评价皮肤刺激性候选蛋白标记物的筛选及蛋白信号转导通路的初步研究

发布时间：2018-03-10 11:49

本文选题：角质细胞　切入点：急性刺激　出处：《暨南大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的：通过蛋白组学技术筛选出用于评价皮肤刺激性的候选生物学标记,并初步探讨相关蛋白的相互作用机制。方法：原代角质细胞暴露于酸碱化合物4h,更换正常培养基,继续培养0、2、4、8和16 h,收集各时间点样品进行检测。应用2-DE/MALDI-TOF/TOF MS的技术筛选出差异表达蛋白,通过Western Blot、Immunofluorescence Staining和real time RT-PCR验证差异表达蛋白在基因和蛋白水平上的表达,以及蛋白在细胞内的定位；检测细胞培养上清中IL-1α的分泌,验证细胞在不同时间点对刺激物的应答强度；最后应用RNAi技术研究HSP27和CFL-1蛋白之间的相关性。结果：(1)化合物刺激后,共有20个蛋白出现有统计学意义的差异表达；(2)酸碱刺激物使角质细胞热休克蛋白27(Heat shock protein 27, HSP27)和铜-锌超氧化物歧化酶1 (Superoxide dismutase[Cu-Zn], SOD1)表达下调,人肌动蛋白素1 (CFL-1, Cofilin-1)表达上调；(3) HSP27、SOD1和CFL1有可能作为评价皮肤刺激性的生物学标记物,其中HSP27的变化最为明显；(4)酸碱刺激物使HSP27出现明显核转位,有助于进一步探讨上述差异蛋白的相关作用机制；(5)酸碱刺激物诱导后,角质细胞IL-1α的分泌峰值出现在撤去刺激物后的0h和8h；(6)干扰HSP27后,CFL-1的表达也随着下调,SDS刺激后,则表达上调。结论：HSP27、SOD1和CFL1可作为评价皮肤刺激性的候选生物学标记；急性刺激能影响角质细胞骨架的改变,HSP27和CFL-1的相互作用参与调节急性刺激反应。
[Abstract]:Aim: to screen candidate biomarkers for evaluating skin irritation by proteomics and to explore the interaction mechanism of related proteins. Methods: the primary keratinocytes were exposed to acid-base compounds for 4 h, then the normal culture medium was changed, and then cultured for 8 and 16 hours. The samples were collected at different time points for detection. The differentially expressed proteins were screened by 2-DE-MALDI-TOF-TOF MS. The expression of differentially expressed proteins at gene and protein levels and the localization of proteins in cells were verified by Western blottison immunofluorescence Staining and real time RT-PCR, and the secretion of IL-1 伪 in the supernatant of cell culture was detected to verify the response intensity of cells to stimuli at different time points. Finally, RNAi technique was used to study the correlation between HSP27 and CFL-1 protein. Results A total of 20 proteins were significantly differentially expressed after stimulation by 1: 1) the heat shock protein 27 shock protein 27 (HSP27) and the copper zinc superoxide dismutase 1 superoxide dismutase [Cu-Zn] (SOD1) were down-regulated by acid-base stimulators in keratinocytes, and the expression of heat shock protein 27 (HSP27) and copper zinc superoxide dismutase 1 (Cu-Zn) were down-regulated in keratinocytes. The expression of human actin 1 / CFL-1 (Cofilin-1) upregulated the expression of HSP27SOD1 and CFL1 may be used as biological markers for evaluating skin irritation, among which the change of HSP27 is the most obvious) the acid-base stimulator can cause obvious nuclear translocation of HSP27. It is helpful to further explore the related mechanism of the above differentially expressed proteins: after the acid-base stimulation, the peak of IL-1 伪 secretion in keratinocytes appears at 0h and 8h after the withdrawal of the stimulator) and interferes with the expression of IL-1 L-1 after HSP27. The expression was up-regulated. Conclusion 1: HSP27SSOD1 and CFL1 can be used as candidate biomarkers for evaluating skin irritation, and acute stimulation can affect the changes of keratinocyte cytoskeleton. The interaction between HSP27 and CFL-1 may play an important role in regulating the acute stimuli response.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

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