| Objective To ascertain whether methylmethacrylate (MMA) monomer has toxic effects on tumor cells, the degree of cytotoxicity and how the MMA monomer influences tumor cells in vitro.Methods 1. A549-cells were subcultured and seeded to 96-well microtitre plates at the rate of 1×104 per well at logarithmic growth phase. The optical density (OD) value was continually detected by MTT test for 7 days and the growth curve was drawed according to the OD values. 2. A549-cells were subcultured and seeded to 96-well microtitre plates at the rate of 1×104 per well at logarithmic growth phase and were divided into nine concentration of MMA groups (0.01μg/mL,0.1μg/mL,1μg/mL,5μg/mL,10μg/mL,25μg/mL,40μg/mL,80μg/mL,120μg/mL) and blank control group(with no MMA). The OD values of different groups were detected by MTT test at 30min,1d,3d and 5d after administration. And the relative growth rates (RGR) were accounted according to the OD values. 3. A549-cells grew on the clean microscopic slides in 6-well microtitre plates and were divided into nine concerntration of MMA groups (0.01μg/mL,0.1μg/mL,1μg/mL,5μg/mL,10μg/mL,25μg/mL,40μg/mL,80μg/mL,120μg/mL) and blank control group(with no MMA). Hematoxylin eosin stain was made 24h after administration. The slide droped with pure MMA was made 30s after administration. The changes on the cellular integrity of A549-cells were observed through light microscope. 4. A549-cells were divided into 6 groups(MMA of 1μg/mL,10μg/mL,40μg/mL,80μg/mL,120μg/mL and blank control group) and subcultured in 25mL-bottles. After MMA was added 24h, the cells of each group were collected and divided into 3 tubes at a rate of 5×105 each tube. Using AnnexinⅤ/PI method to test the percentage of apoptosis and necrosis on flow cytometer (FCM) and observe the cell change under fluorescence microscope. Results 1. A549-cells entered the logarithmic phase in the first 4 days and reached the platform on the 4th day, and then decreased on the 6th day. 2. There is no significant difference of OD values among groups 30min after administration(P>0.05). But at 1d,3d and 5d after MMA added, the differences of OD values amnog different concentrations were significant (P<0.05), especially between blank control group and groups with MMA of 25μg/mL,40μg/mL,80μg/mL and 120μg/mL. And the OD values decreased as the concentration rised. However, the differences among groups that below the concentration of 5μg/mL were not significant (P>0.05). We also find significant differences between 10μg/mL group and 40μg/mL group,80μg/mL group,120μg/mL group(P<0.05). But the difference between 10μg/mL group and 25μg/mL group was not significant(P>0.05). At the 5th day there are significant differences between 25μg/mL group and 80μg/mL group, 25μg/mL group and 120μg/mL group(P<0.05). But the difference between 80μg/mL group and 120μg/mL group is not significant (P>0.05). The most serious degree of cytotoxicity is 2 degree range the groups. It appears in 120μg/mL group at 1d,3d,5d and 40μg/mL group,80μg/mL group at 5d. 3. Hematoxylin eosin stain found condensation of nucleus in 40μg/mL group,80μg/mL group,120μg/mL group 24h after MMA added. And when the cell exposed to pure MMA for 30s, most cells died. The light microscope showed cytoplasm dissolved and cell membranes ruptured that the cells form were not integral. 4. The differences of percentage of apoptosis and necrosis on FCM were significant (P<0.05). The results of 1μg/mL group and blank control group look alike and the percentage of apoptosis and necrosis resemble. The two percentages grew as the concentration rised especially the percentage of necrosis. The percentage of necrosis is two times as the percentage of apoptosis in 10μg/mL group. And the max percentage appears in 120μg/mL. Through fluorescence microscope we can see apoptosis cells in green fluorescence and necrosis cells in red fluorescence.Conclusions 1. Low concentration of MMA and short contact time were not toxic enough to injure the tumor cells. 2. As the concentration and contact time rised the influences to tumor cell became significant. 40μg/mL is a critical cytotoxicity dose. And the degree of cytotoxicity is no more than 2 degree within 120μg/mL. Exposure to pure MMA can cause cell rupture immediately. 3. MMA monomer has the influence to induce apoptosis and necrosis of tumor cells. As the dose of MMA rised the influnce became significant, especially in necrosis. 4. The cytotoxicity of MMA and the ability of inducing apoptosis and necrosis may probably act as one mechanism of PVP in the treatment of vertebral tumor.
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