The Study On Cytotoxicity Biomarkers Screening Of Silver Nanoparticles By Multi-level Biomics Combined Analysis Method

Most biomaterials for clinical use would contact and interact with tissues,therefore,it is very important to ensure the safety and effectiveness of these biomaterials and improve their biocompatibility.The evaluation for biocompatibility is required for all biomedical materials before clinical application.Biomarkers can identify the potential risks of biomaterials before they are implanted into human body,and realize the biological effect assessment earlier,more sensitive and more quickly.In addition,biomarkers can be employed to quickly screen a large number of new materials at the initial stage of material design and reduce the time and investment of research and development.Thus,it is of great significance to study biomarkers of biomaterials.Silver nanoparticle(SNP)is a widely used antimicrobial agent in medical equipment,food,cosmetics and other fields.However,a large number of studies have shown the cytotoxicity of SNPs,so it is necessary to study biomarkers that can evaluate the cytotoxicity of SNPs in earlier stage.At present,the researches on the cytotoxic biomarkers of SNPs were insufficient.On the one hand,previous studies were carried out only by single omics method and lack of joint analysis of biomarkers with multi-omics technologies,which may lead to inconsistency in different levels of biomics studys.On the other hand,biomarkers acquired in most previous researches lacked experimental validation.Since the application of omics technology to detect molecular expression level may lead to deviation in results by technical errors,and bioinformatics method is only a theoretical analyzing method for the function of biomarkers,the application of the screening results in practice is limited.In order to solve the deficiencies above,in this thesis we conducted high-throughout screening of SNPs cytotoxicity biomarkers based on multi-omics(genomics,proteomics,metabolomics)technologies,employed series of verification experiments on the screened biomarkers,and finally obtained biomarkers that can evaluate the cytotoxicity of SNPs.In addition,based on the correlation of different biomarkers,in this thesis weconstructed molecular biological pathways from gene transcription to protein expression to metabolite changes,and carried out corresponding association verification and pathway function verification experiments.Finally,the specific mechanism of different levels of biomarkers in the cytotoxicity of SNPs was clarified.The main contents of this thesis are as follows:1.Preparing and characterizing SNPs: SNPs were prepared by reducing silver nitrate with sodium borohydrate.The maximum absorption wavelength of SNPs measured by spetrophotometer was 389 nm,the particle size of the SNPs characterized by transmission electron microscopy was 20.45±2.72 nm,and the concentration of SNPs measured by plasma emission spectrometer was 874mg/L.2.Screening biomarkers: The expression pattern analysis,biological pathway analysis,multigroup screening and correlation analysis were carried out on genomic,proteomic and metabonomics data that obtained through the earlier omics experiments by the research group.Three genes,three proteins,and two metabolites were selected as the target biomarkers,which meet the following conditions: differential expression was in consistent pattern at the same time point;participated in common cytotoxic related pathways;and significantly differentially expressed metabolites were in downstreams of significantly differentially expressed genes/proteins.3.Verifying the expression levels of biomarkers: RT-PCR,Western blot and LC-MS methods were used respectively to verify the expression levels of the 2 target gene biomarkers,2target protein biomarkers and 2 target metabolite biomarkers.The results showed that the three genes,three proteins and two metabolites were uniformly down-regulated in the validation and omics experiments,which proved the selected genes,proteins and metabolites above could be used as target biomarkers to evaluate the cytotoxicity of SNPs.4.Verifying the function of biomarkers: Functional verification experiments were carried out on target gene biomarkers CPT1 C and PCK2 which were involved in multiple cytotoxicrelated pathways.First,CPT1 C and PCK2 gene knockout/overexpression stable cells were successfully constructed by CRISPR/Cas9 technology;CPT1C's function of regulating energy metabolism and cell growth and proliferation were identified by the ATP content detection and cell proliferation rate evaluation experiments;PCK2's function of regulating citric acid synthesis and glycolysis were identified through the citric acid content and lactic acid metabolism detection experiments.Finally,we ensured that CPT1 C and PCK2 genes can be used as the final cytotoxicity biomarkers of SNPs.5.Analysing the correlation and mechanism of biomarkers: Two molecular biological pathways were constructed based on the upstream and downstream relationships of biomarkers screened in the previous stage.One is based on the fatty acid metabolism pathway: the upstream CPT1 C gene transcription?the change of downstream metabolite Palmitoyl-L-carnitine.The other is based on the tricarboxylic acid cycle pathway: the upstream PCK2 gene transcription?the upstream MDH2/ACO2 protein expression?the downstream metabolite Malic acid change.Constructing CPT1 C and PCK2 gene underexpression/overexpression stable cells by CRISPR/Cas9 technology.Then the relation of these genes,proteins and metabolites biomarkers were verified by Western blot and LC-MS/GC-MS methods.The pathway function were verified by different metabolic pathway kits.The results showed that the expression level of the metabolite Palmitoyl-Lcarnitine was synchronized with the expression level change of CPT1 C gene,and the expression level of the protein ACO2 and the metabolite Malic acid was synchronized with the expression level change of PCK2 gene.Finally,it was ensured that the protein ACO2,the metabolites Palmitoyl-L-carnitineand Malic acid can be used as the final cytotoxicity biomarkers of SNPs.In addition,SNPs could affect the expression of CPT1 C gene biomarker? the change of Palmitoyl-L-carnitine metabolite biomarkers? the change of intracellular acetyl-Co A,leading to the cytotoxicity manifests as abnormal energy metabolism.Meanwhile,SNPs could affect the expression of PCK2 gene biomarker? the change of ACO2 protein biomarker? the change of Malic acid metabolite biomarkers,the resulting cytotoxicity appears as effects onintracellular mitochondrial membrane potential,ATP production and ROS content,cell energy metabolism was blocked,and cell oxidation was impaired.