| Easily mimicing the extracellular matrix and providing appropriate contact guidance to help nerve cells differentiation and neural synaptic growth, nanofibers become one kind of biomaterials of the nerve tissue engineering. For further application, a comprehensive in-depth interpretation of the molecular mechanism of how directional nanofibers affect neural cell differentiation should be known, which suggests the necessary to study the differential expression of all genes, mRNA, proteins, metabolites, and the relationship between them in such a process. The emergence of high-throughput techniques and systems biology approach recently provides a strong technical support for us to study the influence of the molecular mechanism by detecting the differential expression of mRNA, proteins, metabolites. In the end, A comprehensive understanding of the interaction between biomaterials and cells will be showed.This thesis aims to investigating the expression of intracellular metabolites of PC12 cells when they are differentiating on aligned/random levorotatory polylactic acid nanofibers by metabonomics technology and bioinformatics methods, which reveals how aligned/random nanofibers effects on differentiation of PC12 cells at the metabolic level. After that, a conjoint analysis of the previous studies (the transcriptome and proteomics) and the current study was performmed to explain the mechanism of how aligned/random nanofibers effects on differentiation of PC12 cells.The specific work of this paper is as follows:1. Prepare aligned/random PLLA nanofibers by electrostatic spinning and PLLA films by spin coating. The characterization of three kinds of materials was investigated by Scanning electron microscopy(SEM). The diameters of aligned/random PLLA nanofibers were about279.58±6.67 nm and 245.02±7.93nm.2. After predifferentiation for 48h, PC12 cells were planted on the aligned PLLA nanofibers (AF), random PLLA nanofibers (RF) and PLLA films (Control) for 12h,24h, and 36h to collect cell samples for metabolomics experiment. There are six experimental groups (A12, A24, A36, R12, R24 and R36), and three control groups (C12, C24 and C36), with 106～107 cells in each group (n=5).3. Metabonomics means based on liquid chromatography mass spectrometry was performed to investigate the intracellular metabolites of PC12 cells planted on the aligned PLLA nanofibers (AF), random PLLA nanofibers (RF) and PLLA films (Control) for 12h,24h, and 36h. Spectrum peak figures of those six experimental groups and three control groups were obtained. Principal component analysis was performed to investigate the difference between the groups. A combination of VIP value of Partial least squares discriminant analysis with p value of t test was to screen differentially expressed metabolites. As a result, the number of metabolites of differentially expressed in A12 group and R12 compared to C12 group is 53 and 54, respectively. The number of metabolites of differentially expressed in A24 group and R24 compared to C24 group is 50 and 47, respectively. The number of metabolites of differentially expressed in A36 group and R36 compared to C36 group is 32 and 42, respectively.4. Bioinformatics method of the cluster analysis and MetPA analysis was applied to analysis the differentially expressed metabolites and the pathway they participated in. Reaults shows PC12 cells the differentially expressed metabolites of PC12 cells planted on the aligned/random PLLA nanofibers for 12h are quite different from PC 12 cells planted on the aligned/random PLLA nanofibers for 24h and 36h. As for the pathway analysis, phenylalanine, tyrosine and tryptophan biosynthesis and phenylalanine metabolism are pathways associated with the differentiation of neurocyte, which shows high impact factors both in AF group. and RF groups. Arachidonic acid metabolism and taurine, taurine metabolic are also pathways associated with the differentiation of neurocyte,which only showes high impact factors AF group.5. a conjoint analysis of the previous studies (the transcriptome and proteomics) and the current study was performmed, which showes 11 pathways of the differential expression of mRNAs, proteins, and metabolites of AF group participated in and 7 pathways of the differential expression of mRNAs, proteins, and metabolites of RF group participated in. Among these pathways, Only three pathways are associated with the differentiation of neurocyte. Tyrosine metabolism is the one owned by both AF group and RF group. Sphingolipid metabolism and glycerol phospholipid metabolism are pathways only owned by AF group. There are two aspects to explain how aligned PLLA nanofibers affect the differentiation of PC12 cells. First, the expression of Laol and Gstzl affects the expression of tyrosine. The expression of Aoc3 and Aldh3al affects the expression of dopamine. Second, The expression of Pla2g4b affects the expression of phospholipase A2, which promots the release of lysophospholipids and arachidonic acid. Inside the PC12 cells planted on the aligned PLLA nanofibers, the down-regulated expression of phenylalanine and its metabolites and up-regulated expression of arachidonic acid and dopamine,which are beneficial to the differentiation of PC12 cells.To sum up, aligned PLLA nanofibers is a biomaterial surface suitable for PC12 cells to differentiate. |
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