摘要
目的建立荧光定量PCR方法检测Alzheimer病(AD)患者外周血中淀粉样蛋白前体 (APP)的 mRNA水平,并探讨该基因在AD患者外周血中的表达及意义。方法根据APP的基因序列,设计并合成引物和荧光标记探针。将PCR扩增目的片段用AT克隆的方法克隆入T载体,重组质粒经筛选、鉴定后,作为阳性模板,用于标准曲线的制定和样品检测。用该方法检测30例AD患者和 23例正常老年人对照组外周血中APP基因的mRNA水平。结果应用重组质粒制作的定量曲线循环阈值与模板浓度具有良好的线性关系。AD组APP基因平均表达水平为(2．54×105±1．53×105) copies/μgRNA,高于对照组(6．03×104±7．58×105)copies／μgRNA(P<0．001)。结论荧光定量PCR检测AD患者APP mRNA水平的方法较常规PCR技术更为简便、快速、准确。用该法测得APP在AD患者外周血中的mRNA水平有所增加。
Objective To establish fluorogenic quantitative PCR method for detecting amyloid precursor protein（APP） mRNA levels in the peripheral blood of Alzheimer＇ s disease, and observe the gene expression and significance of APP. Methods According to the specific sequence of APP genes, the primers and the fluorogenic probe were designed and synthesized. The fragments generated from PCR as templates were cloned into the T vector with the methord of AT clone. The positive recombinant plasmid would be used as standard quantitative template to make the standard curve for sample detection to detect APP mRNA levels in peripheral blood between 30 cases of patients with AD and 23 normal elder controls. Results The standard curve indicated the linear relationship between CT （cycle threshold） and template concentration. The expression levers of APP was （（2.54×10^5±1.53×10^5）copies/μgRNA RNA in AD patients, it higher than that in controls （6.03×10^4±7.58×10^5）copies/μgRNA RNA（P〈0.001）. Conclusion The methord of using fluorogenic quantitative PCR to detect the mRNA levels of APP in AD patients was constructed. The fluorogenic quantitative PCR is more simple and accurate than common PCR. The mRNA levers of APP in peripheral blood of the AD patients was increased.
出处
《中华神经医学杂志》
CAS
CSCD
2006年第4期325-328,341,共5页
Chinese Journal of Neuromedicine
基金
国家计划委员会项目(国经贸委1998-345)
国家自然 科学基金(30470904)
广东省自然科学基金(010698)
广州市科技攻关重点项目(2002Z2-E0112)
关键词
阿尔茨海默病
淀粉样蛋白前体
荧光定量PCR
基因表达
Alzheimer＇s disease
Amyloid precursor protein
Fluorogenic quantitative PCR
Gene expression
