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

为了阐明慢性脑灌注不足引起痴呆的分子机制，我们从双侧颈总动脉（2VO）永久性闭塞后的大鼠脑中克隆了一些改变mRNA表达的内在因子。其中，对两个被2VO增强表达的新因子进行了进一步的详细研究。其中一个作为2VO后4天表达增强的因子被分离出来，命名为vof-21（加入号：vof-21）。AB089205)。vof- 21,4,885 nt的全序列得到了澄清。估计的氨基酸序列为265个残基。2VO后第7天表达量达到高峰，第35天表达量降至假手术水平。在脑和肺中表达丰富，其次是胸腺。原位杂交结果显示，该基因在脑核座、脑桥核和海马中表达丰富。另一株在2VO后4个月表达增强，称为vof-16（加入号：vof-16）。AB089206)。整个序列长2098 nt，估计的氨基酸序列有73个残基。vof-16在海马、裂谷、梨状皮质和主动脉周围区域大量表达。在2VO诱导的认知损伤大鼠海马中表达丰富。相比之下，vof-16在无损伤的2VO大鼠和假手术大鼠中表达水平较低。RT/PCR结果显示，NG108-15细胞中同时存在vof-16和vof-21 mRNA。为了通过将反义DNA送入细胞抑制其表达，我们建立并报道了ph敏感脂质体方法的条件。利用该脂质体系统研究vof-21的功能意义，特别是对Ca^<2+>等离子通道和/或迁移的影响仍在研究中。研究并报道了这类实验的新方法；改进的5′-RASE方法和基于RT/PCR的义序列测定方法。研究了5-HT2C受体及其变异对NG108-15细胞神经元分化的影响。少

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