A Comparison Of The Effect Of Cisplatin, VP-16, Vincristine, Bleomycin On The Function Of Human Granulosa Cell

Objective:Malignant ovarian germ cell tumors derive from primordial germ cell, typically occur in girls and young women,and the morbidity is following which of the epithelial tumor of ovary. A comprehensive surgical stag-ing with contralateral ovary and uterus,and postoperative chemotherapy with Platinum and etoposide-based regimens has been accepted as stand-ard management is advocated. Chemotherapy can lead diverse adverse side effect,especially premature menopause and infertility paid more and more attention.Ovarian malignant germ tumor cell line PA-1 was interfered with cisplatin,VP-16,vincristine and bleomycin in different concentration.The median lethal dose of cisplatin,VP-16,vincristine and bleomycin on PA-1 was measured by MTT. Human granulosa cells were interfered with the median lethal dose of the four drugs above,and the effect of cisplatin, VP-16, vincristine,bleomycin on the function of human granulosa cell was observed.Methods:1 Ovarian malignant germ cell tumor cell line PA-1 was cultured in MEM culture medium which have 10% FBS and penicillin and streptomycin(100IU/ml) in an invironment of 37℃,5% CO2,constant temperature,saturated humidity in vitro.The cell line which stayed in logarithmic growth phase was digested in pancreatic enzyme.Single-cell suspension was prepared for use.2 The cell line PA-1 was inoculated in 96-well plates at a density of 6.5×104/ml,was interfered with cisplatin, VP-16, vincristine and bleomycin in different concentration for 48h.The median lethal dose of cisplatin,VP-16,vincristine and bleomycin on PA-1 was measured by MTT.3 Granulosa cells were obtained from infertile patients undergoing IVF-ET cycles in the partment of reproductive medical in the second affiliated hospital of HeBei Midicial Univercity.The pathogeny of sterility was obstruction of fallopian tube or male factors. The menstrual cycle,ovulatory and endocrine function were normal.Woman using long period schema over-ovulation stimulants after injected for 34-36h of human chlrionic gonadotrophin was take out of ovum via vagina-ultrasound conducted.4 Granulosa cells were cultured in vitro.and interfered with the median lethal dose of cisplatin,VP-16,vincristine and bleomycin for 72h,the viability of cells was determined by using the method of MTT,The culture medium was collected,and the estradiol(E2) level was detected by chemiluminescence method. Using immunocytochemical technique,the expression of Bcl-2/Bax of granulosa cell was observed.Results:1 The effect of cisplatin,VP-16,vincristine and bleomycin on ovarian malignant germ cell tumor cell line PA-1.1.1 Morphological trait of PA-1 cell lineWe observed the cell morphological trait under inverted phase contrast microscope, The growing state of PA-1 cell line was well.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.1.2 IC50 of cisplatin, VP-16,vincristine and bleomycin on PA-1 cell line by MTTCell line PA-1 was interfered with cisplatin,VP-16,vincristine and bleomycin in different concentration for 48h, inhibition rates of PA-1 cell increased with the concentration augmented.It was revealed that the consecutive IC50 of cisplatin, VP-16,vincristine and bleomycin was 0.55mg/L,1.67 mg/L,1.8μg/L,1.7mg/L2 The effect of the IC50 of cisplatin,VP-16, vincristine and bleomycin (on PA-1) on granulosa cell.2.1 Morphological trait of granulosa cellWe observed the cell morphological trait under inverted phase contrast microscope,Granulosa cell was round inoculated instantly, adherence after 6h,adherence rate was above 90%. Granulosa cell morphological was fusiform regularly,nucleolus was big round. Cytopalsm rich in vacuole and granule.It growed crowding together.2.2 MTT assay of granulosa cellCompared with control group, Absorbance value of granulose cell of the drug groups all decreased,and had significant differences between drug groups apart from vincristine group, there was no significant difference between VP-16 group and bleomycin group.2.3 E2 level of granulosa cellCompared with control group,the E2 level secreted by human granulose of cisplatin,VP-16, vincristine and bleomycin groups decreased slightly,and had significant differences between drug groups apart from vincristine group, there was no significant difference between VP-16 group and bleomycin group.2.4The expression of Bcl-2/Bax using Immunocytochemical techniqueBcl-2/Bax located in cytolymph under light microscope,was browngranule. The control group and every drug groups all express Bcl-2/Bax. Compared with control group, Bax expression was up-regulated, Bcl-2 expression was down-regulated. Apart from vincristine group, there was significant difference between all drug groups and the control group.Conclusion:1 Inbibition rates of PA-1 cell increased with the concentration and time augmented after 48h of VP-16, bleomycin, cisplatin and vincristine on PA-1 cell line. 2 The IC50 of VP-16,bleomycin, cisplatin,vincristine on PA-1 for 48h was bleomycin 1.7 mg/L, VP-161.67 mg/L, cisplatin 0.55mg/L, vincristine 1.8μg/L,separetly.3 It is suggested that the IC50 of cisplatin,VP-16,bleomycin except vincristine on PA-1 all can promot apoptosis of human granulosa cell markedly. Vincristine non-distinct.4 The E2 level secreted by human granulose after 72h of the IC50 of cisplatin,VP-16 and bleomycin on PA-148h decreased, vincristine non-distinct.5 VP-16,bleomycin,cisplatin,vincristine can upregulate the expression of Bax and down regulate the expression of Bcl-2 to accelerate the opotosis of human granulose.6 The IC50 of VP-16 and bleomycin on PA-1 promot apoptosis to the strongest, and there was no significant difference, cisplatin less, vincristine does not play a significant role.