Inhibitory Effect Of Microtubule-associated Protein-2 Transfection On The Melanoma Cell Growth

Objective: To study the effect of Microtubule-associated protein-2 (MAP-2) on the morphology of melanoma cell, and to understand the mechanisms of the inhibitory effect on the melanoma growth, we transfected MAP-2 into mouse melanoma cell lines, B16 and B16C29,This study provided the experimental basis for gene therapy of melanoma.Methods: Our work was composed of 6 parts:(1)Infected HEK293 cell with Ad-MAP-2, and determined the titer by plaque test.(2)Infected B16 cell with Ad-MAP-2 vector at MOI 3.5,and count the cell number every 3 days before and after infection , analyzed the data with statistics software of SPSS 11.5.(3)Infected B16C29 cell with Ad-MAP-2 vector at MOI 3.5,and observed the cell morphology.(4)Detected B16 and B16C29 cells apoptosis after infection by flow cytometry (PI-ANNEXIN-V staining method).(5)Observed the change of dendrites and microtubules in cell with electron microscope.Results:(1)The titer of Ad-MAP-2 was 1 × 1010pfu/ml.(2)4 days after infection,long dendrites formed in B16C29 cell.This change was remarkable on the 5th day after infection.(3)Cell apoptosis was found on the 5th day after infection .Flow cytometry showed that MAP-2 induced significant apoptosis in B16 and B16C29 cells 6 days after transfection .(4)Number of microtubules in B16 cell increased after infection with electron microscope ,and microtubules became longer and thicker ,gathered to form bundles which extending along cell dendrites .(5)Counted and analysed the number of cells ,we found that ,there was no significant difference in the number of B16 cell between the MAP-2 group and negative control group 3 days after infection(t=0.336,P=0.754>0.05);On the 6th day ,the cell number of the MAP-2 group was significantly fewer than negative control group(P=0.006<0.01).On the 9th day after infection ,the cell number was more fewer than control(P=0.001<0.01).Conclusion:(1) The overexpression of MAP-2 inhibits the growth of melanoma B16 cell.(2)The overexpression of MAP-2 induces the cell apoptosis of melanoma B16 and B16C29 cells, these results suggest that apoptosis is one of the reason for inhibition of cell growth.(3)After infection of Ad-MAP-2, melanoma B16C29 cell can reform or partly reform the dendritic shape.(4)The overexpression of MAP-2 can increase the number of microtubules in melanoma B16 cell; and can make microtubule longer and thicker, gather and form bundles which extending along the cell dendrites.Taken together, these results suggest that the overexpression of MAP-2 inhibits melanoma cell growth, the Ad-MAP-2 vector is likely to be a value drug of gene therapy for melanoma.