The Impact Of Fluorescent Markers On Morphology And Biological Behavior Of B-16 Cell

Background The metastasis and invasion is a significant characteristic of cancer.And it also is the leading cause of failure in Clinical treatment and death of patients. The key to cure cancer is to remove the tumor tissue of the body and prevent its recurrence and metastasis.The metastasis mechanism of cancer is one focus in cancer research. Studies have also made many achievements in vitro and in vivo.But due to the lack of effective research means there are still many issues unresolved on the tumor metastasis of the early events.It is very difficult to capture and record the cloning process from a single tumor cell proliferates to multiple tumor cells real-time in the body.The difficulty is that we can not distinguish between normal cells and tumor cells in the body.In recent years the studies of application of fluorescent dyes or fluorescent protein gene markers of tumor cells for observing the invasion, proliferation and metastasis of tumor cells real-time in the body were reported.But we do not know whether the staining of fluorescent protein gene has significant impact on morphology,proliferation,invasion and adhesion of tumor cells in vitro or not, as well as the adhesion state,adhesive and proliferation of the marked tumor cells in the capillary network of the body has not gone further study.Objective To observe the impact of fluorescent markers on morphology,proliferation,invasion and adhesion of B-16 cell.And also to observe adherent state and proliferation of the marked B-16 cell in the capillary network of the body.Methods pEGFP-N3 expression plasmid transfected rat malignant melanoma cell line B16.Then established malignant melanoma cell lines B16/EGFP which can express green fluorescent protein (GFP) gene stably.Fluorescent dye CMFDA tag mouse malignant melanoma cell line B16/CMFDA.Division:B16 cells were as the control group(groupA), malignant melanoma cells marked with fluorescent dyes were as B16/CMFDA group(groupB),and malignant melanoma cells marked with green fluorescent protein were as B16/EGFP group(groupC).We observed the adherent state of tumor cells of group A, group B and group C under a inverted microscope.Counted the number and drew the proliferation curve of the groupA,groupB and groupC.We determined the adhesive rate of the groupA, groupB and groupC by MTT micro-reaction assay. Measured the invasive capacity of groupA, groupB and groupC. After we injected B16 cells with Fluorescent into the vein of the C57 mice tail,we observed the adhesion, proliferation of B16 cells in the capillary network of the body.Results In vitro,both pEGFP-N3 expression plasmid transfected B16/EGFP cell lines and fluorescent dye CMFDA marked B16/CMFDA cells had no significant change in morphology and adherent state under the inverted microscope. Cell culture to seven days, in the first to seventh days the number (×104)of B16 group cells were 5,12,25,70,220,510,810,the number of B16/CMFDA group were 5,11.5,27,80,210,510,880,the number of B16/EGFP were 5,12.1,25.5,71,210,500,830.The cell growth curves of group A,B and C were almost overlap The cells of three group were inoculated in the first day, entered the Health Long-term in the third day.Cells of three group were covered the 25 cm2 bottles fully in the seventh day.Statistical analysis shows that the proliferation of B16 cells, B16/CMFDA cells, B16/EGFP cells had no significant difference (P>0.05)Cell cultured at 37℃,5%CO2 for 60 minutes. The OD values of B16 group,B16/CMFDA group,B16/EGFP group were 0.269±0.029,0.303±0.027,0.319±0.06.Compared with the control group, the adhesion rate of B16/CMFDA group,B16/EGFP group was 3.4 percent,5.0 percent. Statistical analysis show the adhesion of B16 group,B16/CMFDA group,B16/EGFP group had no significant difference (P>0.05)Cell cultured at 37℃,5%CO2 for 48h,the number of B16 group,B16/CMFDA group,B16/EGFP group cells that penetrated the cell membrane respectively were 85.33±7.57,61.33±17.56,73.67±25.32. Statistical analysis that there were no significant difference among B16 group,B16/CMFDA group and B16/EGFP group (P>0.05).Concludes There are no significant effects among B16 group,B16/CMFDA group and B16/EGFP group on the morphology, proliferation,adhesion and invasion.B16-cells of fluorescent markers can normally proliferate in the body,adhere vascular endothelial cell and invade the extracellular matrix,form metastatic in lung. Thus tumor cells with fluorescent markers provide a means and method to study the invasion and the metastasis mechanism of the tumor cells,especially provide a common platform for the related research of early tumor metastasis on morphology, learned molecular biology and cell biology level.