诱导型衰老细胞模型的建立及Klotho启动子截短型突变体的构建和活性检测

发布时间：2018-11-18 16:55

【摘要】：目的:建立诱导型衰老细胞模型并检测抗衰老相关的Klotho基因启动子的不同截短型突变体的活性,为下一步研究奠定基础.方法:原代的人脐带静脉内皮细胞(HUVEC)经过流式细胞术分离鉴定后,通过设置不同过氧化氢的浓度,按时间梯度摸索诱导原代HUVEC细胞衰老的条件并最终通过SA-β-gal染色及定量PCR技术来确定其诱导条件.以全长的Klotho启动子为模版,通过PCR的方法,获得不同长度的Klotho启动子截短型突变体,并通过双萤光素酶报告基因系统检测试剂盒检测不同的启动子截短型突变体在诱导型衰老细胞内的激活程度.结果:培养基中过氧化氢浓度为600μM,处理96 h后,成功构建了诱导型衰老细胞模型;通过PCR的方法成功构建了长度分别为1 711 bp、1 262 bp、785 bp和735 bp的4个长度截短型突变体,并发现735 bp的截短型突变体在诱导型衰老细胞内的激活程度存在明显差异.结论:成功建立了诱导型衰老细胞模型,并成功构建了Klotho基因的启动子截短型突变体,检测和发现这些突变子在诱导型衰老细胞内的激活程度存在明显差异,为下一步治疗衰老相关疾病奠定了良好的工作基础.
[Abstract]:Aim: to establish an induced senescence cell model and to detect the activity of different truncated mutants of anti-aging related Klotho gene promoters in order to lay a foundation for further research. Methods: the primary human umbilical vein endothelial cells (HUVEC) were isolated and identified by flow cytometry. The conditions for inducing senescence of primary HUVEC cells were explored according to the time gradient, and the inducing conditions were determined by SA- 尾-gal staining and quantitative PCR technique. Using the full-length Klotho promoter as template, the truncated mutants of Klotho promoter with different lengths were obtained by PCR. The activation degree of different truncated promoter mutants in induction senescence cells was detected by double luciferase reporter gene system. Results: the induced senescence cell model was successfully established after 96 h treatment with the concentration of hydrogen peroxide 600 渭 m in the medium. Four truncated mutants with the lengths of 1 711 bp,1 262 bp,785 bp and 735 bp were successfully constructed by PCR, and the activation of 735 bp truncated mutants in induced senescence cells was found to be significantly different. Conclusion: the induced senescence cell model was successfully established, and the truncated mutants of Klotho gene promoter were successfully constructed. For the next step in the treatment of aging related diseases laid a good foundation for the work.
【作者单位】：暨南大学生物医药研究院暨南大学细胞生物学系基因工程药物国家工程研究中心广东省生物工程药物重点实验室广东省生物技术药物工程技术研究中心;
【基金】：国家自然科学基金项目(8147313) 广州市科技计划项目(2016201604030039)
【分类号】：R339.38

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