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

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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

基础医学 | 更新时间：2026-01-09

- 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
- 第三军医大学学报 2019年41卷第17期页码：1649-1655
- 作者机构：
  
  1.400016 重庆，重庆医科大学:附属第一医院皮肤科
  2.400016 重庆，重庆医科大学:中医药学院
- 作者简介：
  
  [ 陈爱军，E-mail:1360999716@qq.com CHEN Aijun, E-mail:1360999716@qq.com
- 基金信息：
  
  国家自然科学基金面上项目(81573027);国家自然科学基金面上项目(81874238);重庆市自然科学基金基础与前沿(一般)项目(CSTC2016jcyjA0430)
- DOI：10.16016/j.1000-5404.201903210
  中图分类号： R-332;R322.991;R363.122
- 收稿：2019-03-25，
  
  修回：2019-05-30，
  
  纸质出版：2019-09-15
- 稿件说明：
移动端阅览
刘川, 黄欣, 王萍, 等. 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[J]. 第三军医大学学报, 2019,41(17):1649-1655.

Chuan LIU, Xin HUANG, Ping WANG, et al. Establishment of skin photoaging model and preliminary mechanism of senescence in keratinocytes cell line HaCaT[J]. Journal of Third Military Medical University, 2019, 41(17): 1649-1655.
刘川, 黄欣, 王萍, 等. 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[J]. 第三军医大学学报, 2019,41(17):1649-1655. DOI： 10.16016/j.1000-5404.201903210.

Chuan LIU, Xin HUANG, Ping WANG, et al. Establishment of skin photoaging model and preliminary mechanism of senescence in keratinocytes cell line HaCaT[J]. Journal of Third Military Medical University, 2019, 41(17): 1649-1655. DOI： 10.16016/j.1000-5404.201903210.

摘要

目的

2

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

2

以3

0 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

2

30 mJ/cm

2

UVB辐照后，HaCaT细胞增殖率均较对照组降低(

P

<

0.01)，G

2

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

2

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。

Abstract

Objective

2

To establish an efficient skin photoaging model in HaCaT cells and investigate the possible signaling pathways of photoaging of skin.

Methods

2

After HaCaT cells were exposed to UVB at 30 mJ/cm

2

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

2

Exposure of UVB at 30 mJ/cm

2

resulted in significantly decreased proliferation (

P

<

0.01)

increased proportion of cells at G

2

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

2

UVB-exposure at 30 mJ/cm

2

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

Keywords

references

GILCHREST B A. Skin aging and photoaging[J]. Dermatol Nurs, 1990, 2(2): 79-82.

MOAN J, POROJNICU A C, DAHLBACK A. Ultraviolet radiation and malignant melanoma[J]. Clin Dermatol, 2008, 624(1): 67-104.DOI:10.1007/978-0-387-77574-6_9.

SYED D N, KHAN M I, SHABBIR M, et al . MicroRNAs in skin response to UV radiation[J ] . Curr Drug Targets,2013, 14(10): 1128-1134.

李建民, 徐艳明, 陈巧云, 等.杜仲抗UVB致HaCaT细胞光老化活性部位的初步筛选[J].中医药信息, 2011, 28(1): 24-26. DOI:10.3969/j.issn.1002-2406.2011.01.010.

LI J M, XU Y M, CHEN Q Y, et al . Screening on activity partof eucommia for resistance photoaging from UVB induced damage[J ] . Information Tradit Chin Med, 2011, 28(1): 24-26. DOI:10.3969/j.issn.1002-2406.2011.01.010.

尹颂超, 张云青, 薛晓杨, 等.不同剂量中波紫外线照射对角质形成细胞的氧化损伤作用[J].中国美容医学, 2016, 25(5): 57-59. DOI:10.15909/j.cnki.cn61-1347/r.001151.

YIN S C, ZHANG Y Q, XUE X Y, et al . The oxidative damage on keratinocytes from UVB at different doses[J ] . Chin J Aesth Med, 2016, 25(5): 57-59. DOI:10.15909/j.cnki.cn61-1347/r.001151.

MICHALOGLOU C, VREDEVELD L C W, SOENGAS M S, et al . BRAFE600-associated senescence-like cell cycle arrest of human naevi[J ] . Nature, 2005, 436(7051): 720-724. DOI:10.1038/nature03890.

COLLADO M, SERRANO M. The power and the promise of oncogene-induced senescence markers[J]. Nat Rev Cancer, 2006, 6(6): 472-476. DOI:10.1038/nrc1884.

