| Purpose: The research of methotrexat and ifosfamid's effect to induce apoptosis and mechanism of action for osteosarcoma cell(MG-63)Methods: MG-63 cells were cultivated in Dulbecco's Minimum Essential Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum. Cultures were incubated at 37?C in a humidified atmosphere containing 5% CO2. The logarithmically growing MG63 cells were used.1.Using inverted phase contrast microscope observe the growth of cell morphological Changes regularly: MG63 cells were treated with different concentrations of methotrexat and ifosfamid , the change of morphology was observed by inverted phase contrast microscope in different time and photographed.2.The suppressive of methotrexat on the proliferation of MG63 cells were evaluated in vitro by MTT assay. The cells were plated in 96-well plates at a density. Then treated with different concentrations of methotrexat (0, 10, 50, 100,200 ug/ml)and different concentrations of ifosfamid (0, 5, 25, 50,100 ug/ml) for 24h,48h,72h , 10μl MTT was added to each well and incubated for 4 h.Centrifuging the 96-well plates at 2000rpm,then supernatant liquid were then discarded and 150μl of dimethyl sulfoxide (DMSO) was added. Absorbance was measured at 492 nm using an ELISA reader. The relative cell number of adherent cells was then determined by the MTT method.3.Flow cytometry analysis: MG63 cells were treated with various concentrations of methotrexat and of ifosfamid for 48 hours. Experimental group and control group cells were harvested, using AnnexinV-FITC/PI double staining method detect apoptosis of osteosarcoma cell MG63, Data acquisition and analysis were carried out by using a flow cytometry system.4.Western blot analysis: We observed the expression of PCNA, Cyclin-D1,Cyclin-E,Bcl-2 and Bax-2 in methotrexat and ifosfamid treated MG-63 by western blot.Results:1.we observed that under the inverted phase contrast microscope,the control group cells were fusiform and adherent cells.After the MG-63 cells treated by methotrexat about 48h, the growth of cells was inhibited, the volume of some cells became round and small, the cells refraction became strong, after 48h, lot of cells became round and floated,much cell fragment can be seen suspended in medium.When it came to 72h,most of the cells floated and the change became significant. The cells treated by ifosfamid about 48h have no morphological Changes.2. The MTT results showed that after we treated MG-63 cells in different time 24h,48h and 72h and with methotrexat in different concentration, the growth of cells was inhibited significantly in a time- and dose-dependent(p<0.05).The IC50 of MG-63 cells of 48h and 72h were 136.029 and 54.018 ug/ml. After we treated MG-63 cells 72h,The cells inhibitory rate could reach to 74.48 % . The cells treated by ifosfamid in different concentration and in different time 24h,48h and 72h,the OD values showed increasing tency. after we treated MG-63 cells with 25ug/ml in different time 24h,48h and 72h ,the OD values were (0.374±0.016),(0.562±0.049),(0.629±0.006),The cells treated by ifosfamid was not inhibited.3. The analysis of by FCM showed that after we treated MG-63 cells with methotrexat in different concentration at 48h, earlier period apoptosis percentage was(10.7±0.6)%,(20.4±1.9)%,(14.8±2.3)%. advanced stage apoptosis percentage was (7.4±0.3)%,(22.8±3.6)%,(55.7±3.1)%. Comparing with control group cells, the inhibitive effectiveness in a dose-dependent(p<0.05). After we treated MG-63 cells with ifosfamid of 100ug/ml at 48h, we couldn't find the peak of apoptosis in front of G0/G1 phase , the proportion of G1 phases was 41.0%,the proportion of G2 phases was 17.9%,the proportion of S phases was 41.1%,compared with the control group cells ,the cell cycle without significant change in the proportion.4. Western blot's results: Compared with the control group,by methotrexat treated osteosarcoma cells showed PCNA, Cyclin-D1,Cyclin-E ,Bcl-2 expression was down regulated,Bax expression was up regulated.Conclusions:1. In this study,as was observed under inverted microscope osteosarcoma cells showed morphological changes in the situation within a certain range of the increase over time of drugs on cells gradually. The growth of cells treated by methotrexat was inhibited significantly in a time- and dose-dependent. The cells treated by ifosfamid phases cycle without significant change in the proportion.2. The MTT results showed that after we treated MG-63 cells in different time and with methotrexat in different concentration, the drugs could significantlyretrain the multiplication of MG-63,and the effectiveness depended on time of experiment and dose of drugs(p<0.05).3. The analysis of by FCM showed that compared with the control group, MG63 cells apoptosis percentage increased when methotrexat concentration increased. The cells treated by ifosfamid phases cycle without significant change in the proportion.Ifosfamid had no anti-tumor activity in vitro.4. Proliferating cell nuclear antigen(PCNA),is a reflection of cell proliferation status of a good indicator, PCNA in this experiment methotrexat in group expression inosteosarcoma compared with the control group decreased significantly,indicating that methotrexat on osteosarcoma cell lines significantly inhibited proliferation.5. In this experiment after we treated MG-63 cells with IC50 methotrexat,we could find that cyclinD1 expression was slightly down regulated, CyclinE expression was significantly up regulated. Bcl-2 gene family play a fundamental role in controlling cell apoptosis. In this experiment , methotrexat group Bax expression increased compared with control group,Bcl-2 expression slightly decreased.
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