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目的:观察PCNA泛素化修饰对Hela细胞损伤敏感性的影响。方法:Western blot法检测His-PCNA及His-mutant PCNA(mPCNA,K164R)在Hela细胞中的表达。DNA损伤剂苯并芘(BaP)和依托泊苷(VP-16)分别处理Hela细胞后,MTT法检测不同细胞系对DNA损伤药物的敏感性;Western blot法检测细胞PCNA的泛素化修饰。结果:Western blot结果显示His-PCNA和His-mPCNA在Hela细胞中稳定高表达。MTT结果显示,苯并芘损伤后,稳定高表达mPCNA的细胞系与野生型及高表达PCNA细胞系相比,其细胞存活率呈明显下降趋势,而VP-16作用后,三种细胞存活率无明显差异。Western blot结果显示苯并芘损伤可特异性诱导PCNA发生泛素化修饰。结论:苯并芘损伤能够诱导PCNA发生泛素化修饰,从而降低Hela细胞对苯并芘损伤的敏感性。
Objective: To observe the effect of ubiquitination of PCNA on the injury sensitivity of Hela cells. Methods: The expression of His-PCNA and His-mutant PCNA (mPCNA, K164R) in Hela cells was detected by Western blot. The sensitivity of DNA damage drugs to different cell lines was tested by MTT assay after DNA damage agents BaP and VP-16 were treated respectively. The ubiquitination of PCNA was detected by Western blot. Results: Western blot results showed that His-PCNA and His-mPCNA were stably overexpressed in Hela cells. MTT results showed that the cell survival rate of the cell lines stably expressing mPCNA was significantly decreased compared with the wild-type and high-expressing PCNA cell lines after the injury of benzopyrene, while the survival rate of the three cell lines after VP-16 treatment No significant difference. Western blot results show that benzopyrene injury can specifically induce ubiquitination of PCNA. CONCLUSION: Benzopyrene injury can induce ubiquitination of PCNA and reduce the sensitivity of Hela cells to benzopyrene injury.