A Study On Cell Cycle Arrest Of GCs In Human Ovary Induced By Epirubicin

Objective:with the development of younger patients and survival rate of lymphoma and breast cancer,iatrogenic damage caused by chemotherapeutic drugs has been paid more attention.Ovary is the most sensitive part of the reproductive system to chemotherapy.High dose and long time chemotherapy can destroy ovarian tissue,leading to premature ovarian failure.Epirubicin,a representative drug of anthracycline antibiotics,is a first-line chemotherapy for female breast cancer.It was found that 6 courses of breast cancer patients with epirubicin based chemotherapy were diagnosed with premature ovarian failure with amenorrhea(premature ovarian failure),the probability of POF symptoms was 45-83%,and about 60% of the patients had no complete recovery of ovarian function in one year.Previous studies in the research group confirmed that epirubicin could induce ovarian function decline and estrous cycle disappeared in mice.Therefore,we studied the damage mechanism of epirubicin on human ovarian granulosa cells,and provided a cure for chemotherapy induced premature ovarian failure.Methods: 1,Ficoll density centrifugation was used to isolate and cultivate human ovarian GCs;2,the expression of GCs surface specific receptor(Follicle-stimulation hormone receptors,FSHR)of human ovary was detected by cell immunohistochemical staining method for identification of GCs;3,ovarian GCs and three different concentrations of epirubicin were completed.The total medium(group G: 0 mg/l,GP1: Group 0.0125 mg/l,GP2 group: 0.025 mg/l)was incubated with 24 h after 24 h,and PI monstaining flow cytometry was used to detect the change of G1 and G2 phase ratio of the ovarian GCs cell cycle in each drug concentration group,4,and the complete medium containing three different concentrations of epirubicin(0,0.0125,0.0125).Mg/l and GP2 group: 0.025 mg/l)the human ovarian GCs was incubated with Western blot,and the changes in the expression of GCs Gadd45 beta,Cdc2 and cyclin B1 protein in the human ovary were measured at three concentrations.Result: 1.Identification of human ovarian GCs cells: cell immuno histochemical staining was used to identify the isolated cells.The staining sites were located in the cell membrane,the process brown,the number of stained cells >80%,the strong positive,and the identification of the cultured cells as GCs;2,the flow cytometry was used to detect the changes of the human ovary GCs cell cycle: from the G group to the GP2 group,in G The 1 phase ratio decreased,from(66 + 1.15)% to(59.5 +1.83)%,then(51.07 + 1.05)%,G2 phase ratio increased,from(4.75 + 0.45)% to(5.94+ 0.69)%,and then(8.93 + 0.81)%,P<0.05,the results of different concentrations were not exactly the same,SNK analysis indicated that there were significant differences in each group.The proportion of G2 phase in the group cell was statistically significant with the increase of drug concentration.It was suggested that epirubicin stars led to the block of GCs G2/M phase,and the block was more and more obvious with the increase of drug concentration.3,the use of Western blot to detect the cyclin and related proteins of human ovarian GCs: target protein Cyclin B1,Cdc2 The expression of GADD45 beta was gradually increased from group G to GP2,and the value of gray value from group G to GP2 group was 171716.133 + 1243.12,170834 + 637.592 and 164211.67 + 182.719(Cyclin B1).P<0.05 and SNK showed that there was no statistical difference between GP1 and G group.The changes of protein expression were statistically significant;152500 + 1255.930,150293 +791.595,139746.33 + 741.756(Cdc2),P<0.05,SNK analysis suggested that there were significant differences between each group,and the changes of protein expression were statistically significant;195393.33 + 220.185,189663.33 + 1343.251,182365 + 290.184(GADD45 beta),P< 0.05,SNK analysis showed significant differences among groups,and the change of protein was statistically significant.Conclusion: 1,the extracted cells were human ovarian GCs;2,the human ovarian GCs of epirubicin remained in the G2 phase;3,epirubicin maybe increase the GADD45 beta of the human ovarian GCs,leading to the increase of the periodic protein Cyclin B1 and Cdc2 to cause the G2/M phase block of the GCs cell cycle.