced at 4 days after 2VO, and named vof-21 (accession No. AB089205). Whole sequence of vof-21, 4,885 nt, was clarified. The eatimated amino acid sequence was 265 residues. The expression was shown a peak at 7 days after 2VO, and thendecreased to the sham level at 35 days. The expression was abundant in brain and lung followed by in thymus. The results of in situ hybridization revealed that the expressions in brain were rich in locus coreuleus, pontine nucleus and hippocampus. Another one was enhanced the expression at 4 months af … More ter 2VO and called vof-16 (accession No. AB089206). The whole sequence was 2,098 nt and the eatimated amino acid sequence was 73 residues. The vof-16 was abundant in the hippocampus, the tenia tecta, the piriform cortex and the area around the aorta. The expression was abundant in the hippocampus of rats with cognitive impairment induced by 2VO. In contrast, the expression levels of vof-16 were lower in the 2VO rats with no impairment and in sham-operated rats. The RT/PCR show that both vof-16 and vof-21 mRNA also existed in NG108-15 cells. To inhibit the expression by delivering the antisense DNA into the cell, the conditions of pH-sensitive liposome methods were established and reported. The functional sygnificance of vof-21 was still studying by using of this liposome system especially on Ca^<2+> and other ion channel and/or mobility. New methods for these kinds of experiment were also developed and reported ; the improved methods of 5'-RASE and determination methods of sense sequence based on RT/PCR. Involvement of 5-HT2C receptor and the variant on neuronal differentiation of NG108-15 cells was also studied by this grant. Less

为阐明慢性脑低灌注性痴呆的分子机制，采用差异显示技术，从永久性结扎双侧颈总动脉(2VO)的大鼠脑内克隆了几种改变其基因表达的内源性因子。其中，对2VO增强表达的两个新因子进行了进一步的详细研究。其中1株为2VO后4d表达增强因子，命名为VOF-21(登录号：VOF-21)。AB089205)。VOF-21的全序列为4885个核苷酸。氨基酸序列为265个残基。2VO后7d达高峰，35d降至假手术水平。该基因在脑和肺中大量表达，在胸腺中次之。原位杂交结果显示，脑内核仁组成区、桥核和海马区表达丰富。另一组在…后4个月时表达增强更多2VO，称为VOF-16(登录号：AB089206)。全长2,098个核苷酸，氨基酸序列为73个残基。VOF-16在海马区、顶盖延髓、梨状皮质和主动脉周围区域表达丰富。在2VO致认知障碍大鼠的海马区有丰富的表达。而未损伤的2VO大鼠和假手术大鼠的VOF-16表达水平较低。RT/PCR结果表明，NG108-15细胞中同时存在vof-16和vof-21基因的表达。为了通过将反义DNA导入细胞来抑制其表达，建立并报道了pH敏感的脂质体方法的条件。VOF-21的功能意义仍在研究中，尤其是对Ca~(2+)、Lt~(2+)、Gt~(2+)等离子通道和/或流动性的影响。并报道了这类实验的新方法：改进的5‘-rase方法和基于RT/PCR的正义序列测定方法。本研究还研究了5-HT2C受体及其变异体对NG108-15细胞神经分化的影响。较少