| objectiveTo study the interactions and its mechanism of mouse bone marrow-derived mesenchymal stem cells with B16, a mouse malignant melanoma cell line, which further prove the resources of vascular endothelial cell on vascularization mechanism of malignant melanoma, and to investigate the expression of some cytokine in malignant melanoma and whether the interaction with mesenchymal stem cells affects the expression of some cytokine. The study could enrich and support the three stage of the vascularization mechanism theory of Professor Sun, but also provides the theoretical basis about autocrine mechanisms and positive feedback loop of vascular endothelial growth factor (VEGF) in malignant melanoma, which made us have more comprehensive understanding about biological functions of MSC & VEGF.MethodsMethods such as long bone separation, medullary cavity clysis and cell adherence selection, with cell culture conditions as appropriate serum concentration, cell density of subculture, make cell separation, extraction and amplification of mouse bone marrow-derived mesenchymal stem cells in vitro, then through the methods of immunohistochemistry testing CD44, CD 105, CD34 and CD45 staining, and other indicators to identify phenotype. Using the control mono-culture of MSC and the indirect co-culture with B16 cell in vitro, we observe the variance of MSC between the control mono-culture group and the indirect co-culture group in the cell morphous with light microscope, in the immunohistochemistry staining results, which observed by light microscopy and laser scanning confocal microscopy and analyzed by statistics, of seven indicators, such as CD44, CD105, CD34, CD45, VIII,VEGFR-1, VEGFR-2, and in the ultrastructure with transmission electron microscope. And there are also the immunohistochemistry staining results of VEGF and its receptors of B16 cells in the indirect co-culture group, we could make some judgments whether the B16 cells take the affect on biology characteristics of MSC on the basic of the results which we observed. Using the control mono-culture of B16 cells, the indirect co-culture of B16 cells with MSC and the direct co-culture of B16 cells with MSC in vitro, we observe the variance of B16 cells among the control mono-culture group, the indirect co-culture group and the direct co-culture group in the cell morphous with light microscope and in the immunohistochemistry staining results, which observed by light microscopy and laser scanning confocal microscopy and analyzed by statistics, such as VEGF-a, VEGFR-1 and VEGFR-2 etc, which further maybe detect the biology characteristics of B16 cells and whether the MSC cells take the affect on biology characteristics of MSC.ResultsThe results showed that after taking cell separation, extraction and amplification of mouse bone marrow-derived cells we get a kind of cells that are adhering growth closely the wall of culture dish, cloning growth and fibrocyte-like. Then the immunohistochemistry staining results of cell phenotype identification and the data through statistical analysis of the staining results shows that CD44 strong positive (average positive rates is 93.1%), CD 105 positive (average positive rates is 79.2%), CD34 negative (average positive rates is 4.7%, immunofluorescence negative), CD45 negative (average positive rates is 6.2%, immunofluorescence negative), VIII factor negative (average positive rates is 5.5%, immunofluorescence negative), VEGFR-2 negative (average positive rates is 4.5%, immunofluorescence negative), VEGFR-1 negative (average positive rates is 5.8%, immunofluorescence negative).Through the control mono-culture of MSC and the indirect co-culture with B16 cell, the results of experiments showed that: CD With light microscope observed, in some intensive region of the indirect co-culture group of MSC with B16, there are a few more cells that have a typical monolayer kind and paving-stone mosaic configuration;?The immunohistochemistry staining results of MSC and the data through statistical analysis of the staining results shows that MSC in the control mono-culture group is CD44 strong positive (average positive rates is 93.1%), CD 105 positive (average positive rates is 79.0%), CD34 negative (average positive rates is 4.8%, immunofluorescence negative), CD45 negative (average positive rates is 6.1%, immunofluorescence negative), VIII factor negative (average positive rates is 5.5%, immunofluorescence negative), VEGFR-2 negative (average positive rates is 4.5%, immunofluorescence negative), VEGFR-1 negative (average positive rates is 5.8%, immunofluorescence negative);MSC in the indirect co-culture group is CD44 positive (average positive rates is 73.9%), CD 105 positive (average positive rates is 62.3%), CD34 positive (average positive rates is 50.9%, immunofluorescence positive), CD45 weakly positive (average positive rates is 17.2%, immunofluorescence weakly positive, part of negative), VIII factor positive (average positive rates is 42.2%, immunofluorescence positive), VEGFR-2 weakly positive (average positive rates is 31.8%, immunofluorescence positive), VEGFR-1 positive (average positive rates is 29.7%, immunofluorescence positive);the statistical analysis of data shows that there are significant differences (P<0.01) in the seven indicators, CD44, CD105, CD34, CD45, VIII factor, VEGFR-1 and VEGFR-2 between the indirect co-culture group and the control group;?With transmission electron microscope observed, in some region of the indirect co-culture group of MSC with B16, We find that Weible-Palade body on round transverse section couldbe seen numbers of cross-section of microtubule, and on oval-shap longitudinal section seen vertical section of microtubule along the major axis inside the cell membrane;@The immunohistochemistry staining results of B16 cell and the data through statistical analysis of the staining results shows that B16 cell in the indirect co-culture group is VEGF-a strong positive (average