| As a common medical material, nickel-titanium (NiTi) alloys have a certain application in artificial bones and teeth, cardiovascular stents, and other plastic surgery or cardiovascular fields. In spite of this, there are about 54.5%-56.3% nickel elements in NiTi alloys, which are supposed to be teratogenetic and carcinogenic. Therefore, the study of biocompatibility of NiTi alloys and Ni2+ is necessary. Current research on the biocompatibility of Ni2+ are mostly at the integral, cell and molecular level, while mainstream methods in molecular level studies are traditional approaches(ELISA, RT-PCR, etc.), and results of major studies are small amounts of DNA, RNA, protein, metabolite, and other molecule affected by Ni2+. Even though there have been several studies using high-throughput omics technology(gene expression profile microarray, LC-MS, etc.) to investigate the toxic mechanism of Ni2+ on a whole set of one kind of molecule, combining more than one kind of omics technologies to explore cytotoxicity mechanism of nickel ions from the perspective of systems biology have not been reported.The first purpose of this paper is exploring the toxic mechanism in protein and metabolite level using proteomic technology and metabolomics technology, combining various bioinformatics methods. And the second purpose is integrating the analysis results of proteomics, metabolomics and genomics, and elaborating the toxic mechanism from the perspective of system biology.The main contents of this paper are as follows:1. Proteomics and metabolomics experiments:Cultivating L929 cells with normal complete medium as a control groups, and culturing L929 cells with different times and different mediums which containing different concentrations of nickel ions as experimental groups. Then cell samples (including the control and experimental groups) are collected with pancreatin or lysate, and sent to companies for proteomics and metabolomics experiments.2. Analysis of proteomics results:158,1633,1587 differentially expressed gene are found in 12h, 24h,48h in the concentration of 100μM, and 66,1292,1784 differentially expressed gene are found in 12h,24h,48h in the concentration of 200μM. And there are 22 proteins in all of these experimental groups,19 of which are functional proteins. DAVID and GenMAPP were respectively used for GO functional analysis and biological pathways analysis, and many GO functional categories are found influenced by nickel ions in all experimental groups, and when nickel ion concentration is 200μM and the reaction time is 48h, the influenced GO functional categories in cells are the most. There are 16 biological pathways containing differentially expressed gene in all of 6 experimental groups, and most of them are related with cell adhesion and migration, cell growth and proliferation, intracellular expression of genes and proteins, energy metabolism, inflammation, and so on. All of these pathway analysis results are identical to the results of previous genomics results.3. Validation tests of proteomics experiment:Western blot experiments are used to verify the protein detection results, and the results of western blot experiment are similar to the results of proteomics, which confirms the validity of proteomics based on iTRAQ technology. ATP energy metabolism tests are used to validate the conclusion in proteomics analysis that nickel ions would affect energy metabolism within cells, and the conclusion is proved to be reliable.4. Analysis of metabolomics results:40,64,79 differentially expressed metabolites are found in 12h, 24h,48h in the concentration of 100μM, and 41,77,64 differentially expressed metabolites are found in 12h,24h,48h in the concentration of 200μM. MetaboAnalyst is used to analyze metabolic pathways based on the differentially expressed metabolites in each experimental group. The results show that there are only 3 pathways existed in every group, and they are ’phenylalanine metabolism pathway’,’arginine and proline metabolism pathway’(amino acid metabolism), and ’nitrogen metabolism pathway’(energy metabolism). It is proved that Ni2+ can severely affect the process of protein synthesis and energy metabolism in cells.5. Conjoint analysis of three kinds of omics experiments results:The pathway analysis results of genomics, proteomics and metabolomics experiments are integrated for comparison, and there are three pathways contain differentially expressed genes, proteins and metabolites at the same time, namely ’glutathione metabolism pathway’,’TCA cycle’ and ’biosynthesis of unsaturated fatty acids’. All these results show that nickel ions can affect the metabolic processes of the three main substances (protein, carbohydrate and lipid) within the cell, and further impact synthesis, energy metabolism, cell membrane stability and other aspects of cell functions.6. Conjoint analysis of proteomics and metabolomics experiments results: The pathway analysis results of proteomics and metabolomics experiments are integrated for comparison, and 19 pathways are found to contain differentially expressed proteins and metabolites at the same time, which belong to amino acid metabolism, nucleotide metabolism, lipid metabolism, carbohydrate metabolism, and other types of biological function. All of these results confirm the cytotoxicity of nickel ions from many aspects. |
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