人端粒酶反转录酶对人胚胎皮质神经元损伤的保护  

Human telomerase reverse transcriptase protects human embryonic cortical neurons

作　　者：魏桂芬[1] 刘妍[2] 李艳玲[2] 张惠爱[2] 孔令平[2] 

机构地区：[1]广州医学院从化学院病原生物学教研室,广东省从化市510925 [2]广州医学院从化学院解剖学与组织胚胎学教研室,广东省从化市510925

出　　处：《中国组织工程研究》2013年第28期5229-5235,共7页Chinese Journal of Tissue Engineering Research

基　　金：广东省教育厅科技创新项目(2012KJCX0089);广东省医学科研基金项目(A2010222)~~

摘　　要：背景:端粒酶可维持端粒长度,避免细胞复制性衰老和凋亡,其催化亚基端粒酶反转录酶还具有抗凋亡和调节细胞生存的作用。目的:观察人端粒酶反转录酶对β淀粉样蛋白1-40引起的人胚胎皮质神经元损伤的影响。方法:分离和培养12-16周龄人胚胎皮质神经元,将人端粒酶反转录酶基因重组腺病毒转染至神经元。免疫细胞化学法检测人端粒酶反转录酶基的表达,端粒重复序列扩增酶联免疫吸附法检测端粒酶活性。转染后第3天,给予10μmol/Lβ淀粉样蛋白1-40作用24h后,应用MTT检测细胞活力。荧光探针2’7’-二乙酰二氯荧光素标记检测细胞内活性氧水平,比色法测定细胞匀浆中谷胱甘肽含量。结果与结论:转染后第3天,人端粒酶反转录酶的表达最高,并重建了其端粒酶活性;10μmol/Lβ淀粉样蛋白1-40显著降低神经元的细胞活力和谷胱甘肽的含量(P<0.05和P<0.01),升高活性氧水平(P<0.05)。转染了人端粒酶反转录酶基因的神经元能显著对抗β淀粉样蛋白1-40的毒性作用,增加细胞的活力和谷胱甘肽含量(P<0.05和P<0.01),降低活性氧水平(P<0.05)。结果表明,人端粒酶反转录酶对β淀粉样蛋白1-40引起的人胚胎皮质神经元的损伤有明显保护作用。METHODS： Human cortical neurons derived from 12-16 weeks old aborted fetuses were transfected with recombinant adenovirus vector encoding human telomerase reverse transcriptase. Expression of human telomerase reverse transcriptase was evaluated by immunocytochemical staining. Telomerase activity was measured using a PCR-based telomeric repeat amplification protocol enzyme-linked immunosorbent assay kit. Human embryonic cortical neurons were treated with 10 umol/L ol/L amyloid β1-40 after transfected for 3 days. Cell viability, reactive oxygen species levels and glutathione contents in human embryonic cortical neurons were respectively detected by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide assay, fluorescent probe 2＇,7＇-dichlorodihydrofluorescein diacetate and chromatometry. RESULTS AND CONCLUSION： Expression of human telomerase reverse transcriptase reached peak at 3 days after transfection, and the telomerase activity was rebuilt; 10 umol/L amyloid β1-40 could significantly reduce the cell viability of neurons and glutathione content （P 〈 0.05 and P 〈 0.01 ）, and increase the reactive oxygen species levels （P 〈 0.05）. The neurons transfected with human telomerase reverse transcriptase gene could be significantly against the toxicity of amyloid β1-40 and increase the cell viability and glutathione content （P 〈 0.05and P 〈 0.01 ）, and decrease the reactive oxygen species levels （P 〈 0.05）. The results indicate that human telomerase reverse transcriptase can protect amyloid 131 40-induced human embryonic cortical neurons injury

关 键 词：组织构建 组织构建细胞学实验 端粒酶 人端粒酶反转录酶 皮质神经元 13淀粉样蛋白 阿尔茨海默病 省级基金 

分 类 号：R318[医药卫生—生物医学工程]