| Objective: Malignant ovarian germ cell tumor is the malignant tumorderived from embryonic gonad and which occurred frequently in children andadolescents who have fertility requirements. The combined postoperativechemotherapy is effecient. But the ovarian function damage afterchemotherapy influence the quality of life and the fertility function seriously.Scholars dedicated to the study of protection against ovarian function injurycaused by chemotherapy. Many studies showed that the GnRH analogues playthe role of ovarian protection in chemotherapy. Ginkgo flavone can resistoxidation, inhibit apoptosis and reverse the chemotherapy resistance ofcarcinoma. These effects are also verified in multiple experiments. Amifostineis used as a cell protective agent in clinic. But there is rarely study on theovarian protection.The BEP, EP and VIP are the commonly scheme in therapyin MOGCT, cisplatin is the main drug to injure ovarian function.Rats are usedas models of ovarian function damage in this trial. The protective effectsagainst the cisplatin-induced ovarian impairment of Ginkgo flavone,Amifostine and Leuprorelin are being explored. The influence of the threedrugs on the inhibitiory of Cisplatin on PA-1cells in vitro are being detectedsimultaneously.Methods:1Establish the rats model of ovarian function damage induced bycisplatin. To determine the E2and FSH levels in serum and count ovarianfollicles in order tu determine the dosage and period of cisplatin.2To determine the protective effects against the cisplatin-inducedovarian impairment of Ginkgo flavone, Amifostine and Leuprorelin: count ovarian follicles, weigh both overis, TUNEL to detect apoptosis index,Western-blot to detect cytoplasm c and Apaf-1intracytoplasmic proteincontents and real-time quantitative PCR to detect the expression of Cyt-c andApaf-1mRNA.3To study the influence of the three drugs on the inhibition of Cisplatinon PA-1cells in vitro: CCK-8method to detect cell proliferation inhibitionrate, Western-blot to detect Cyt-c and Apaf-1intracytoplasmic proteincontents and real-time quantitative PCR to detect the expression of Cyt-c andApaf-1mRNA.Results:1E2and FSH levels:The E2level was significantly lower in Group D, Group E, Group F andGroup G compared with group A(3.17±6.78pg/ml,32.80±2.46pg/ml,28.60±2.78pg/ml,22.37±2.91pg/ml vs73.87±13.95pg/ml).The FSH level was significantly higher in Group E, Group F and GroupG compared with group A (2.20±0.36IU/L,3.23±0.31IU/L,4.13±0.40IU/L vs1.08±0.15IU/L).Combined with follicles count, the Group E is selected as the model.2The protective effects showed by follicles count:Group A: primordial follicles37.9±2.9, growing follicles24.3±1.3,mature follicles13.2±1.3, total follicles was75.4±3.1. Group B: primordialfollicles35.2±4.0, growing follicles8.9±1.3, mature follicles5.5±1.1, totalfollicles49.6±4.5. Group C：primordial follicles39.2±3.1. growing follicles13.6±1.3， mature follicles9.2±1.1, total follicles60.5±3.9. Group D:primordial follicles36.7±2.1. growing follicles15.8±2.1， maturefollicles11.3±2.0, total follicles63.8±5.1. Group E: primordial follicles58.4±2.5. growing follicles6.4±1.0，mature follicles2.7±2.2, total follicles67.7±3.5.3The results of the ovarian apoptosis index: The ovarian apoptosis index of Group A, B, C, D and E is3.6±0.7,65.3±2.9,.35.7±2.0,37.4±1.6and30.5±2.9respectively.4Cyt-c and Apaf-1intracytoplasmic protein contents in rats:The Cyt-c and Apaf-1intracytoplasmic protein contents of Group B,Group C and Group D was significantly higher compared with Group A; theCyt-c and Apaf-1intracytoplasmic protein contents of Group C, Group D andGroup E was significantly lower compared with Group B; the Cyt-c andApaf-1intracytoplasmic protein contents of Group E was significantly lowercompared with Group C and Group D. The difference between Group D andGroup C is not statistically significant (P>0.05). The difference betweenGroup E and Group A is not statistically significant too(P>0.05).5The expression of ovarian Cyt-c and Apaf-1mRNA in rats:The expression of ovarian Cyt-c and Apaf-1mRNA of Group B, GroupC and Group D was significantly higher compared with Group A. Theexpression of ovarian Cyt-c and Apaf-1mRNA of Group C, Group D andGroup E was significantly lower compared with Group B. The expression ofovarian Cyt-c and Apaf-1mRNA of Group E was significantly lowercompared with Group C and Group D. The difference between betweenGroup E and Group A is not statistically significant (P>0.05).6The proliferation inhibition rate of PA-1cells in five groups:The proliferation inhibition rate of Group C, Group D and Group E wassignificantly higher compared with Group A and Group B(56.3%,64%and61.5%vs0and44.7%).7Cyt-c intracytoplasmic protein contents in PA-1cells:The Cyt-c intracytoplasmic protein contents in PA-1cells of Group B,Group C, Group D and Group E was significantly higher compared withGroup A(1.56±0.09,1.87±0.03,2.91±0.07and2.31±0.05vs1). The Cyt-cintracytoplasmic protein contents in PA-1cells of Group D and Group E wassignificantly higher compared with Group B. 8Apaf-1intracytoplasmic protein contents in PA-1cells:The Apaf-1intracytoplasmic protein contents in PA-1cells of Group B,Group C, Group D and Group E was significantly higher compared withGroup A(1.72±0.09,2.66±0.10,4.35±0.30and2.46±0.17vs1). The Apaf-1intracytoplasmic protein contents in PA-1cells of Group D and Group E wassignificantly higher compared with Group B.9The expression of Cyt-c mRNA in PA-1cells:The expression of Cyt-c mRNA in PA-1cells of Group B, Group C,Group D and Group E was significantly higher compared with GroupA(2.23±0.45,3.46±0.35,7.56±0.66and4.22±0.42vs1). The expression ofCyt-c mRNA in PA-1cells of Group D and Group E was significantly highercompared with Group B.10The expression of cytoplasm Apaf-1mRNA in PA-1cells:The expression of Apaf-1mRNA in PA-1cells of Group B, Group C,Group D and Group E was significantly higher compared with GroupA(1.91±0.23,2.72±0.45,5.77±0.45and3.07±0.34vs1). The expression ofApaf-1mRNA in PA-1cells of Group C and Group D was significantlyhigher compared with Group B.Conclusion:1Cisplatin-induced ovarian impairements in rats is obvious. Rats thoseinjected the dosage of Cisplatin2mg/(kg· d) intraperitoneally for seven dayswill gain the ovarian impairements as experimental requirements.2Given Ginkgo flavone, Amifostine and Leuprorelin before cisplatin canprotect the ovarian function partly. The effection of Leuprorelin is stongerthan that of Ginkgo flavone and Amifostine. The effection of Ginkgoflavone is equal with Amifostine.3Cisplatin can inhibit the proliferation of PA-1cells in vitro obviously.Given Ginkgo flavone, Amifostine and Leuprorelin before cisplatin canenhance the inhibition, upregulate the expression of Cyt-c and Apaf-1mRNA and increase the cytoplasmic protein contents of cytoplasmic Cyt-c andApaf-1. The effect of Ginkgo flavone is the strongest among the three drugs.The effect of Leuprorelin is stronger than that of Ginkgo flavone. |
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