AD7c-NTP在AD诊断中的临床及实验研究
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摘要
本研究旨在探寻AD的早期无创性诊断方法。应用ELISA法测定各组中AD7c-NTP含量,诊断AD的敏感性为90.7%,特异性为89.3%,提示AD7c-NTP含量检测作为一种临床工具,对于AD的早期诊断及临床评价具有一定的实用性,肯定了其在AD诊断中的临床参考价值。研究pIRES-AD7c-NTP质粒的构建、表达及其对神经细胞的损伤,应用MTT、流式细胞技术、TUNEL染色、RT-PCR、Western blot等方法,发现AD7c-NTP的过度表达导致PC12细胞发生氧化损伤,体外实验证明AD7c-NTP在AD的发病中起着重要的作用。本实验得出的结论及其意义：1.对比研究AD、VD及MCI患者尿中AD7c-NTP含量变化,对于鉴别诊断AD与VD以及对MCI向AD转化的风险评估具有重要意义；2.构建重组pIRES-AD7c-NTP质粒并转染至PC12细胞内,研究PC12细胞发生的氧化损伤,证明AD7c-NTP的过度表达介导了与AD神经退行性变化有关的细胞凋亡级联反应,进一步证实自由基损伤和氧化应激是AD神经元凋亡的主要原因；3.确定了AD7c-NTP在AD病理变化中的作用,进一步证明.AD7c-NTP检测对于AD诊断的重要意义。同时寻找AD7c-NTP的拮抗物质可能为AD治疗提供新方法。
Alzheimer's disease (AD) is a chronic progressive degenerative disease of the central nervous system, characterized by the impairment of cognition and memory. So far, there are no exact methods for the early diagnosis. When the patients are diagnosed as definite AD, the obvious neuronal damage has occurred. The preventive treatment in the pre-clinical period, i.e. prior to the pathological changes, should be implemented as early as possible. In the recent years, the markers for early diagnosis of AD have been studied intensively.
Objective:To test if the level of AD7c-NTP in urine can be a marker for the diagnosis of AD; To elucidate the role of recombinant plasmid pIRES-AD7c-NTP in the pathogenesis of AD.
Methods:The direct competitive enzyme-linked immunosorbent assay (ELISA) was used to detect urinary AD7c-NTP level for 258 cases of elderly people, including 46 cases of AD, 40 cases of MCI,50 cased of VD,122 cases of aged control (CN). The data were analyzed by SPSS. In the other part of study, the recombinant plasmids pIRES-AD7c-NTP was constructed and studied in PC 12 cell. NOS and SOD activity in damaged cells was checked. Additionally, apoptosis was detected by flow cytometry and TUNEL staining; the mRNA level of apoptosis-related protein bcl-2 and bax expression was measured by RT-PCR; and the expression of AD7c-NTP, Caspase3, NF-κB was analyzed by Western blot.
Results:The level of AD7c-NTP in urine of AD group, MCI group, VD group and CN group was 33.35±1.61,24.85±1.46,18.19±1.41, and 18.30±1.45 ug/mL, respectively. AD7c-NTP level in AD group was significantly higher than the other three groups (P<0.001); and AD7c-NTP level in MCI group was also higher than the CN group and the VD group (P 0.005). The level of AD7c-NTP in 91.4% of AD and 48.7% of MCI cases was increased (> 22 ug/mL), while the level of AD7c-NTP in 91.2% of VD and 91.0% of CN cases was normal(≤22 ug/mL). In addition, we found that recombinant plasmid pIRES-AD7c-NTP was involved in the oxidative damage and apoptosis of PC 12 cells, as demonstrated by increased NOS activity, decreased SOD activity, obvious apoptosis, lower bcl-2 expression, higher bax expression, higher AD7c-NTP and Caspase3 expression, as well as lower NF-κB expression compared with the control group.
