Impact Of Ginkgetin On PA-1 Cell Line Of Human Ovarian Teratoma And Human Granular Cell

Objective: Human ovarian teratoma is the most common style in malignant ovarian germ cell tumors(MOGCT). It occurs mainly in young women even girls, because of its sensitivity to chemotherapy, we offen take the fertility-sparing surgery combined with effective chemotherapy or chemotherapy to improve the life quality to these patients. Though chemotherapy is important, it brings injury to normal cells.Ginkgetin is extracted from ginkgo leaf, has clinical value in some diseases, such as arteriosclerosis, hypertensionand and so on. Recent years, the effect of ginkgetin on tumors has caused more and more attention.Our experiment was designed to investigate the impact of ginkgetin on the proliferation of PA-1 cell line of human ovarian malignant teratoma and human granular cell in vitro.Methods: The human ovarian malignant teratoma cell line PA-1 was cultured in MEM culture solution which containing 10% heat-inactivated FBS under a humidified 5% CO2 atmosphere, at 37℃, PA-1 cells were applied to the experiment when they entered to the logarithmic phase.1 We treated PA-1 cells with ginkgetin at different concentrations and used light microscope to observe the cells morphological changes.2 MTT assay: the cultured cells were initiated in 96-well plates at a density of 6.5×10⁴/ml. Cells were treated for different times(24h, 48h, 72h ) with various concentrations(0, 150mg/L, 175mg/L, 200mg/L, 225mg/L) of ginkgetin with three replicate wells for each concentration. We choosed 0mg/L as the control group. MTT assay was used to determine cell proliferation. 3 FCM labed with PI was used to detect the opototic rates and cell cycle phase of PA-1 cells in different experimental groups after treated 48h. Cells were divided into groups as follows: (1)Control group: cells were only cultured in MEM supplemented with 10% heat-inactivated FBS. (2)Drug groups: cells were treated with various concentrations(175mg/L, 200mg/L, 225mg/L) of ginkgetin.4 The change of Bcl-2 and Bax were detected by munohistochemistry. PA-1 cells were divided into groups as follows: (1)Control group: cells were only cultured in MEM supplemented with 10% heat-inactivated FBS. (2)Drug groups: cells were treated with various concentrations(175mg/L, 200mg/L, 225mg/L) of ginkgetin.5 Human granular cells were treated with ginkgetin in different concentrations(0,175mg/L,200mg/L,225mg/L), the inhibate rate was evaluated by MTT assay.6 Human granular cells were treated with ginkgetin in different concentrations(0,175mg/L,200mg/L,225mg/L), the level of E₂ was assayed by chemiluminescence method.Results:1 We treated PA-1 cells with ginkgetin in different concentration and observed the cell morphological changes under inverted phase contrast microscope. The shape of some cells began to change gradually, the long cells turned to round. As the concentration increased, the cells began to shrink, the number of round cells increased gradually. Then the volume of cells turned smaller and smaller than before, and the cytoplasm condensed, morphology became unnormal, membrane became incomplete. At last, the cell adhesion weakened and cell floated.2 The MTT results showed that inhibition rates of PA-1 cell increased according to the concentration of ginkgetin and time.3 (1)The opotosis rates of PA-1 cells were detected by PI labed FCM. With the concentration of ginkgetin increased, the opotosis of PA-1 cells in different congcentration groups increased and the differences compared with control group are significant(p<0.05).(2)The cell cycle phase of PA-1 cells were detected by PI labed FCM. The results of FCM showed that ginkgetin could stop the growth of PA-1 cells at G₀-G₁ period. There are significant differences in drug groups compared with control group(p<0.05).4 The munohistochemistry showed that activation of Bax increased with increased doses of ginkgetin, while the activation of Bcl-2 decreased.5 We treated human granular cells with ginkgetin in different concentrations and the inhibition rates were detected by MTT, the MTT results showed that ginkgetin had rare effect on human granular cells, there are no significant differences between drug groups and control group(p>0.05).6 After treating the human granular cells with ginkgetin in different concentration, the level of E₂ assayed by chemiluminescence method showed that there are no significant differences between drug groups and control group(p>0.05).Conclusion:1 Ginkgetin can inhibit the growth of human ovarian malignant teratoma cell line PA-1 apparently in vitro.2 Ginkgetin can arrest the cell cycle of PA-1 cells at G₀-G₁ period, thus regulate cell cycle progression of PA-1 negatively.3 Ginkgetin can upregulate the expression of Bax and down regulate the expression of Bcl-2 to accelerate the opotosis of PA-1.4 Ginkgetin has rare effect on the proliferation of human granular cells.5 Ginkgetin do not influence the secretion of human granular cells.6 Ginkgetin brings no injgury to human granular cells.