Dissertation > Excellent graduate degree dissertation topics show

Heterogeneous Expression of AFP and CK19 in Different Subpopulations of Hepatocellular Carcinoma Cells

Author: ZhouHongBing
Tutor: ZuoKeFeng
School: Fourth Military Medical University
Course: Surgery
Keywords: Hepatocellular carcinoma Marginalized groups AFP Cytokeratin Flow cytometry
CLC: R735.7
Type: Master's thesis
Year: 2008
Downloads: 119
Quote: 0
Read: Download Dissertation

Abstract

Tumorigenesis is currently a hot research field, the current in the academic process of its formation has not yet been unified understanding. In recent years, some scholars have proposed tumor cells and stem cells have a naive phenotype and many similarities, and thus raise cancer stem cell theory. Studies have found that acute myeloid leukemia cells can be isolated from bone marrow hematopoietic stem cells and stem cell surface markers with the same cancer cells, these cells can be induced in immunodeficient mice with acute myeloid leukemia; This indicates that the tumor tissue may be there is both a stem cell characteristics, but also has characteristics of cancer stem cells in tumors. Currently, there are reported in the literature in a variety of solid tumors were found to have the presence of cancer stem cells, such as breast cancer, brain tumors. With further research, there are a variety of methods can be applied to tumor tissue with stem cell-like properties of tumor cells isolated and purified. Using stem cells for a variety of dyes and alien substance has exclusive specificity feature flags, excited by the ultraviolet light source combined with fluorescence-activated cell sorting by flow cytometry method (FACS), can be low fluorescence sorting and aggregation showed borderline the edge of the group of cells (side population; referred SP cells). After a more in-depth study, the subgroup cells have been initially confirmed enriched with stem cell-like characteristics with cells. At present, this separation and enrichment methods stem-like cells have been successfully applied in various tissues of adult stem cells in the separation process, this method compared with the original magnetic activated sorting and fluorescence-activated sorting more efficient. Tumor cells and stem cells in the unlimited proliferation, differentiation and self-renewal of multi-directional mechanism has similarities. Tumor heterogeneity theory has been widely confirmed. HCC is one of common malignant tumors, because the prognosis is poor, so the higher mortality rate. On hepatoma cells specific subsets of cells were isolated, it may be the occurrence of liver cancer recurrence, metastasis, drug resistance form the best way. Alpha-fetoprotein (AFP) is embryonic antigen, or as a pathological state of embryonic gene expression product, it is accompanied by strong embryonic development and cell division process. In addition, AFP also has many functions in the body and play a variety of important physiological and pathological effect. Organized CK19 expression and differentiation specificity, is screened and highly expressed in the liver stem cell-specific marker for better is one of the bile duct epithelium-specific markers. CK19 in normal adult liver expressed only in the bile duct epithelial cells, but not expressed in the liver cells. CK19 expression was positive hepatocytes are considered stem cells derived from the liver, and is located in an early differentiation. Studies have found that when the liver cells become cancerous, some cancer cells may express CK19, and its expression level and cell differentiation was negatively correlated. This study intends to flow through the UV excitation of fluorescence-activated cell sorting method to obtain the two hepatoma cell subsets - side population cells (SP) and non-edge group of cells (Non-SP), and by indirect immunofluorescence approach to understanding cell subsets in HCC AFP and CK19 expression in the existence of significant differences, through the two subpopulations cell cDNA gene amplification understand SP cells CK19mRNA transcript levels of AFP and whether the Non-SP cells differ . In order to further understand whether SP cells rich in clonal expansion, the formation of tumors and self-renewal ability, more primitive stem cell-like tumor cells, and further reveal hepatoma cells with stem cell-like occurrence of liver cancer, recurrence, metastasis and resistance Relationship between drug. Finally, the use of immunohistochemical methods observed liver tissue expression of AFP and CK19, revealing its two early recurrence and the relationship between the clinical phenomena. Objective: ultraviolet light source excitation fluorescence-activated flow cytometry sorting sorting method in hepatoma cell lines MHCC97 edge groups and non-edge group two subsets of cells were observed by indirect immunofluorescence level of AFP and CK19 protein expression changes and content, using semi-quantitative RT-PCR analysis of AFP and methods CK19mRNA transcript levels to explore different cell lines in MHCC97 subsets differentially expressed AFP and CK19 significance. Observed by immunohistochemical methods HCC AFP and CK19 