两种纳米陶瓷颗粒对细胞DNA损伤反应的研究

Investigation of damage pathway and DNA reaction induced by two ceramic nanoparticles

目的探讨羟基磷灰石(HAP)和β-磷酸三钙(β-TCP)两种纳米陶瓷颗粒(NPs)对细胞DNA损伤应答的机制。方法选用原代大鼠腹腔巨噬细胞,运用RT-PCR的方法,在分子水平上检测HAP和β-TCP两种纳米颗粒对DNA损伤通路中P53、P21、gadd45以及HSP70这些关键蛋白转录水平的影响。结果 P53、P21及HSP70的表达均随着HAP NPs浓度的增加而升高,并在100μg/ml条件下P53、P21及HSP70有显著性表达(P<0.05),而当HAP NPs达到200μg/ml,只有P21的表达反而有所降低,同时各浓度的HAP NPs对gadd45表达无明显影响(P>0.05)。而β-TCP NPs在20μg/ml的浓度条件下能引起以上四种基因的明显表达(P<0.05),并随着浓度的升高基因表达有所下降。结论本研究发现100μg/ml HAPNPs能引起DNA的明显损伤,而β-TCP NPs在更低浓度的条件下(20μg/ml)就能引起DNA的损伤;同时HAP NPs倾向于通过阻止细胞分裂、给细胞提供足够时间的方式来修复损伤,而β-TCP NPs则是同时启用诱导核苷酸切除和阻滞细胞周期的这两种方式全面修复受损的DNA。并且当这两种纳米颗粒浓度达到200μg/ml时,受损的DNA均已不易被修复。由此说明,分子水平上通过对纳米颗粒致细胞损伤机理的探讨,能解释不同生物陶瓷材料的纳米级颗粒对DNA的损伤应答存在不同的途径。

Objective To study the mechanisms of DNA damage induced by hydroxyapatite（HAP） and tricalcium phosphate（β-TCP） nanoparticles（NPs）.Methods Primary rat peritoneal macrophages were cultured the expression of P53,P21,GADD45,and HSP70 were examined in rat peritoneal macrophages after incubation with NPs by RT-PCR. Results The results showed that the expression of P53,P21,and HSP70 increased with increasing concentrations of HAP NPs.The expression of P53 and HSP70 was clearly higher than the negative control at 100μg/ml HAP NPs（P0.05）, but P21 expression decreased at 200μg/ml HAP.HAP NPs had no effect on GADD45 expression（P0.05）.Furthermore, 20μg/mlβ-TCP NPs could markedly induce the expression of all four genes（P0.05）,although their expression decreased with increasing concentration ofβ-TCP NPs.Conclusions These studies confirm that only 20μg/mlβ-TCP NPs could induce DNA damage compared with 100μg/ml HAP NPs.HAP NPs induced cell cycle arrest to allow enough time for DNA repair,whileβ-TCP NPs simultaneously promoted removal of damaged nucleotides and cell cycle arrest to repair damaged DNA.DNA damage was irreversible when the concentration of these NPs was greater than 200μg/ml.Therefore, HAP andβ-TCP NPs induce DNA damage at the molecular level and induce different DNA damage responses.