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目的:探讨人正常肝细胞内蛋白磷酸酶2A-B56γ亚基(PP2A-B56γ,由PPP2R5C基因编码)高表达对镉诱导相关基因转录水平和DNA损伤效应的影响,并探讨其作用机制。方法:构建PP2A-B56γ高表达L02-2R5Cc和空白载体对照L02-pBabe细胞模型;两种细胞分别给予CdCl_2(Cd)、TNFα单独处理及联合处理后,实时荧光定量-PCR(QRT-PCR)检测不同处理组PPP2R5C和金属硫蛋白1B(MTIB)mRNA转录水平;彗星试验(SCGE)检测细胞DNA断裂损伤情况;并按照3×2的析因设计分析不同处理之间是否存在联合作用及作用类型。结果:与L02-pBabe细胞相比较,L02-2R5Cc细胞中PPP2R5C、外源性PPP2R5C-FLAG mRNA显著高表达(P<0.05),细胞株构建成功。3种因素单独处理时,与阴性对照组相比,Cd降低PPP2R5C、升高MT1B mRNA表达,PPP2R5C基因高表达诱导的效应与Cd处理相反,TNFα降低PPP2R5C和MT1B mRNA表达(均P<0.05);析因分析表明,在PPP2R5C和PPP2R5C-FLAG mRNA检测中,PPP2R5C高表达与Cd、与TNFα之间均存在协同性交互效应(P<0.05);在MT1B mRNA检测中,PPP2R5C高表达与cd和TNFα之间均表现为拮抗作用,PPP2R5C高表达与TNFα为协同抑制作用(均P<0.05)。SCGE检测表明,与阴性对照相比.Cd诱导彗星尾长、尾矩、尾部DNA含量和拖尾细胞阳性率显著增高(P<0.05),TNFα、PPP2R5C高表达单独作用均不引起DNA损伤(P>0.05),但两两联合作用的析因分析结果表明,TNFα预处理与Cd处理对DNA损伤具有协同作用,PPP2R5C高表达与Cd处理存在拮抗作用,交互作用均具有统计学意义(P<0.05)。结论:Cd抑制肝细胞PPP2A-B56γ编码基因的转录并显著诱导DNA损伤,PP2A-B56γ高表达抑制镉诱导的DNA损伤,而炎症因子可增强镉对细胞DNA的损伤。
AIM: To investigate the effect of high expression of protein phosphatase 2A-B56γ subunit (PP2A-B56γ, encoded by PPP2R5C gene) on the transcriptional level of Cd-induced genes and DNA damage in normal human hepatocytes and to explore its mechanism. Methods: L02-2R5Cc with high PP2A-B56γ expression and L02-pBabe cell model with blank vector were constructed. The two cells were treated with CdCl2, TNFα alone or in combination, and then detected by QRT-PCR The transcriptional levels of PPP2R5C and metallothionein 1B (MTIB) mRNA in different treatment groups were detected. The damage of DNA damage was detected by comet assay (SCGE). The 3 × 2 factorial design was used to analyze whether there was a combination of different treatments and the type of action. Results: Compared with L02-pBabe cells, PPP2R5C and exogenous PPP2R5C-FLAG mRNA were highly expressed in L02-2R5Cc cells (P <0.05), and the cell lines were constructed successfully. Compared with the negative control group, Cd decreased PPP2R5C and increased MT1B mRNA expression when compared with the negative control group. The effect of PPP2R5C gene overexpression was opposite to that of Cd treatment. TNFα decreased the expression of PPP2R5C and MT1B mRNA (all P <0.05). The analysis of factorial analysis showed that both PPP2R5C and PPP2R5C had a synergistic interaction between PPP2R5C and TNFα in PPP2R5C and PPP2R5C-FLAG mRNA detection (P <0.05) Showed antagonism, PPP2R5C high expression and TNFα synergistic inhibition (all P <0.05). The results of SCGE showed that compared with the negative control, the tail length, tail moment, tail DNA content and the positive rate of tail cells were significantly increased (P <0.05), while the high expression of TNFα and PPP2R5C alone did not cause DNA damage > 0.05). However, the factorial analysis of the combined effect of each pairwise showed that TNFα pretreatment and Cd treatment had a synergistic effect on DNA damage. PPP2R5C overexpression had antagonism with Cd treatment and the interaction had statistical significance (P <0.05 ). CONCLUSION: Cd inhibits the transcription of PPP2A-B56γ gene in hepatocytes and induces DNA damage significantly. High expression of PP2A-B56γ inhibits cadmium-induced DNA damage, whereas inflammatory cytokines enhance the DNA damage induced by cadmium.