| BackgroundGlioblastoma multiforme (GBM) is the most common and most malignant adult intracranial primary tumor, with high recurrence rate, poor prognosis and short survival and high mortality. Even the current standard of care is surgical resection to the range of maximum safe, following by adjuvant radiotherapy plus temozolomide (TMZ), patients still have a high recurrence rate and poor prognosis.Large sample clinical data has been confirmed that the patients with GBM whose care is surgical resection to the extent feasible, followed by adjuvant radiotherapy plus TMZ have a clinically meaningful and statistically significant survival benefit overall survival and two-year survival rate compared with radiotherapy alone. The tumor cells in patients with TMZ resistance is one of the main reasons for poor prognosis.Although invested a lot of research for TMZ resistance on GBM, TMZ specific resistance mechanism is still not clear.Current research on TMZ drug-resistant mechanism is mainly a single protein, and there are a lot of proteomics researches on TMZ resistance mechanism, but it’s still not clear. Proteomics is a large-scale, high-throughput, systematic research for all proteins by biochemical methods. It contains protein expression level, the protein modification after translation and the interaction between protein and protein. Thus, we can aquire comprehensive understanding for the disease and cells metabolism. However, with the deepening of the research, it was found that the whole proteome is dynamic, and there are specific to the time and space diversity. Under the influence of the external environment, different cells of the same species or the same cells in different periods which the proteome is changing. Therefore, the subcellular proteomics is the inevitable trend of development of proteomics. Subcellular proteomics research not only can reduce the complexity of whole cell proteome analysis, but also can enrich the low-abundance proteins, and improve the detection rate of low-abundance proteins, and deepen our understanding of the structure and function of the subcellular component, which can reflect some protein quantitative, qualitative change and migration of protein sensitively caused by some pathological factors.And therefore it will help us better understand the life activities. The analysis of the different expression of the subcellular proteome in different stages and studying its function provide a powerful means for us to explore the related mechanism and screen the specificial biomarkers. Therefore, in this paper, we adopt the subcellular proteomics research strategy to investigate the resistance mechanism of TMZ for GBM.Reviewing of previous literatures, the mechanism TMZ resistance researchs are mainly drug-resistant cell lines (natural or induced resistance) in comparisonwith sensitive cell lines. But it is rare to focus on the process that the GBM cells transform sensitive for TMZ into resistance for it, and the one we found in clinic. U87 cells were incubated with 200μM of TMZ for 1 week, and the majority of cells have died, but there are still a small number of survival cells.We found that the survival cell morphology changed significantly.The cell body and nuclei were bigger, and the cell atypia became more obvious. However, with the duration incubated withTMZ extending, the cell morphology gradually reverted to a previous normal state. This phenomenon implies this time may be the critical period which the U87 cells resisted for TMZ. So we choose the cells incubated with TMZ for 1 week, and then applied subcellular proteomics methods to screen differential proteins to discuss the the mechanism TMZ resistance. At last, we hope we can find some new biological markers to increase the sensitive to TMZ or inhibit the TMZ resistence.Therefore, in this research, U87 cell lines are studied. When the U87cells are incubated with TMZ for 1 week, the cells are collected and then extracted cytosolic proteins and nuclear proteins. We use subcellular proteomics label-free method to identify and quantify the level of protein expression, and the carry on significant difference analysis of protein by bioinformatics to screen differential proteins. At the same time, GO annotation and enrichment analysis, KEGG pathway annotation and enrichment analysis and clustering to make to find significant differential protein. At the end, we hope we can find some new biological markers to reveal the TMZ resistant mechanism and increase the sensitive to TMZ or inhibit the TMZ resistence and provide new theoretical foundation with targeting therapy of GBM. In order to further in-depth explore the function of these screened proteins, good cell model is the foundation of the following study. Primary cells are the most commom tools to study tumor pathogenesis, resistance to chemotherapy, because its nature and characteristics are closer to the original state of humans. However, traditional glioma primary cell culture method is time-consuming, complicated operation, low success rate, so we improve it, and culture enough primary glioma cells, which provides a good tool to study the function of these differential proteins in cellular level.Chapter 1 Subcellular proteomics identify and quantitate differential proteins after TMZ induced U87 cells for 1 weekPurpose:Use Subcellular proteomics identify and quantitate differential proteins from cytosolic proteins and nuclear proteins after TMZ induced U87 cells for 1 week, and then screen the differential proteins and related pathway associated with TMZ resistance, to find some new biological markers to increase the sensitive to TMZ or inhibit the TMZ resistence, to explore the mechanism of TMZ resistance.Methods:U87 cells were incubated with 200 μM of TMZ for 1week, and those treated with medium containing DMSO served as controls. Cell morphology was observed and test the number of live cells and cell vitality above two groups. Cytosolic proteins and nuclear proteins were extracted, respectively. The mean of Bradford was used to quantitate each protein sample, and Western-Blot verifies the purity of cytosolic proteins and nuclear proteins. Silver stain initially tested whether cytosolic proteins and nuclear proteins expressed differentially or not.Selecting the protein samples (repeat 3 times) and then using subcellular proteomics label-free method identify and quantify the level of protein expression, and the carry on significant difference analysis of protein by bioinformatics to screen differential proteins(standard Ratio>+/-2.0 and P value<0.05). At the same time, GO annotation