摘要
目的研究氢醌(hydroquinone,HQ)诱导下L-02肝细胞PCNA、RAD6B及RAD18基因mRNA表达的时间效应,探讨PCNA、RAD6B及RAD18基因在HQ所致肝细胞毒性过程中的分子作用机制。方法体外培养的L-02肝细胞经40μmol/L的HQ诱导不同时间(6、12、24和48 h)后,用Trizol试剂从L-02肝细胞中分离总RNA,而后反转录为cDNA;利用Primer 3软件设计PCNA、RAD6B及RAD18基因及内参照β-ACTIN基因的引物,并进行标准曲线分析和熔解曲线分析,建立和优化定量检测各目的基因在mRNA水平上表达的反应体系和反应条件;最后采用实时荧光定量PCR法检测不同时间点PCNA、RAD6B及RAD18基因mRNA的表达状况。结果在各时间处理组(6、12、24和48 h)中,染毒组L-02肝细胞内PCNA、RAD6B及RAD18基因在mRNA水平上的表达均高于未染毒组。在24 h的时间范围内,PCNA基因在mRNA水平上的表达无论是染毒组(40μmol/L)还是未染毒组(0μmol/L),均具有随时间的延长而增加的趋势;到24 h时,相对表达量达到最大值。RAD6B和RAD18基因在未染毒组中的表达在48 h范围内均有随时间的增加而升高的趋势,它们均在48 h时间点上表达最高,其相对表达量分别为2.66和4.32;但在染毒组中,RAD6B基因和RAD18基因在48 h处的表达则有所下降,其相对表达量分别由3.74和4.83降至2.78和4.61。结论HQ可以诱导PCNA、RAD6B及RAD18基因mRNA的表达上调,并且呈现一定的时序性。
Objective To explore the time-effect of PCNA, RAD6B and RAD18 mRNA expressions induced by hydroquinone （HQ） in L-02 hepatic cells, and the molecular mechanism of toxic effects of HQ in L-02 hepatic cells. Methods L-02 hepatic cells were exposed to 40 μmol/L HQ for 6, 12, 24 and 48h respectively, then the total cellular RNA was isolated from L-02 hepatic cells using Trizol reagent and inversely transcripted to cDNA. Expression of PCNA, RAD6B and RAD18 mRNA in L-02 hepatic cells were measured by RT-qPCR in different time-points. Results Levels of expression of PCNA, RAD6B and RAD18 mRNA in L-02 hepatic cells treated with HQ were higher significantly than that of the control （0μmol/L）. The expression of PCNA mRNA in L-02 hepatic cells were increased along with time within 0--24 h in both treated groups （40 μmol/L） and control group, which reached the maximum at 24h. The expressions of RAD6B and RAD18 mRNA in control group were all increased time-dependently, which reached 2.66 and 4. 32 at 48 h, respectively; while the expression of RAD6B and RAD18 mRNA in treated group were decreased at 48h. Conclusion HQ might induce the up-regulation of expressions of RAD6B, RAD18 and PCNA mRNA, and with some time-dependent manner.
出处
《中国工业医学杂志》
CAS
北大核心
2009年第2期83-86,90,共5页
Chinese Journal of Industrial Medicine
基金
国家重点基础研究发展计划(973)项目(2002CB512903
2002CB512904)