Conclusions:(1) Oxidative stress plays an important role in the development of AD, AD7c-NTP could cause intracellular calcium overload by the generating of OH, further activate Caspase-3 and induce apoptosis in PC 12 cells; (2) AD7c-NTP could activate NFκB, trigger Caspase cascade, and finally activate Caspase-3 to cause apoptosis in PC 12 cells; (3) In AD group, AD7c-NTP levels were significantly increased than in CN group, which is related to the severity of the disease; and in MCI group, AD7c-NTP levels were also higher than CN group, suggesting in MCI group, some cases might develop to AD; (4) 22 ug/mL AD7c-NTP in urine could be used as the diagnostic point for the clinical evaluation of AD and MCI patients; (5) As a marker to diagnosis of AD, AD7c-NTP level in urine had the high sensitivity and specificity, which were 90.7% and 89.3%respectively.
Alzheimer's disease (AD) is a chronic progressive degenerative disease of the central nervous system, characterized by the impairment of cognition and memory. So far, there are no exact methods for the early diagnosis. When the patients are diagnosed as definite AD, the obvious neuronal damage has occurred. The preventive treatment in the pre-clinical period, i.e. prior to the pathological changes, should be implemented as early as possible. In the recent years, the markers for early diagnosis of AD have been studied intensively.
Objective:To test if the level of AD7c-NTP in urine can be a marker for the diagnosis of AD; To elucidate the role of recombinant plasmid pIRES-AD7c-NTP in the pathogenesis of AD.
Methods:The direct competitive enzyme-linked immunosorbent assay (ELISA) was used to detect urinary AD7c-NTP level for 258 cases of elderly people, including 46 cases of AD, 40 cases of MCI,50 cased of VD,122 cases of aged control (CN). The data were analyzed by SPSS. In the other part of study, the recombinant plasmids pIRES-AD7c-NTP was constructed and studied in PC 12 cell. NOS and SOD activity in damaged cells was checked. Additionally, apoptosis was detected by flow cytometry and TUNEL staining; the mRNA level of apoptosis-related protein bcl-2 and bax expression was measured by RT-PCR; and the expression of AD7c-NTP, Caspase3, NF-κB was analyzed by Western blot.
Results:The level of AD7c-NTP in urine of AD group, MCI group, VD group and CN group was 33.35±1.61,24.85±1.46,18.19±1.41, and 18.30±1.45 ug/mL, respectively. AD7c-NTP level in AD group was significantly higher than the other three groups (P<0.001); and AD7c-NTP level in MCI group was also higher than the CN group and the VD group (P 0.005). The level of AD7c-NTP in 91.4% of AD and 48.7% of MCI cases was increased (> 22 ug/mL), while the level of AD7c-NTP in 91.2% of VD and 91.0% of CN cases was normal(≤22 ug/mL). In addition, we found that recombinant plasmid pIRES-AD7c-NTP was involved in the oxidative damage and apoptosis of PC 12 cells, as demonstrated by increased NOS activity, decreased SOD activity, obvious apoptosis, lower bcl-2 expression, higher bax expression, higher AD7c-NTP and Caspase3 expression, as well as lower NF-κB expression compared with the control group.
Conclusions:(1) Oxidative stress plays an important role in the development of AD, AD7c-NTP could cause intracellular calcium overload by the generating of OH, further activate Caspase-3 and induce apoptosis in PC 12 cells; (2) AD7c-NTP could activate NFκB, trigger Caspase cascade, and finally activate Caspase-3 to cause apoptosis in PC 12 cells; (3) In AD group, AD7c-NTP levels were significantly increased than in CN group, which is related to the severity of the disease; and in MCI group, AD7c-NTP levels were also higher than CN group, suggesting in MCI group, some cases might develop to AD; (4) 22 ug/mL AD7c-NTP in urine could be used as the diagnostic point for the clinical evaluation of AD and MCI patients; (5) As a marker to diagnosis of AD, AD7c-NTP level in urine had the high sensitivity and specificity, which were 90.7% and 89.3%respectively.
引文
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