positive rates is 93.6%, immunofluorescence positive), VEGF-b negative (average positive rates is 4.6%, immunofluorescence negative), VEGFR-2 positive (average positive rates is 89.4%, immunofluorescence positive), VEGFR-1 positive (average positive rates is 83.0%, immunofluorescence positive).Through the control mono-culture of B16 cells, the indirect co-culture of B16 cells with MSC and the direct co-culture of B16 cells with MSC, The immunohistochemistry staining results of B16 cell and the data through statistical analysis of the staining results shows that: ?B16 cell in the control mono-culture group is VEGF-a strong positive (average positive rates is 91.7%, immunofluorescence positive), VEGFR-2 positive (average positive rates is 90.4%, immunofluorescence positive), VEGFR-1 positive (average positive rates is 81.8%, immunofluorescence positive), VIII factor positive (average positive rates is 90.3%, immunofluorescence positive), VEGF-b negative (average positive rates is 5.1%, immunofluorescence negative);(2)B16 cell in the direct co-culture group is VEGF-a weakly positive (average positive rates is 31.5%, immunofluorescence positive), VEGFR-2 weakly positive (average positive rates is 22.4%, immunofluorescence positive), VEGFR-1 weakly positive (average positive rates is 26.1%, immunofluorescence positive), VIII factor weakly positive (average positive rates is 33.8%, immunofluorescence positive), VEGF-b negative (average positive rates is 5.8%, immunofluorescence negative);(3)B16 cell in the indirect co-culture group is VEGF-a strong positive (averagepositive rates is 91.0%, immunofluorescence positive), VEGFR-2 positive (average positive rates is 87.5%, immunofluorescence positive), VEGFR-1 positive (average positive rates is 86.2%, immunofluorescence positive), VIII factor positive (average positive rates is 90.2%, immunofluorescence positive), VEGF-b negative (average positive rates is 4.9%, immunofluorescence negative);@The comparison of indicators testing results among the control mono-culture group, the indirect co-culture group, and the direct co-culture group, show that there are significant differences (PO.01) in the four indicators, VEGF-a, VEGFR-2, VEGFR-1, VIII factors, and no differences in VEGF-b (P>0.05) between the indirect co-culture group and the control group;there are significant differences (P<0.01) in the four indicators, VEGF-a, VEGFR-2, VEGFR-1, VIII factors, and no differences in VEGF-b (P>0.05) between the indirect co-culture group and the direct co-culture group;and there are no differences (P>0.05) in total five indicators, VEGF-a, VEGF-b, VEGFR-2, VEGFR-1, VIII between the indirect co-culture group and the control groupsConclusion1 After successfully taking cell separation, extraction and amplification of mouse bone marrow-derived cells, we get mouse bone marrow-derived mesenchymal stem cells that are adhering growth closely the wall of culture dish, cloning growth and fibrocyte-like, whitch have the phenotype and the characteristic of mesenchymal cells and stem cells.2 The results of the control mono-culture experiments of MSC and the indirect co-culture experiments with B16 cell in vitro can initially confirm that parts of the MSC transform thier phenotype and differentiate to vascular endothelial cell in the indirect co-culture experiments. According to a group factor secretion, such asVEGF-a etc of B16 cells in the indirect co-culture B16 cells, we presume that B16 cells in indirect co-culture system to produce VEGF-a such factors could play a role in promoting MSC differentiation, resulting in the direction MSC to endothelial cell. By our research, the induced differentiation theory of MSC by tumors is proposed that in vitro MSC could be affacted by VEGF etc various factors tumor secreted, and could differentiate to vascular endothelial cells. And the vascular endothelial cells, which MSC differentiate to, may be involved in the vasculogenesis or angiogenesis of tumor. Although the activation way and the molecular mechanisms of MSC differentiated to vascular endothelial cells, and the exact mechanism of MSC and those vascular endothelial cells forming new blood vessels are not yet very clear. Through the induced differentiation theory of MSC by tumors, we re-create awareness of the role of MSC and the vasculogenesis in vascularization of tumor, further to enrich vascularization theory of tumor. And the in-depth research and understanding on process of vascularization and the regulatory mechanism of vascularization of tumor could take us a new idea on effective treatment of tumor. 3 The results of the control mono-culture experiments of B16 cell and of the indirect co-culture and the direct co-culture experiments with MSC in vitro can initially demonstrate that with hypoxia circumstances of cultivation environment in vitro, B16 cells express VEGF which can way promote the proliferation of cells and the expression of multiple factors by autocrine mechanisms. The autocrine mechanisms is mediated by VEGFR-2 and VEGFR-1, but also there may exist the positive feedback loop among VEGFR-2, VEGFR-1 and VEGF in malignant melanoma cells;MSC provide oxygen to tumor cells in vitro and take other functions to improve the condition in the lack of oxygen, which depress the expression levels of VEGF in malignant melanoma cells, disrupt the positive feedback loop of VEGF,and then lead to the decline of expression levels of various factors related to the positive feedback loop. The research for autocrine mechanisms and the positive feedback loop of VEGF in tumor, and the study on the variance, which affected by MSC, of the expression levels of various factors which tumor produce in vitro could allow us to have a comprehensive and innovative awareness of VEGF biological function and give us the sufficient theoretical supports on targeting VEGF and its receptors for the treatment of anti-vascularization of tumor. |
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