Role Of Stem Cells Fusion In Gastric Carcinoma Caricinogenesis

Role of bone marrow derived stem cells fusion during the development and progression of gastric carcinoma was studied during this experiment. Chimeric mouse model was established through sex mismatch bone marrow transplantation by using female transgenic C57/BL-GFP mouse as donor and male C57/BL mouse as receiver. Then gastric carcinoma was induced by multiple carcinogens attack and cell fusion was assesed through Y chromosome fluorescence in situ hybridization (FISH). Mean while, gastric epithelial cells were isolated from mouse glandular stomach with collagenese digestion, and bone marrow derived stem cells were isolated from mouse bone marrow with MACS separation kit. Then cell fusion with polyethylene glycol (PEG) was performed between these cells. Cord blood mesenchymal stem cells (CB-MSCs) were also isolated and collected from our hospital and in vitro cell fusion was performed between these cells, and fusion cells were sorted with FACSVanture. Then morphology of the fusion cells was observed, and phenotypes of the cells were examined with immunohistochemistry (IHC) and flow cytometry (FCM). Then migration, invasion, and proliferation ability of the fusion cells were also assessed through cell scratch assay, transwell assay, and MTT cell proliferation assay.Part I Establishment of Chimeric Mouse Model and Induction of Gastric CarcinomaObjective Role of bone marrow derived stem cell in gastric carcinoma development and progression were still obscured, and through what mechanism it participated in this process was still under debate between transdiffertiation and cell fusion. In the present study, sex mismatch chimeric mouse was used to induce gastric carcinoma, and gastric sample was collected and examined for cancer induce rate and cell fusion, to investigate the role of bone marrow derived stem cells during the development and progression of gastric carcinoma.Method Chimeric mouse model was established through sex mismatch bone marrow transplantation by using female transgenic C57/BL-GFP mouse as donor and male C57/BL mouse as receiver. Then bone marrow and peripheral blood were collected on week1and week4to examine by FCM, and frozen slide of different organs were also collected to study the distribution of GFP+cells in different organs. Chimeric mouse was then used to induce gastric cancer by multiple carcinogens attack, and stomach samples were collected for pathology study and Y chromosome FISH for examination of tumor induce rate and cell fusion.Result All the mice underwent bone marrow transplplantation survived, and FCM results showed chimeric mouse bone marrow GFP+cells was7.48±1.38%and73.92±5.57%at week1and week4respectively. In the peripheral blood, only small portion of cells were GFP+on week1and recovery of CD8+cells was faster than that of the CD4+cells, while GFP+cells increased on week4but the amount of CD4+cells was still low.72.9%of chimeric mouse survived after multiple carcinogens attack. For those mice survive through carcinogens attack,18.2%of mice showed precancerous lesion and25.0%of mice were successfully induced with gastric cancer, with2adenocarcinoma and1squamous cell carcinoma. Great amount of GFP expression was detected for stomach sample with severe dysplasia or gastric cancer, and GFP expression was also detected in other different organs such as kidney, brain, liver, and spleen, but no GFP expression was detected in the heart. Great amount of GFP expression was detected for stomach sample with severe dysplasia, and most of the cells showed both GFP and Y-chromosome, which indicated that fusion had occurred (Figure3C). Great amount of GFP expression was also dectected in both adenocarcinoma and squamous cell carcinoma (Figure3D-E). Most of the cells in adenocarcinoma sample showed cells with Y-chromosome in the neucleus and these cells also expressed GFP in the cytoplasm, which indicated that cell fusion had occured. For squamous cell carcinoma, GFP expression was mainly detected in the interstitial or keartine perals, but no sign of fusion was detected. Normal chimeric mouse showed little GFP expression in the interstitial, but no fusion was detected. Great amount of GFP expression was detected for stomach sample with severe dysplasia, and most of the cells showed both GFP and Y-chromosome, which indicated that fusion had occurred.Conclusion Sex mismatch chimeric mouse model was successfully established. Mouse gastric inflammation, severe dysplasia, and gastric cancer were successfully induced through multiple carcinogens attack. BMSCs involve in the renewal of vessel endothelial cells, and some of the cell participated through cell fusion. BMSCs participated in the renewal of gastric mucosa during gastric inflammation and dysplasia through cell fusion. When tumor occurred, fusion cell or BMSCs have become the main cells in tumor with little amount of fusion cells been detected.Part Ⅱ Fusion of Mouse Bone Marrow Derived Stem Cells and Gastric Epithelial CellsObjective Role of cell fusion during tumor development and progression was worth of attention, whereby it might enhance the metastasis ability of tumor cells or potentially be the source of cancer stem cell. In the present study, mouse gastric epithelial cells and mouse BMSCs were isolated and collected. Cells collected were used for in vitro cell fusion to investigate the possibility for BMSCs fusion to be the primary mechanism of neoplasia during the development and progression of gastric tumor.Method Mouse gastric epithelial cells were isolated and