| Objective: Toexplore a simple,rapid and non-invasive method for the diagnosis of colorectal cancer.Rapid and non-invasive identification of benign and malignant colorectal exosomes by optical instrument Raman spectroscopy was carried out by means of isolated exosomes.Secondly,through the bioinformatics analysis of human colorectal cancer exosomes miRNA,the differential expression of exosome miRNA and its regulatory enrichment pathway were studied.Methods: The colorectal cells are cultured and the culture medium of the cells is collected,the exosomes produced by the cells are extracted by ultra-high speed centrifugation,and exosomes are identified by a series of recognized methods.Then,the extracted exosomes were analyzed by Raman spectroscopy for the exosomes of benign and malignant cells.The bioinformatics method was used to analyze and screen the differentially expressed genes transcribing miRNAs and their enrichment pathways.And preliminarily understand the internal mechanism of miRNA in regulating the occurrence and evolution of colorectal cancer.The first part of the thesis: exosomes originate from the release of cells,contain genetic material such as proteins,nucleic acids,lipids and other components that distinguish the released cells,and become an important substance for liquid biopsy.The target cells are cultured and the cell culture medium is collected,and the exosomes are extracted using an optimized ultracentrifugation protocol.Characterization and identification of biophysical features such as morphology and size of exosomes and their surface proteins by physical methods such as transmission electron microscopy(TEM),nanoparticle tracking analyzer(NTA),flow cytometry(FCM),and Western-Bloting methods CD9,CD63,CD81,TSG101,etc.The second part of the thesis: Raman spectroscopy in liquids without labeling can directly measure the composition of exosomes.The combination of optical instruments and computer can directly screen and identify the characteristic spectra of benign and malignant extracellular bodies.The target cells are cultured and the cell culture medium is collected,and exosomes of each cell are obtained by modified ultra-high speed centrifugation,respectively.Raman spectroscopy was performed after passing the first part of the exosome identification method.Raman spectroscopy waveforms of three kinds of tumor cell exosomes and one normal cell exosome were used to analyze the Raman spectrum waveform,and each peak and its possible corresponding material composition were analyzed and compared,and the characteristic spectrum of the material was summarized and analyzed.In addition,principal component discriminant analysis was performed on these exosome Raman spectroscopy data,and cluster analysis was performed based on the principal components of the spectra.The third part of the thesis: Exosomes miRNAs use different mechanisms to sort out the genetic material in the mother cells,which may become an important substance regulating the signal pathway of the target gene.The target cells were cultured and the cell culture medium was collected,and exosomes were obtained after ultracentrifugation and correlated.Exosomal samples were qualified for sequencing for exosome miRNA sequencing.The sequencing data of the miRNA is first tested for the quality and length of the miRNA tag fragment,and the classification and annotation analysis is performed through different databases.Then,a significant differential expression of the miRNA and its corresponding predicted target gene was found by comparison between the tumor cell exosomes and the normal exosome group.The GO and KEGG enrichment analysis of predicted target genes was performed by gene function and pathway database,and the common pathways of enrichment pathways between the groups were screened to find out the signaling pathways of exosome miRNA regulation.Results:1The cell culture supernatant extracted from the experiment was subjected to transmission electron microscopy at the Experimental Center of Guangxi Medical University,and CT26,Mc38,and Ana-1 electron micrographs were obtained.Electron microscopy of exosomes extracted by ultracentrifugation showed that the exosome background was clear,the distribution was uneven,and the size was acceptable.The measurement size was 40-200 nm,and the morphology was a typical double concave disc-like structure with a complete lipid package membrane structure.The electron micrograph of the exosomes extracted by the kit showed that the exosomes were relatively full and nearly round,but the background was quite mixed and unclear,and sometimes the exosomes could not be identified.2Detection of exosomes by nanoparticle tracking analyzer shows the size of benign and malignant extracellular bodies is basically the same,generally distributed between 40-200 nm,more between 100-150 nm,and the median value is distributed at 115.5-165.5nm.3Labeling exosomes with CD9-FITC and CD63-APC with fluorescently labeled antibodies for flow cytometry showed that