The Study On The Cytotoxic Biomarkers Of Ni²⁺with Multi-omics Technologies

It is important to ensure safety of biological materials in clinical applications.Before the biomaterials are implanted,the potential risks of biomaterials can be identified by biomaterial biomarkers.The research of biomaterial biomarkers can establish an efficient,rapid,sensitive and accurate evaluation index.In addition,a large number of new materials can be screened efficiently and rapidly by choosing material preliminarily according to biomaterial biomarkers,which can help people save both cost and time.In conclusion,the research of biomarker is important for predicting the potential toxicity of biomaterials.Because of the shape memory effect,the alloy of nickel and titanium is widely used in lots of fields,especially in medical realm.It is often used in artificial bones,orthodontic wires and cardiovascular implant devices.Because the release of Ni²⁺can lead to cell toxicity after implantation,it is necessary to study the biomarkers that can evaluate the Ni²⁺cytotoxicity.At present,researcheres analyzed the cytotoxic biomarkers of Ni²⁺with one kind of omics technology,which lacked conjoint analysis of multi-omics technologies.This led to the inconsistencies in biomarkers which screened by omics technologies.Moreover,bioinformatics analysis in omics only theoretically analyzes the function of biomarkers,which lacks a series of verification to determine the biomarkers,thus leads to invalid biomarkers guidance to the development of biomaterials.The aim of this thesis is to initiatively conduct high-throughput screening with multi-omics technologies and validation experiments for the cytotoxic biomarkers of Ni²⁺,and to further investigate the biomarkers'mechanisms of action.At the same time,the thesis hope to reveal the relations among these confirmed genes,proteins,and metabolites biomarkers in the impacts of Ni²⁺on cells by using correlation analysis and validation,and finally identify the pathway from gene transcriptionto protein expressiontometabolite changes.The main contents of this thesis are as follows:1.The test of Ni²⁺cytotoxicity:The cytotoxicity of L929 cells which were treated by Ni²⁺?100?M 200?M,12h 24h 48h 72h?can be evaluated through MTT method.The experiment showed that Ni²⁺could affect the proliferation of L929 cells2.Genomics experiment:RNA-Seq was employed to study the genes of L929 cells after growing in 100?M and 200?M Ni²⁺for different periods?12h 24h 48h 72h?.The L929 cells cultured in normal medium were used as control group,and the cells from each group were collected before sending to company.3.Screening biomarkers:By analyzing the results of genomic experiment,proteomics experiment and metabonomics experiment,340 differentially expressed genes,114differentially expressed proteins,9 differentially expressed metabolites were found in several experimental groups;8 genes/proteins were found by conjoint analysis of genomics and proteomics as candidate genes/proteins biomarkers;The screening of the 8 candidate genes/proteins biomarkers and 9 differentially expressed metabolites involved in pathways for cytotoxicity was performed by the biological pathway analysis.Finally,4 genes/proteins and 5metabolites were chosen as the target biomarkers.4.Verifying the expression levels of biomarkers:4 gene/proteins biomarkers were verified by RT-PCR and Western blot methods to find the biomarkers which results of omics experiments and verification experiments were consistent.According to related literatures,UQCRB and CSF1 were considered to have the biology functions of associated with the cytotoxic biomarkers of Ni²⁺;5 candidate metabolites biomarkers were verified by LC-MS/GC-MS method,and the Sphinganine,Uridine and Hypoxanthine were determined as potential metabolites markers to evaluate the cytotoxic biomarkers of Ni²⁺.5.Verifying the function of biomarkers:Constructing Uqcrb and Csf1 gene interference/overexpression stable cells by RNA interference and gene overexpression techniques;By the oxidative stress and mitochondrial membrane potential experiments,Uqcrb's function of regulation ROS and electron transport were identified,finally UQCRB gene/protein can be used as the cytotoxicity biomarker of Ni²⁺;Cell proliferation experiments had proved that CSF1 gene/protein was not necessary for cell proliferation,and CSF1gene/proteinwas not the cytotoxicity biomarker of Ni²⁺.6.Verifying the relations among genes,proteins,and metabolites biomarkers:By analyzing the results of genomic experiment,proteomics experiment and metabonomics experiment,the biomarkers which differentially expressed in some experimental group and involved in pathways for cytotoxicity were found;Constructing Acot2 and Aldh2 gene interference/overexpression stable cells by RNA interference and gene overexpression techniques;Proteins/metabolites biomarkers were verified by Western blot and LC-MS/GC-MS methods to reveal the relation of these genes,proteins,and metabolites biomarkers.The results showed that Ni²⁺can influence the expression of Acot2,Aldh2 genes biomarkers,thus lead to the variation of ACOT2,ALDH2 proteins biomarkers expression,and then change metabolites biomarkers.Finally the series of reaction to Ni²⁺may affect the function of cell membrane,intracellular ROS level and ATP generation.