SD大鼠偏侧帕金森病模型差异表达cDNA文库的构建(英文)

Construction of differentially expressed cDNA library of rat brain in Parkinson′s disease

背景帕金森病（Parkinson＇s disease，PD）是好发于中老年人的神经退行性疾病，随着社会的老龄化，其患病率及致残率呈上升趋势。根据流行病学调查，神经病理学研究及基因水平分析，一般认为PD的病因及其病理损伤过程是多因素的，包括遗传因素、环境毒素、氧化应激、兴奋性氨基酸毒性作用、线粒体功能障碍、细胞凋亡和神经营养因子水平降低等。对疾病分子水平变化的研究，不但为阐明疾病的发生发展提供新的资料，而且还能为其治疗开辟新的途径。然而目前对PD分子水平所知尚少，因此，揭示PD分子病理变化，具有十分重要的意义。本研究旨在构建偏侧帕金森病SD大鼠纹状体组织差异表达基因消减cDNA文库，并筛选出一些可能参与PD病理损伤过程的基因，为PD的基因治疗提供依据。方法行SD大鼠纹状体区立体定向注射6-OHDA制备偏侧帕金森病模型；用抑制消减杂交方法分离差异表达基因的cDNA片段，连接T载体构建文库，转化扩增后随机挑取白色克隆行菌落PCR鉴定；利用反Northern杂交对重组质粒进行筛选；通过序列测定后与Genebank进行同源性比较。结果消减文库扩增获得1500余个白色阳性克隆，随机挑取95个行PCR扩增，81％的克隆中有100～600bp的插入片段，经反Northern杂交筛选出12个差异表达基因。结论利用立体定向技术成功制备偏侧帕金森病SD大鼠模型；利用SSH法及T／A克隆技术成功构建偏侧帕金森病大鼠纹状体组织差异表达基因消减cDNA文库。

Background To construct and analyze the differentially expressed gene library in the SD rat＇s brain of Parkinson＇s disease. Methods The lateralization Parkinson＇s disease model of SD rat was established by stereotaxis injection of 6- OHDA in lateralization corpora striatum. Suppression subtractive hybridization was utilized to isolate the cDNA fragments in both control and experimental groups. Then the cDNA fragments were directly bound to pMDIS-T to set up the subtractive library. Amplification of the library was carried out by transformation of E. coli with high voltage electroperforation. Ninety five positive bacteria clones were randomly picked up and identified by colony PCR. Results The amplified library contained more than 1500 positive bacteria clones. Ninety-five clones were analyzed randomly by colony PCR. And there were 81% clones contained 100 -600 bp DNA fragments. They might be the cDNA fragments of differentially expressed genes of Parkinson＇s disease. Conclusions A differentially expressed genes subtracted cDNA library of Parkinson＇s disease of SD rat was constructed successfully with SSH and T/A cloning techniques. The library is efficient and it was useful in screening and cloning new and specific differential expression genes of Parkinson＇s disease.