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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 Parkinson’s disease (PD) is a neurodegenerative disease that occurs in the elderly. As the society ages, its prevalence and disability are on the rise. According to epidemiological investigation, neuropathological study and gene level analysis, the etiology and pathological damage of PD are generally considered to be multifactorial, including genetic factors, environmental toxins, oxidative stress, excitotoxicity of amino acids, mitochondrial dysfunction, Apoptosis and decreased levels of neurotrophins. The research on the change of disease molecular level not only provides new information for elucidating the occurrence and development of the disease, but also opens up new avenues for its treatment. However, little is known about the molecular level of PD. Therefore, it is of great significance to reveal the pathological changes of PD. This study aims to construct subtractive cDNA library of differentially expressed genes in striatum of Parkinson’s disease (SD) rats and to screen out some genes that may participate in the pathological process of PD, providing the basis for the gene therapy of PD. METHODS: A rat model of hemiparkinsonism was established by stereotactic injection of 6-OHDA into striatum of SD rats. CDNA fragments of differentially expressed genes were isolated by suppression subtractive hybridization and ligated with T vector to construct a library. After transformation and amplification, white The colonies were identified by colony PCR. The recombinant plasmids were screened by reverse Northern blotting. The sequences were compared with that of Genebank. Results A total of 1 500 white positive clones were obtained by amplification library subtraction. 95 random amplified polymorphic DNA fragments (PCRs) were amplified by PCR, and 100 ~ 600bp insertions were found in 81% of the clones. Twelve differentially expressed genes were screened by reverse Northern blotting. Conclusion SD rat model of unilateral Parkinson’s disease was successfully established by stereotactic technique. The subtracted cDNA library of differentially expressed genes in striatum of rats with unilateral Parkinson’s disease was successfully constructed by SSH and T / A cloning techniques.