以蛋白質體學方法研究皮膚細胞經UVB傷害後之分子機制

A Study of Proteomic Analysis of HaCat Cell after UVB Radiation

10.30001/JIES.201103.0003

楊子妍(Zu-Yen Yang)；陳國蕙(Kuo-Hui Chen)；林政頡(Chen-Gi Lin)；陳正吉(Chen-Gi Chen)；吳裕仁(Yu-Jen Wu)

紫外光 ； 蛋白質體學 ； UV radiation ； proteome

美容科技學刊

8卷1期（2011 / 03 / 01）

25 - 40

繁體中文

皮膚過度的照射紫外光會造成急性或是慢性的傷害，短暫的照射紫外光會造成曬傷及產生紅斑，長期性的照射則會增加癌症及加速老化的產生，在細胞研究中也指出過度的照射紫外光會增加活性氧物質(reactive oxygen species; ROS)的產生，引發皮膚細胞產生複雜的訊息傳遞的代謝途徑，減低穀胱甘肽(glutathione)的含量，並且造成一些保護性酵素如超氧歧化酶(superoxide dismutase)、觸酶(catalase)等活性降低，進而使皮膚膠原蛋白(collagen)的產生減少，造成皮膚光老化現象發生。 紫外光過量的照射在生物體或是細胞實驗中都發現其所造成的傷害，但是以往的實驗都是檢測某些抗氧化酵素的活性變化，或是分子層次的改變，但是無法觀察出其蛋白質的變化，同時也無法觀察經由紫外光過度的照射，其生物體內整體的蛋白質變化。此研究目的是利用蛋白質體(proteome)的方法，觀察角質細胞(keratinocyte)經由紫外光照射後其總體蛋白質的變化，經由二維電泳(two-dimentional electrophoresis, 2DE)圖譜分析，總共有16個蛋白質經由LC/MS/MS分析，有16個蛋白質身分被鑑定出來，其中有13個蛋白質表現量漸增，分別為keratin1，keratin9，MTHSP75，Lamin B2、stomatin (EPB72)-like 2、thioredoxin peroxidase，1個BIP蛋白質表現量下降，將部分蛋白質分別進行RT-PCR及western blot分析，以驗證在mRNA及蛋白質層次之差異表現。並且建立此一系統，對於研發皮膚抗氧化物質的篩選，可以多一種整體性的研究方法，並可了解抗氧化物質在皮膚細胞中的功效，對於化妝品中抗氧化成分的研究，有更多的細胞實驗的證據。

Overdose of UV radiation could cause acute or chronic damages to the skin. Short- term UV radiation could cause sun burn and erythema; long-term UV radiation could cause cancers and accelerated aging. High-dose radiation causes the generation of reactive oxygen species which could trigger the activation of complex signaling pathways in skin cells, reduce the amount of glutathione, and reduce the activities of some protective enzymes such as superoxidase and catalase. The consequences could retard the synthesis of collagen in skin to cause skin photoaging. Conventionally the studies on damages caused by overdose UV radiation are carried out by examining the activities of oxidases or their changes at the molecular level. The changes at the total protein levels has not been examined. The purpose of this research is to establish a system which employ proteomics to observe the overall protein changes in skin cells caused by UV radiation. By using two- dimentional electrophoresis analysis, sixteen protein spots have been identified by LC/MS/MS. The levels of thirteen protein spots including keratin1, keratin 9, MTHSP75, Lamin B2, stomatin (EPB72)-like 2, and thioredoxin peroxidase increased. The level of one BIP protein decreased. RT-PCR and western blot analysis have been carried out on some proteins to confirm the expression difference in mRNA and protein level. The developed system could be used to evaluate potential antioxidants through the cell level reflecting their effectiveness closer to real conditions. The function of the antioxidants in the cells can be observed and the screening of the potential antioxidants used for cosmetics formulations could be done efficiently.

1. Bayerl C.,Lauk J.,Moll I.,Jung E. G.(1997).Immunohistochemical characterization of HSP, α-MSH, Merkel cells and neuronal markers in acute UV dermatitis and acute contact dermatitis in vivo.Inflamm. res.,46,409-411.
2. Broers, Jos L. V.,Machiels, Barbie M.,Kuijpers, Helma J. H.,Smedts, Frank,van den Kieboom, Ronald,Raymond, Yves,Ramaekers, Frans C. S.(1997).A- and B- type lamins are differentially expressed in normal human tissues.Histochem Cell Biol,107,505-517.
3. Chaturvedi, Vijaya,Qin, Jian- Zhoneg,Denning, Mitchell F.,Choubey, Divaker,Diaz, Manuel O.,Nickoloff, Brian J.(1999).Apoptosis in Proliferating, Senescent, and Immortalized Keratinocytes.Biological Chemistry.,274(33),23358-23367.
4. Dudek, J.,Benedix, J.,Cappel, S.,Greiner, M.,Jalal, C.,Muller, L.,Zimmermann, R.(2009).Functions and pathologies of Bip and its interaction partners.Cell. Mol. Life Sci.,66,1556-1569.
5. Green, Jasper B,Peter, J.,Young, W.(2008).Slipin: ancient origin, duplication and diversification of the stomatin protein family.BMC Evolutionary Biology,8,44.
6. Hatsell, Sarah J.,Kelsell, David P.(2000).The diffuse palmoplantra keratodermas.Dermatovenerologica,9(2)
7. Novoselov, S. V.,Peshenko, I. V.,Popov, V. I.,Novoselov, V. I.,Bystrova, M. F.,Evdokimov, V. J.,Kamzalov, S. S.,Merkulova, M. I.,Shuvaeva, T. M.,Lipkin, V. M.,Fesenko, E. E.(1999).Localization of 28- kDa peroxiredoxin in rat epithelial tiaaues and its antioxidant properties.Cell Tissue Res.,298,471-480.
8. Planko, Laura,Bohse, Karsten,Hohfeld, Jorg,Betz, Regina C.,Hanneken, Sandra,Eigelshoven, Sibylle,Kruse, Roland,Nothen, Markus M.,Magin, Thomas M.(2007).Identification of a keratin- associated protein with a putative role in vesicle transport.European Journal of Cell Biology,86,827-839.
9. Rafferty, Teresa S.,Mckenzie, Roderick C.,Hunter, John A. A.,Howie, A. Forbes,Arthur, John R.,Nicol, Fergus,Beckett, Geoff J.(1998).Differential expression of selenoproteins by human skin cells and protection by selenium from UVB- radiation- induced cell death.Biochem. J.,332,231-236.
10. Schafer, Matthias,Werner, Sabine(2008).Oxidative stress in normal and impaired wound repair.Pharmacological Research,58,165-171.
11. Stevens, A.、Lowe, J.、尹相姝編譯(2006)。臺北:藝軒圖書出版社。
12. Venables, RS,MCLean, S,Luny, D.,Moteleb, E.,Morley, S.,Quinlan, RA,Lane, EB,Hutchison, CJ(2001).Expression of individual lamins in basal cell carcinomas of the skin.British Journal of Cancer,84,512-519.
13. Zhang, B. O.,Wang, Yan,Yongping(2009).Peroxiredoxins, a novel target in cancer radiotherapy.Cancer Letters
14. 石宗憲(2003)。蛋白質體學。藥物基因生物技術教學資源中心。
15. 吳裕仁(2006)。。
16. 陳宜嫻、黃宜純、黃淑桂、傅如嶽、溫慧萍、楊佳璋、鄭智交(2002)。皮膚生理學。臺中:華格那企業。