expression levels. Method: 1 MHCC97 on human hepatoma cell line in the edge of the base cell sorting and cell differentiation capacity of marginalized groups detection; 1) conventional cell culture; 2) 0.25% trypsin digestion of cells mild, routine centrifuged and washed with HBSS cells; 3) SP cells and Non-SP cell detection and sorting preparation Hoechst33342 stained liver single cell suspension, the control group on the basis of added calcium channel antagonist molecule ABC transporter ABCG2 protein family of active efflux effect. Hepatoma cells by flow cytometry Hoechst33342 fluorescence, ultraviolet excitation light source, reflected by the dichroic filters and filters in different combinations in the hepatoma cell fluorescence signal into two parts to HoechstRed the X-axis, Hoechst Blue for the Y axis a two-dimensional scatter plots, the low low Hoechst Red and Hoechst Blue and the control group missing area is set to SP cells of the \4) According to AFP provided Genebank (AFP) and cytokeratin 19 (CK19) gene sequences, primer design software using Primer5 respectively, two semi-quantitative gene primers were designed, synthesized and sent to the company. Respectively, marginalized groups and non-side population cells in both cell clusters cell cDNA as a template, the line semi-quantitative detection. 5) The two groups of cells were seeded coverslips, AFP and CK19 were performed two-color immunofluorescence. Using the Image-Pro Plus5.0.2 SP cells were detected by the software and non-SP cells seeded average integrated optical density, the cells were compared AFP and CK19 expression. Using SPSS 11.0 statistical package using a paired t-test to analyze the data. 2 Purchase HCC tissue microarray, AFP and CK19 were performed immunohistochemical staining. Compare the two groups of cells in tissue specimens AFP and CK19 expression. Result: a dual-wavelength fluorescence flow cytometry analysis showed that there is an edge on the two-dimensional scatter plot group of cells present. MHCC97 cell lines marginalized groups account for nearly 0.25% of cells. After adding the calcium channel blocker group of cells was significantly reduced, because the disappearance of low fluorescence characteristics of non-marginalized groups into the cell populations, suggesting that SP cells MHCC97 cell lines and cell surface phenotype active transporter molecules active efflux of ABCG2 function on; 2 SP cells and Non-SP cells AFP and CK19 gene semi-quantitative RT-PCR analysis using the agarose gel electrophoresis bands of AFP and CK19 mRNA and G3PDH mRNA optical density optical density values ??for statistical analysis showed that the ratio of SP cells group AFP and CK19 mRNA transcript levels were significantly higher than the Non-SP cells group. 3 SP cells and Non-SP cells AFP and CK19-color immunofluorescence and Non-SP SP cells line cells seeded cells were cultured for 4 hours were observed adherent stretch, AFP and CK19 were performed two-color immunofluorescence and laser of Focus microscope, SP and Non-SP cells were seen within the cytoplasm of the red fluorescent protein expressing AFP particles, CK19 green fluorescent protein particles. Application of Image-Pro Plus5.0.2 SP cells were detected by the software and non-SP cells seeded average integrated optical density, the cells were compared AFP and CK19 expression. With SPSS11.0 statistical package using a paired t-test to analyze the data. MHCC97 cell lines showed that non-marginalized groups marginalized groups and between cells AFP and CK19 expression at the protein level there is a significant difference. 4 liver cancer tissue microarray AFP and CK19 protein expression detected by immunohistochemistry on tissue microarray HCC AFP and CK19 staining were performed under a fluorescence microscope visible part of the organization intracytoplasmic brown particles were compared cells in tissue specimens AFP and CK19 expression. Results vast majority of HCC expressed AFP, partial liver tissue expression CK19, a few hepatocellular carcinoma expressing both AFP, but also expressed CK19. Conclusion: a dye through the use of flow cytometry Hoechst33342 confirmed human hepatoma cell lines exist side population cells MHCC97. 2 MHCC97 source side population cells co-expressed AFP and CK19, suggesting that the edge of the group of cells does have some bi-directional differentiation capabilities. This shows that the edge of the group of cells with some original features. SP cells enriched further elucidate phenotypic naive, clonal expansion, tumor formation and self-renewal ability, more primitive stem cell-like tumor cells, and suggesting that the incidence of HCC recurrence, metastasis and resistance to the formation of relationships . 3 Most HCC expressed AFP, partial liver tissue expression of CK19, small hepatocellular carcinoma expressing both AFP, but also expressed CK19. Since normal liver cells do not express CK19, CK19 expression in hepatoma cells suggested may be derived from certain progenitor cells in hepatocellular carcinoma.

Heterogeneous Expression of AFP and CK19 in Different Subpopulations of Hepatocellular Carcinoma Cells

Abstract

Related Dissertations