and enrichment analysis, KEGG pathway annotation and enrichment analysis and clustering to make to find significant differential protein.Results:After U87 cells treated with TMZ and DMSO respectively for 1 week, a number of cells are dead in the TMZ group, and the morphology of remained alive cells changed significantly, both cell bodies and nucleus became much larger, and almost all the alive cells extended some long protrusion from their cell bodies. Cell viability decreased obviously, compared with DMSO group, number of live cells was also significantly reduced. This result is consistent with previous experimental results. The cells are collected and then extracted cytosolic proteins and nuclear proteins, respectively. And then the proteins are quantified, the total proteins meet the demands of subsequent experiments. Western Blot results show that there is no pollution between cytosolic proteins and nuclear proteins, and nuclear/Cytosol fractionation succeed. The result of silver staining displays that each group is significantly different. Mass spectrometry analysis showed that the distribution of sample signal peak and mass spectrum signal strength are similar in the group, and which is obviously different between groups. Label free method is used to identify and quantify the changes of cyoplasmic protein and nuclear protein expression in each group, and the total number of proteins is 3910 from the original data. According to the screening criteria, there are 304 differential proteins to be screened. Among them, the number of differential proteins is 148 in the cyoplasmic protein, and 90 kinds of proteins are upregulated,58 kinds of proteins are down regulated in them.156 kinds of proteins are screened in the nuclear proteins, and the up-regulated proteins are 76, and the down-regulated proteins are 80 among them. GO enrichment analysis of differential proteins shows that the up-regulated differential proteins in the cytoplasma are mainly distributed in the ribosome in cellular component, and from the molecular function, its main function is involved in ribosome structure component, and from the biological processes,these aproteins are related to translation and post-translational modifications. While the up-regulated differential proteins in the nucleus are located in the nucleus, the molecular functions are related to the replication and transcription of DNA, and mainly participate in the metabolism of base compound and chromatin remodeling. However, the down-regulated differential proteins in the cytoplasma and nucleus don’t appear obvious central tendency which are related to the resistence of TMZ. KEGG pathway enrichment analysis found that the up-regulated differential proteins in the cytoplasma are mainly associated with the transport of RNA signaling pathway, ribosome signaling pathway. In the nucleus proteins, the up-regulated differential proteins are closely related to mismatch repair signaling pathway, transcriptional misregulation signaling pathway and spliceosome signaling pathway. We found that both them are associated with PI3K-Akt signaling pathway in the down-regulated differential proteins of cytoplasma and nucleus. In addition, the down-regulated differential proteins in the nucleus are also related to MicroRNAs in cancer signaling pathway. By GO enrichment analysis and KEGG pathway enrichment analysis, we identify 5 kinds of differential proteins which may be closely associated with the resistence of TMZ.Conclusion:This experiment used the subcellular proteomic Label free technology to study the changes of proteins of cytoplasma and nucleus when U87 cell were incubated with TMZ for 1 week, and screen differential proteins of cytoplasma and nucleus respectively. By the GO enrichment analysis of differential proteins, we found that the up-regulated differential proteins in the cytoplasma are mainly located in the ribosome, and participate in translation and post-translation modifications, which suggest that the translation and post-translation modification process are closely related to the resistence of TMZ, and they are worthy of study further. And the down-regulated differential proteins in the nucleus are mainly located in the nuclesus, and associated with replication and transcription of DNA, and participate in the metabolism of base and chromatin remodeling process. These results are in accordance with the DNA damage repair of tumor cells caused by TMZ, so we think the nuclear proteins may play a important role to participart in the resistance of TMZ. However, the down-regulated differential proteins in the cytoplasma and nucleus don’t appear obvious correlation with the resistence of TMZ, so the next step analysis of differential proteins don’t take them into consideration. As the same time, by the KEGG pathway enrichment analysis, we select the signaling pathways which are closely telated to the drug resistence of TMZ, and analyse the differential proteins participating in these signaling pathway. Based on the result of GO enrichment analysis and KEGG signaling pathway analysis, we have selected 5 kinds of protein may be closely related to TMZ differences, which laid a foundation for further investigate TMZ resistance mechanism.Chapter 2 The improved rapid enzymatic digestion primary cell culture method of human glioma cellsPurpose:To explore an improved enzymatic digestion primary cell culture method of human glioma cells, and to provide the support for the later experiments in the primary cell level verification protein function.Methods:Using the new method to culture, purify and pass the glioma cells from 32 glioma patients of different grades. The morphology was inspected by a inverted phase contrast microscope, The cells were identified by immunofluorescence technique, and the growth curves (the method of CCK) were analyzed to investigate the growth character of cells in vitro culture.Result:The primary human glioma cells by the new method were successfully cultured and transferred. The successful rate is 87.5%, and the cell morphology varied from different gliomas. These cells which glial fibrillary acidic protein (GFAP) was positive were determined to be the glioma cells. In addition, cell counting kit-8(CCK-8) assay indicated that the cultured cells had the proliferative ability, and the higher grade of the glioma was, the stronger of the proliferative ability would be.Conclusion:Modified enzyme digestion method is simple, efficient and has a high success rate, and can provide a good guarantee for the latter experiment. |
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