DUMONT P. Increasing the complexity of respiratory syncytial virus infection: Reactive oxygen species, DNA damage, and premature senescence[J]. Virulence, 2016, 7(4): 372-375. DOI:10.1080/21505594.2016.1162370.

KENNEDY A L, MCBRYAN T, ENDERS G H, et al . Senescent mouse cells fail to overtly regulate the HIRA histone chaperone and do not form robust senescence associated Heterochromatin Foci[J ] . Cell Div, 2010, 5: 16. DOI:10.1186/1747-1028-5-16.

LEE S H, UM S J, KIM E J. CBX8 antagonizes the effect of Sirtinol on premature senescence through the AKT-RB-E2F1 pathway in K562 leukemia cells[J]. Biochem Biophys Res Commun, 2016, 469(4): 884-890. DOI:10.1016/j.bbrc.2015.12.070.

BAKER D J, WIJSHAKE T, TCHKONIA T, et al . Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders[J ] . Nature, 2011, 479(7372): 232-236. DOI:10.1038/nature10600.

ZHOU F, HUANG X, PAN Y, et al . Resveratrol protects HaCaT cells from ultraviolet B-induced photoaging via upregulation of HSP27 and modulation of mitochondrial caspase-dependent apoptotic pathway[J ] . Biochem Biophys Res Commun, 2018, 499(3): 662-668. DOI:10.1016/j.bbrc.2018.03.207..

REDDY K K, GILCHREST B A. The molecular basis of cutaneous aging[J]. Expert Rev Dermatol, 2011, 6(5): 525-536. DOI:10.1586/edm.11.48.

GILCHREST B A. Photoaging[J]. J Invest Dermatol, 2013, 133: E2-E6. DOI:10.1038/skinbio.2013.176.

MOH M C, ZHANG T, LEE L H, et al . Expression of hepaCAM is downregulated in cancers and induces senescence-like growth arrest via a P53/p21-dependent pathway in human breast cancer cells[J ] . Carcinogenesis, 2008, 29(12): 2298-2305. DO I:10.1093/carcin/bgn226..

KANG J Y, KIM J J, JANG S Y, et al . The p53-p21(Cip1/WAF1) pathway is necessary for cellular senescence induced by the inhibition of protein kinase CKⅡ in human colon cancer cells[J ] . Mol Cells, 2009, 28(5): 489-494. DOI:10.1007/s10059-009-0141-9.

EL-DEIRY W S, HARPER J W, O'CONNOR P M, et al . WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis[J ] . Cancer Res, 1994, 54(5): 1169-1174.

LEHMAN T A, MODALI R, BOUKAMP P, et al . P53 mutations in human immortalized epithelial cell lines[J ] . Carcinogenesis, 1993, 14(5): 833-839. DOI:10.1093/carcin/14.5.833.

DATTO M B, LI Y, PANUS J F, et al . Transforming growth factor beta induces the cyclin-dependent kinase inhibitor p21 through a p53-independent mechanism[J ] . Proc Natl Acad Sci USA, 1995, 92(12): 5545-5549. DOI:10.1073/pnas.92.12.5545.

CMIELOVÁ J, REZÁČOVÁ M. P21Cip1/Waf1 protein and its function based on a subcellular localization[corrected][J]. J Cell Biochem, 2011, 112(12): 3502-3506. DOI:10.1002/jcb.23296.

ZHOU B P, LIAO Y, XIA W, et al . Cytoplasmic localization of P21Cip1/WAF1 by Akt-induced phosphorylation in HER-2/neu-overexpressing cells[J ] . Nat Cell Biol, 2001, 3(3): 245-252. DOI:10.1038/35060032.

PARRY D, BATES S, MANN D J, et al . Lack of cyclin D-Cdk complexes in Rb-negative cells correlates with high levels of P16INK4/MTS1 tumour suppressor gene product[J ] . EMBO J, 1995, 14(3): 503-511.

BURD C E, SORRENTINO J A, CLARK K S, et al . Monitoring tumorigenesis and senescence in vivo with a P16INK4a-luciferase model[J ] . Cell, 2013, 152(1/2): 340-351. DOI:10.1016/j.cell.2012.12.010..

WESTHOFF J H, HILGERS K F, STEINBACH M P, et al . Hypertension induces somatic cellular senescence in rats and humans by induction of cell cycle inhibitor P16INK4a[J ] . Hypertension, 2008, 52(1): 123-129. DOI:10.1161/HYPERTENSIONAHA.107.099432.

RAYESS H, WANG M B, SRIVATSAN E S. Cellular senescence and tumor suppressor gene p16[J]. Int J Cancer, 2012, 130(8): 1715-1725. DOI:10.1002/ijc.27316.

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