中华皮肤科杂志 ›› 2014, Vol. 47 ›› Issue (4): 259-262.

• 论著 • 上一篇    下一篇

RNA干扰p53基因对中波紫外线诱导的早衰和光致癌相关基因表达的影响

陈文琦1,毕志刚2,戴洁1,张晓荣许惠娟1   

  • 收稿日期:2013-07-01 修回日期:2013-08-19 出版日期:2014-04-15 发布日期:2014-04-01
  • 通讯作者: 许惠娟 E-mail:xuhuijuan@medmail.com.cn
  • 基金资助:
    国家自然科学基金;江苏省自然科学基金;南京市卫生局青年科技人才启动项目

Influence of RNA interference in p53 gene on the expressions of genes involved in ultraviolet B-induced premature senescence and photocarcinogenesis in human skin fibroblasts

  • Received:2013-07-01 Revised:2013-08-19 Online:2014-04-15 Published:2014-04-01
  • Supported by:
    ;Natural Science Foundation of Jiangsu Province of China

PDF

8

赞

摘要： 【摘要】 目的 探讨RNA干扰p53基因对中波紫外线（UVB）诱导的人皮肤成纤维细胞（HSF）早衰和光致癌相关基因表达的影响。 方法　利用已构建成功的RNA干扰抑制p53表达的HSF细胞系，加以反复多次亚毒性剂量UVB照射，衰老相关的β半乳糖苷酶（SA β-gal）染色法检测细胞衰老，定制实时定量PCR芯片，检测多种光致癌发生相关基因的表达，包括：参与细胞衰老通路的p53、p21、p19、p16、pRb，衰老相关基因纤维结合素（FN）、骨结合素（ON）、平滑肌22（SM22），p53依赖的细胞凋亡相关基因bax和bcl-2，UVB诱导的癌基因缺氧诱导因子1α（HIF-1α）、血管内皮生长因子（VEGF）以及负性调控p53的人双微球蛋白2基因（hdm2）。使用SPSS10.0软件对各组间数据进行配对t检验。 结果　抑制p53表达的HSF经UVB照射后SA β-gal活性（19.70% ± 0.85%）较照射前（12.77% ± 0.81%）有所增加（t = 6.45，P ＜ 0.05），但显著低于正常HSF经UVB照射后（50.48% ± 5.30%，t = 7.86，P < 0.05），与正常HSF组（18.50% ± 0.45%）差异无统计学意义（t = 2.57，P > 0.05）。基因检测结果表明，抑制p53表达可抑制衰老基因p21、p19、FN、ON、SM22等的表达，但p16通路并不受之影响，在UVB作用下，p16、pRb的表达显著增加，分别上调。抑制p53的表达可使抑凋亡基因bcl-2上调及促凋亡基因bax下调，癌基因HIF-1α、VEGF、hdm2表达明显增加。UVB照射对这些基因表达改变无明显影响。 结论　抑制p53表达能够延缓UVB诱导的早衰。UVB诱导的早衰具有p53依赖的相关肿瘤抑制作用。

关键词: 基因，p53, RNA干扰, 衰老，过早, 紫外线, 成纤维细胞

Abstract: Chen Wenqi *, Bi Zhigang, Dai Jie, Zhang Xiaorong, Xu Huijuan. *Department of Dermatology, Nanjing First Hospital, Nanjing Medical University, Nanjing 210006, China Corresponding author: Xu Huijuan, Email: xuhuij320@163.com 【Abstract】 Objective To evaluate the effect of RNA interference in p53 gene on the expressions of genes involved in ultraviolet B （UVB）-induced premature senescence and photocarcinogenesis in human skin fibroblasts （HSFs）. Methods A previously established HSF cell clone with repressed expression of p53, which was named as HSF-p53, was cultured and irradiated with a subcytotoxic dose （10 mJ/cm2） of UVB once a day for five consecutive days. The HSFs with normal expression of p53 served as the control. Subsequently, β-galactosidase （SA-β-gal）-staining was performed to estimate the degree of senescence, quantitative real-time PCR array was performed to determine the mRNA expressions of photocarcinogenesis- and senescence-associated genes, including p53, p21, p19, p16, pRb, fibronectin, osteonectin, smooth muscle 22 （SM22）, bax, bcl-2, hypoxia-inducible factor-1 α （HIF-1α）, vascular endothelial growth factor（VEGF）, and human double minute-2 （hdm2）. Statistical analysis was carried out by Student′s t test using the software SPSS 10.0. Results The percentage of SA-β-gal-positive cells in irradiated HSF-p53 was 19.70% ± 0.85%, significantly higher than that in unirradiated HSF-p53 （12.77% ± 0.81%, t = 6.45, P ＜ 0.05）, but lower than that in irradiated control HSFs （50.48% ± 5.30%, t = 7.86, P ＜ 0.05）, and similar to that in unirradiated control HSFs （18.50% ± 0.45%, t = 2.57, P > 0.05）. Compared with the control HSFs, the HSF-p53 showed decreased expressions of p21, p19, fibronectin, osteonectin, SM22 and bax genes （all P ＜ 0.05）, but increased expressions of bcl-2, HIF-1α, VEGF and hdm2 genes （all P ＜ 0.05）, and a similar expression of p16 gene （P > 0.05）; the repeated UVB radiation significantly promoted the expressions of p16 and pRb genes （both P ＜ 0.05）, but had no obvious effect on the expressions of the other genes in HSF-p53 compared with unirradiated HSF-p53 （all P > 0.05）. Conclusions The inhibition of p53 expression may decelerate the UVB-induced premature senescence in HSFs, which may be involved in the p53-dependent tumor suppression.

Key words: Genes, p53, RNA interference, Aging, premature, Ultraviolet rays, Fibroblasts