exosomes of each cell contained CD63 and CD9 proteins..4There are various proteins on the surface of exosomes.Western-Bloting experiments revealed that CD9,CD63 and TSG101 showed corresponding bands in the molecular weight of the corresponding antibodies,in which CD9 was stronger,while CD81 showed almost no corresponding bands.Quantitative analysis of the gray values of three benign and malignant extracellular bodies showed that the expression levels of different surface proteins on the same exosomes were different,and the expression of a surface protein on different exosomes was not the same.5The Raman spectral data plots three connected peaks in the Raman shift of 1200-1800 cm-1,in which the Raman peak of malignant extracellular body and the benign cell exosome,the Raman peak of PBS Significant differences are formed and these differential spectra are likely to be characteristic Raman spectra.At the same time,the Raman spectra of the exosomes were analyzed by principal component discriminant analysis,and it was found that the tumor exosomes were clearly separated from the normal exosomes,and the two tumor exosomes overlapped largely and could not be separated.6Data quality evaluation of 12 sample exosomal miRNA sequencing data showed that the miRNA fragment yield rate was 98.8637%-99.1810%,the length distribution of the sequence fragment had a length of 22 bp,and a length of 29 bp.The miRNAs in the different databases GenBank,Rfam sequence annotations show a higher proportion and abundance,and the identification annotations show the relevant gene loci.7Screen miRNA sequencing data according to accepted criteria and find 8 significant differentially expressed miRNAs(hsa-miR-105-5p,hsa-miR-127-5p,hsa-miR-129-5p,hsa-miR-204-5p)hsa-miR-296-3p,hsa-miR-338-3p,hsa-miR-767-5p,hsa-miR-99a-5p).The miRNAs differentially expressed by cluster analysis,heat map analysis and principal component discriminant analysis showed that the exosomal miRNAs of the three samples of the same cell line clustered close to each other in the coordinate system;and the miRNA distance of the four different cell clusters of the same species is unequal,and the exosomal miRNAs of the three tumors are closer to each other than the benign exosomal miRNA.Target gene prediction,functional enrichment and KEGG pathway enrichment analysis and screening were performed through miRTarBase database and TargetScan database database,and the common signal pathway between each group was obtained to obtain the most probable mTOR enrichment signal pathway.Conclusion: 1 Culture the cells and collect the supernatant of the cells.The exosomes should be separated and extracted by ultracentrifugation.The exosomes can be identified by electron microscopy,NTA,flow cytometry and Western blotting.2Ramanspectroscopy can detect benign and malignant extracellular bodies in a liquid state and form a characteristic Raman spectrum,so as to achieve the purpose of distinguishing between benign and malignant exosomes.3Sample miRNA sequencing data quality assessment meets the requirements.Cluster analysis,heat map analysis and principal component analysis show the similarity of miRNAs in each sample.Further,8 significant differentially expressed miRNAs are screened by significant differences between groups.According to these expressed miRNAs,target gene prediction,GO function enrichment and KEGG pathway enrichment analysis were performed.4Through literature analysis and the characteristics of colorectal cancer,the most probable mTOR enrichment signaling pathway is obtained,which may be an important signaling pathway for exosome miRNA regulation.However,the experimental sample types and sample sizes are small,and the conclusions obtained have one limitation.Conclusion:1Culture the cells and collect the supernatant of the cells.The exosomes should be separated and extracted by ultracentrifugation,and the exosomes can be identified by electron microscopy,NTA,flow cytometry,Western blotting,etc.2Raman spectroscopy can detect benign and malignant extracellular bodies in a liquid state and form a characteristic Raman spectrum,so as to achieve the purpose of distinguishing between benign and malignant exosomes.3The quality evaluation of sample miRNA sequencing data meets the requirements.The similarity of miRNAs in each sample is shown by cluster analysis,heat map analysis and principal component analysis.Eight significant differentially expressed miRNAs are further screened by significant differences between groups.Target gene prediction,GO function enrichment and KEGG pathway enrichment analysis were performed based on these expressed miRNAs.4Through literature analysis and the characteristics of colorectal cancer,the most probable mTOR enrichment signaling pathway is obtained,which may be an important signaling pathway for exosome miRNA regulation.However,the experimental sample types and sample sizes are small,and the conclusions obtained have one limitation. |
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