老化相关的自发荧光可作为细胞衰老的标志物

张静 杨璐萌 陈晓春*

doi:10.3969/j.issn.0529-1356.2014.01.001

解剖学报 ›› 2014, Vol. 45 ›› Issue (1) : 1-6. DOI: 10.3969/j.issn.0529-1356.2014.01.001

神经生物学

老化相关的自发荧光可作为细胞衰老的标志物

张静¹ 杨璐萌¹陈晓春^1,2*

作者信息 +

Aging related autofluorescence can be used as a marker of cell senescence

ZHANG Jing¹ YANG Lu-meng¹CHEN Xiao-chun ^1,2*

Author information +

文章历史 +

摘要

目的观察衰老的脑组织内自发荧光的波长特点，探讨如何合理地利用和消减老化相关的自发荧光。方法选取3月龄和24月龄自然老化的C57BL/6J雌、雄性小鼠。以衰老相关β-半乳糖苷酶染色（SA-β-Gal）和脂褐素的沉积评估老年鼠脑组织衰老的程度。以激光共焦显微镜分析老化相关自发荧光的波长特点，免疫组织化学和免疫荧光的方法标记星形胶质细胞。结果老年鼠海马和下丘脑第三脑室室周旁出现强SA-β-Gal染色、脂褐素的聚集及自发荧光。老化相关的自发荧光发射波长介于500～565nm，下丘脑第三脑室室周旁的星形胶质细胞不仅高表达SA-β-Gal，而且出现很强的自发荧光。采用针对脂褐素的自发荧光消除剂处理脑组织后，老化相关的自发荧光几乎完全消失。结论老化相关的自发荧光可以作为特定表型细胞衰老的标志物；恰当的处理老化相关的自发荧光，可极大的提高衰老脑组织免疫荧光检测的特异性。

Abstract

Objective To describe the emission spectra of autofluorescence derived from lipofuscin in aged brain sections and to investigate how aging related autofluorescence is used reasonably and abolished properly. Methods Three months old and 24 months old C57BL/6J male and female mice were used in this study. Staining of senescenceassociated beta galatosidase and accumulation of lipofuscin were used to evaluate the degree of brain aging. Laser confocal microscopy was used to analyze the emission spectra of autofluorescence. Immunohistochemistry and immunofluorescence was used to identify astrocyte. Results Strong SA-β-Gal staining and autofluorescence and accumulation of lipofuscin appeared in the CA3 of hippocampus and the periventricular area of the third ventricle in 24 months old mice. The emission wavelength of aging related autofluorescence was between 500-565 nm. Astrocytes in periventricular area of the third ventricle were of not only strong SA-β-Gal staining, but also strong autofluorescence. Abolition of background autofluorescence from lipofuscin following immersion in autofluorescence eliminator reagent was observed in aged brain tissue. Conclusion Aging related autofluorescence can be used as a marker of cell senescence with a specific phenotype. Reduction of background autofluorescence from lipofuscin can improve the specificity of immunofluorescence detection in aged brain tissue samples.

导出引用

张静杨璐萌陈晓春*. 老化相关的自发荧光可作为细胞衰老的标志物[J]. 解剖学报. 2014, 45(1): 1-6 https://doi.org/10.3969/j.issn.0529-1356.2014.01.001

ZHANG Jing YANG Lu-meng CHEN Xiao-chun*. Aging related autofluorescence can be used as a marker of cell senescence[J]. Acta Anatomica Sinica. 2014, 45(1): 1-6 https://doi.org/10.3969/j.issn.0529-1356.2014.01.001

参考文献

［1］Dowson JH. The evaluation of autofluorescence emission spectra derived from neuronal lipopigment［J］. J Microsc，1982，128(Pt 3):261-270.
［2］Sun A, Liu M, Bing G. Improving the specificity of immunological detection in aged human brain tissue samples［J］. Int J Physiol Pathophysiol Pharmacol, 2009,2(1):29-35.
［3］Debacq-Chainiaux F, Erusalimsky JD, Campisi J, et al. Protocols to detect senescence-associated beta-galactosidase (SA-betagal) activity, a biomarker of senescent cells in culture and in vivo［J］. Nat Protoc,2009,4(12):1798-1806.
［4］Sedelnikova OA, Horikawa I, Zimonjic DB, et al. Senescing human cells and ageing mice accumulate DNA lesions with unrepairable double-strand breaks［J］. Nat Cell Biol, 2004,6(2):168-170.
［5］Krishnamurthy J, Torrice C, Ramsey MR, et al. Ink4a/Arf expression is a biomarker of aging［J］. J Clin Invest, 2004,114(9):1299-1307.
［6］Dimri GP, Lee X, Basile G, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo［J］. Proc Nati Acad Sci USA, 1995,92(20):9363-9367.
［7］Geng YQ, Guan JT, Xu XH, et al. Senescence-associated beta-galactosidase activity expression in aging hippocampal neurons［J］. Biochem Biophys Res Commun, 2010,396(4):866-869.
［8］Narita M, Nunez S, Heard E, et al. Rb-mediated heterochromatin formation and silencing of E2F target genes during cellular senescence［J］. Cell, 2003,113(6):703-716.
［9］Kennedy AL, McBryan T, Enders GH, et al. Senescent mouse cells fail to overtly regulate the HIRA histone chaperone and do not form robust senescence associated heterochromatin foci［J］. Cell Divn, 2010,5:16.
［10］Wang C, Jurk D, Maddick M, et al. DNA damage response and cellular senescence in tissues of aging mice［J］. Aging cell, 2009,8(3):311-323.
［11］Brunk UT, Terman A. Lipofuscin: mechanisms of age-related accumulation and influence on cell function［J］. Free Radic Biol Med, 2002,33(5):611-619.