基于生物组学方法的银纳米粒子细胞毒性机理研究

Mechanism study of cytotoxicity of silver nanoparticles based on biomics methods

目的本论文的目的是应用基因组学和蛋白质组学技术结合生物信息学方法研究银纳米粒子对人皮肤成纤维细胞的毒性机理。方法首先采用水相合成法制备20 nm的银纳米粒子,然后应用MTT法和流式细胞术评价银纳米粒子对人皮肤成纤维细胞的细胞毒性及对细胞周期和细胞凋亡的影响。接着应用基因表达谱芯片、蛋白质组学方法获得银纳米粒子作用人皮肤成纤维细胞后发生差异表达的基因和蛋白质,并进一步对其进行生物信息学分析。结果 MTT实验结果表明,200μM、20 nm银纳米粒子与人皮肤成纤维细胞作用1 h、4 h、8 h后并未产生明显细胞毒性,流式细胞仪结果显示银纳米粒子与人皮肤成纤维细胞后处于S期的细胞增多而G0/G1期、G2/M期的细胞减少。应用基因表达谱芯片筛选出了三个时间点不重复的差异表达基因1593个,应用蛋白质组学方法筛选出三个时间点均发生差异表达的功能蛋白质25个。对差异表达基因和蛋白质的生物信息学分析结果表明,银纳米粒子可能会影响细胞骨架、细胞膜、能量代谢和正常的基因表达调控机制,进而对细胞增殖、凋亡和细胞周期等生理过程产生影响。结论基因组学和蛋白质组学方法是全面深入研究生物材料与细胞作用分子机理的有效手段。

Objective The aim of this paper was to investigate the mechanism of cytotoxicity of silver nanoparticles on human dermal fibroblasts-fetal（HDF-f） based on genomic,proteomic combining bioinformatics analysis.Methods 20 nm silver nanoparticles were prepared by sodium borohydride reducing silver nitrate at first.Then methylthiazoltetrazolium（MTT） assay and flow cytomtery were used to evaluate cytotoxicity and the influence on the cell cycle and apoptosis by silver nanoparticles on HDF-f.Finally,differentially expressed genes and proteins in HDF-f treated with silver nanoparticles were obtained using cDNA microarray,proteomics technology and were analyzed by the integration of bioinformatics method.Results MTT assay showed that 200 μM,20 nm silver nanoparticles caused no significant cytotoxicity to HDF-f cells after being treated for 1 h,4 h and 8 h.Flow cytometry experiment results suggested silver nanoparticles resulted in the increase of the proportion of HDF-f cells in S phase and decrease of the proportion of cells in G0/G1 and G2/M phase.1593 differentially expressed genes in at least one group and 25 proteins differentially expressed in all three groups were identified.Bioinformatics analysis results suggest that silver nanoparticles may cause the disruption of cytoskeleton and cellular membrane,disturbance of energy metabolism and regulation mechanism of gene expression,and then influence cellular physiological processes such as cell proliferation,apoptosis,and cell cycle and so on.Conclusions Genomics and proteomics methods are convenient and efficient for depth study of the molecule mechanism of interaction between biomaterials and cells.