镉对HepG2细胞DNA损伤作用以及对gadd基因表达的影响

The Effects of the cadmium chloride on the DNA damage and the expression level of gadd Gene in HepG2 cell line

目的探讨氯化镉对HepG2细胞DNA损伤作用以及对gadd153和gadd45β启动子和mRNA表达的影响。方法应用彗星试验检测氯化镉对HepG2细胞的DNA损伤作用；分别构建含有gadd153和gadd45β启动子和荧光素酶报告基因的载体pGADD153-Luc和pG45-Luc，荧光素酶活性检测反映gadd153和gadd45β启动子的活性，生物发光检测荧光素酶活性；反转录-聚合酶链反应（RT-PCR）检测gadd153和gadd45β基因mRNA的表达。结果彗星试验结果显示，在100、300μmol/L氯化镉处理细胞24h后，Olive尾矩（分别为0．78±0．06、1．10±0．12）明显高于对照组（0．53±0．08），差异有统计学意义（P〈0．05），并有良好的剂量-效应关系（r=0．9761，P〈0．05）；报告基因分析显示，1、5、10μmol/L氯化镉处理组gadd153启动子表达1分别为（9．45±1．26）、（11．76±1．12）、（21．14±1．47）RIU／μgPro]均明显高于对照组[（7．02±0．82）RIU／μgPro]差异均有统计学意义（P〈0．05），并有良好的剂量-效应关系（r=0．8755，P〈0．05）；5、10μmol／L氯化镉处理组gadd45β启动子表达明显高于对照组，差异有统计学意义（P〈0．05），并有良好的剂量一效应关系（r=0．8856，P〈0．05）；RT—PCR结果显示，1、5、10μmol／L氯化镉处理组gadd153mRNA表达均明显高于对照组，5、10μmol/L氯化镉处理组gadd45βmRNA表达明显高于对照组，差异有统计学意义（P〈0．05）。结论氯化镉可诱导HepG2细胞DNA损伤，较低剂量氯化镉即使未引起明显的DNA损伤，亦可促进HepG2细胞gadd153和gadd45β启动子和mRNA的表达。

Objective To investigate the effects of the cadmium chloride on the DNA damage and the expression of the gadd153 and gadd45β promoter and mRNA in HepG2 cells. Methods DNA damage induced by cadmium chloride was detected by comet assay. The plasmids （pGADD153-Luc and pG45-Luc ） containing DNA damage and repair inducible gene 153 and 45 （gadd153 and gadd45β） promoter and luciferase and gadd45β reporter gene were constructed. The activity of gadd153 and gadd45βpromoter were represented by the luciferase activity, the inducible luciferase activities was detected by bioluminescence. The expression of gadd153 and gadd45β mRNA was detected by RT-PCR. Results The results of comet assay indicated that Olive Tail Moment induced by the cadmium chloride increased significantly at the dose of 100, 300 μmol/L, compared with the control （P〈0.05）. The luciferase activity analysis showed that the expression levels of gadd153 promoter increased significantly in 1, 5, 10 μmol/L treatment group, compared with the control （P〈0.05）. The expression levels of gadd45β promoter in 5, 10μmol/L treatment group were significantly higher than that in control group （P〈0.05）. The expression levels of gadd153 mRNA induced by cadmium chloride at the doses of 1, 5, 10 μmol/L and the expression levels of gadd45β mRNA induced at the doses of 5, 10μmol/L were significantly higher than thoae in control group （P〈0.05）. Conclusion The cadmium chloride can induce the DNA damage and increase the expression levels of the gadd153 and gadd45β promoters in HepG2 ceils.