皮肤角质形成细胞光老化模型的构建及老化机制的初步研究  

Establishment of skin photoaging model and preliminary mechanism of senescence in keratinocytes cell line HaCaT

作　　者：刘川[1] 黄欣[2] 王萍[1] 潘芸[1] 曹迪[1] 刘依依 陈爱军[1] LIU Chuan;HUANG Xin;WANG Ping;PAN Yun;CAO Di;LIU Yiyi;CHEN Aijun(Department of Dermatology, the First Affiliated Hospital of Chongqing Medical University,Chongqing Medical University, Chongqing, 400016, China;College of Traditional Chinese Medicine, Chongqing Medical University, Chongqing, 400016, China)

机构地区：[1]重庆医科大学附属第一医院皮肤科,重庆400016 [2]重庆医科大学中医药学院,重庆400016

出　　处：《第三军医大学学报》2019年第17期1649-1655,共7页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81573027,81874238);重庆市自然科学基金基础与前沿(一般)项目(CSTC2016jcyjA0430)~~

摘　　要：目的建立有效的皮肤光老化HaCaT细胞模型,初步探索可能涉及的老化相关信号转导途径。方法以30 mJ/cm^2中波紫外线(ultraviolet B,UVB)辐照HaCaT细胞,采用CCK-8检测细胞增殖能力,流式细胞仪分析细胞周期,衰老相关β-半乳糖苷酶(SA-β-gal)染色分析各组阳性细胞百分比,qRT-PCR和Western blot检测衰老相关蛋白p53、p21和p16的表达,细胞免疫荧光检测p21蛋白胞内定位。结果 30 mJ/cm^2 UVB辐照后,HaCaT细胞增殖率均较对照组降低(P<0.01),G2期细胞比例较对照组增加(P<0.05),细胞SA-β-gal染色阳性率明显高于对照组(P<0.01)。qRT-PCR和Western blot检测结果均显示:辐照后,HaCaT细胞中p53、p16表达明显升高, p21表达明显下降(P<0.05,P<0.01)。细胞免疫荧光结果显示:给予30 mJ/cm^2 UVB辐照导致p21发生明显的核移位。结论 30 mJ/cm^2 UVB辐照可成功构建光老化HaCaT细胞模型,其机制可能是通过增强p21核定位及激活p16通路诱导了细胞衰老的进程。Objective To establish an efficient skin photoaging model in HaCaT cells and investigate the possible signaling pathways of photoaging of skin. Methods After HaCaT cells were exposed to UVB at 30 mJ/cm2, cell proliferation and cell cycle was detected by CCK-8 assay and flow cytometry. The percentages of positive cells were detected by SA-β-gal staining. The expression of p53, p21 and p16 at mRNA and protein levels were detected by qRT-PCR and Western blotting respectively. The subcellular localization of p21 was elucidated by immunofluorescence assay. Results Exposure of UVB at 30 mJ/cm2 resulted in significantly decreased proliferation(P<0.01), increased proportion of cells at G2 phage(P<0.05), and more positive cells to SA-β-gal(P<0.05) when compared with normal control cells. The results of qRT-PCR and Western blotting showed that UVB exposure up-regulated the expression levels of p53 and p16, and down-regulated that of p21 in HaCaT cells(P<0.05, P<0.01). Immunofluorescence assay indicated obvious nuclear localization of p21 in the HaCaT cells after UVB exposure. Conclusion UVB-exposure at 30 mJ/cm2 successfully establish skin photoaging model in HaCaT cells, which may be due to senescence induced by increasing nuclear localization of p21 and activating p16-dependent pathway.

关 键 词：光老化 细胞模型 P53 P21 P16 

分 类 号：R-322[医药卫生] R322.991