| 摘要: |
| 目的 研究癌基因H-RasV12过度表达或激活与自噬活性的关系,在人成纤维细胞中观察Ras过度表达对自噬活性的影响。方法 在BJ人成纤维细胞中转染原癌基因H-RasV12或其空载体,通过形态学、细胞生长曲线、β-半乳糖苷染色、衰老和自噬相关蛋白表达、流式细胞分析、siRNA(small interfering RNA,siRNA)抑制自噬关键基因5(ATG5),来分析Ras过度表达的细胞效应。结果 与对照细胞相比,H-RasV12过度表达的BJ细胞出现明显的早期衰老,其自噬活性受到抑制,表现为蛋白p62和微管相关蛋白1轻链3Ⅱ( Light chain 3Ⅱ,LC3Ⅱ)的显著蓄积,在研究时间区间内这种抑制作用保持稳定,同时细胞凋亡率增加;利用siRNA抑制ATG5表达,抑制自噬可进一步加重衰老。结论 人成纤维细胞稳定表达癌基因H-RasV12后,可出现自噬活性受到抑制的现象,且自噬抑制处于自噬过程的后期。这一现象可能与Ras相关性癌症的癌变机制有关。 |
| 关键词: 自噬 H-RasV12 过早衰老 成纤维细胞 |
| DOI：10.3724/SP.J.1008.2014.01191 |
| 投稿时间：2014-06-04修订日期：2014-10-19 |
| 基金项目:国家自然科学基金(81102475);南方医科大学南方医院高层次课题匹配基金(81102475). |
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| Autophagy activity is inhibited in human fibroblast cells stably overexpressing H-RasV12 |
| WANG Ling1△,ZHAO Dan3△,YU Le1,LI Yi-lei2* |
(1. School of Pharmaceutical Science, Southern Medical University, Guangzhou 510515, Guangdong, China;
2. Department of Pharmacology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong, China;
3. Department of Pharmacology, No.85 Hospital of PLA, Shanghai 200052, China
△Co-first authors.
*Corresponding authors) |
| Abstract: |
| Objective To investigate the relationship between oncogenic H-RasV12 overexpression/activation and the autophagic activity by observing the effect of Ras overexpression on autophagic activity in human fibroblast cells. Methods Human BJ fibroblast cells were transfected with H-RasV12 or control vector, and then the cellular responses to H-RasV12 overexpression were analyzed by observing the morphology, cell growth curve, senescence-associated β-Gal staining, Western blotting analysis, flow cytometry, and suppression of autophagy-related protein 5 (ATG5) by siRNA. Results Compared with control group, BJ cells overexpressing H-RasV12 developed prominent premature senescence and inhibited autophagic activity, as manifested by significant accumulation of p62 and light chain 3Ⅱ (LC3Ⅱ). The autophagy inhibition by H-RasV12 remained stable during the study period; the apoptosis rate was increased in H-RasV12 overexpressing BJ cells compared with that in the control cells. Suppression of ATG5 by siRNA led to more severe senescence in Ras-overexpressing BJ cells. Conclusion Our results suggest that the autophagy activity is inhibited in human fibroblast cells stably overexpressing oncogenic H-RasV12, and the inhibition is in the later stage of autophagy, which may be related to H-RasV12-related tumorigenesis. |
| Key words: autophagy H-RasV12 premature aging fibroblasts |