collected with collagenese digestion, and phenotype of isolated cells was examined with IHC. Mouse BMSCs were isolated and collected with MACSs separation kit, and cells were examined with FCM. These cells were used for in vitro PEG cell fusion, and fusion rate were examined with FCM and fusion cells were sorted with FACSVantage.Result Mouse gastric epithelial cells were successfully isolated with collagenese digestion. For each mouse,5.27±2.92×106cells were isolated from glandular stomach and cell viability was97.05±2.20%. Proliferation of mouse gastric epithelial cells reached climax on7d, and IHC examination showed that93.21±3.35%of cells expressed cytokeratine-18(CK-18).8.88±4.22×107cells were isolated from tibia and femur bone and cell viability was98.35±1.23%.1.36±1.03×106BMSCs (Lin-) cells were isolated from4.43±2.47×107marrow cells. Before MACS separation,76.21±4.89%of marrow cells were Lin+. After MACS separation,69.23±5.79%of marrow cells were Lin+and93.93±3.35%of marrow cells were Lin-. After staining with CFSE or PKH26, mouse gastric epithelial cells and marrow cells (Lin+or Lin-) expressed either green or red fluorescence, and co-culture of these cells showed no sign of cell fusion. After in vitro PEG cell fusion assay, fusion cells can be observed under fluorescence microscope and fusion rate was5.77±1.91%when examined with FCM. Fusion cells showed no proliferation after FCM sorting.Conclusion Mouse gastric epithelial cells were successfully isolated with collagenese digestion, and proliferation reached climax at7d after isolation. Mouse were successfully isolated from mouse bone marrow with MACS. In vitro PEG cell fusion of these cells was successfully. Proliferation of fusion cells sorted with FCM was unsuccessful, which might be due to that both mouse gastric epithelial cells and BMSCs were of primary culture, therefore the daughter cells after cell fusion did not transformed or gain the phenotype of BMSCs to continuously proliferation. On the other hand, the low fusion rate decreased the chance for fusion cells to go through phenotype transformation or gaining the phenotype of mother cells.Part III Fusion of Human Cord Blood Mesenchimal Stem Cells and Human Gastric Epithelial CellsObjective Proliferation of fusion cell between mouse gastric epithelial cells and mouse BMSCs was unsccesful on our previous experiment. In the present study, immortalized human gastric mucosal epithelial cell line (GES-1) and cord bold mesenchymal stem cells (CB-MSCs) were used for in vitro PEG cell fusion to investigate the possibility of epithelial malignant transformation induced by cell fusion and to explore the origin of cancer stem cells.Method GES-1cells were culture and CK-18expression was determined by IHC. CB-MSCs were isolated from cord blood collected in our hospital, and then the growth characteristics of was monitored. Mesenchymal stem cell-specific phenotype of Thy-1(CD90)、SH2(CD105)、SH4(CD73)、HLA-ABC、HLA-DR、 CD34、CD45were identified by flow cytometry. GES-1cells were labeled with PKH26and CB-MSCs were labeled with CFSE, and fusion of these cellswere induced with PEG. Fusion rate was then determined with FCM and double positive fusion cells were sorted. Then growth characteristics of fusion cells were monitored and CK-18expression was determined by IHC. Cell surface antigen CD90and DNA Ploidy Analysis of fusion cells were determined by FCM. Then migration, invasion, and proliferation ability of fusion cells were assessed by cell scratch test, twanswell assay, and MTT cell proliferative assay. Tumorigenicity of fusion cells were assessed through BALB/c nude mouse subcutaneous injection.Result CB-MSCs were successfully isolated from cord blood, and the first generation of CB-MSCs obtained an oval or spindle shape. After passaging, cells became polygonal with strong adherent ability and self-renewal capacity, and CB-MSCs could be cultured for25passages in vitro. CB-MSCs showed no expression of hematopoietic marker CD34, CD45, HLA-DR, but expressed the typical MSC marker proteins SH2(CD105), SH4(CD73), HLA-ABC. However, the intensity of expression of CD90was significantly below that of other markers. In vitro cell fusion between CB-MSCs and GES-1was successfully performed, and fusion cells were sorted with FCM with fusion rate of8.7%. CK-18was highly expressed within the cytoplasm of fusion cells. Morphological changes of fusion cells included:greater heterogeneity in the size and shape of cells and nuclei, increased nuclear:cytoplasmic ratio as characteristics of malignant cells. GES-1did not express CD90while the fusion cells acquired the markers of CD90. DNA ploidy analysis showed that fusion cells consisted of polyploid and aneuploid cells and with the increase of passages the proportion of aneuploid cells increased. Proliferation, migration and invasion ability of the fusion cells were increased. Subcutaneous mass was observed after injection of fusion cells, and HE with IHC results showed mass with glandular structure of epithelial origin.Conclusion CB-MSCs were isolated, collected, and successfully fused with GES-1under PEG stimulation. Fusion cells were morphological different from typical MSCs and GES-1cells, and showed characteristics of both parental cells, such as high expression of CK-18in plasma and is also obtained the surface antigen CD90. Proliferation, migration and invasion ability of the fusion cells were increased, indicating that fusion between MSCs and epithelial cells can result in enhance metastasis ability. Compare to GES-1and MSCs, fusion cells were able to form subcutaneous mass.