| Choriocarcinoma(GC)is a gestational trophoblastic tumor(GTT)secondary to hydatidiform mole or non-hydatidiform mole pregnancy.Choriocarcinoma is a highly aggressive tumor with rapid growth and high metastatic potential.Hematogenous metastasis is the main metastasis method of choriocarcinoma and the most common metastatic site is lung,with a metastasis rate of 80%.The current chemotherapy regimens have significant therapeutic effects on most choriocarcinomas,but the development of chemotherapy resistance and high toxic and side effects have brought great difficulties to the treatment of choriocarcinoma.Dihydromyricelin(DMY),also known as snake grapein,is a flavonoid active ingredient extracted from vine tea plants.A number of studies have confirmed that dihydromyricelin has pharmacological properties such as liver protection,blood pressure and lipid lowering,anti-lipid peroxidation,anti-inflammatory and antibacterial,etc.Dihydromyricetin is especially promising as a new type of low-toxic and highly effective anti-tumor drug,which can play an important role in inhibiting the key processes of tumor cell proliferation,invasion and metastasis.Previous research by our research group confirmed that dihydromyricetin inhibited the growth of choriocarcinoma in vitro and in vivo,and inhibited the invasion and metastasis of choriocarcinoma in vitro.However,whether dihydromyricetin affects the metastasis of choriocarcinoma in vivo has not been reported in the literature.In this study,we successfully established the human choriocarcino ma JAR cell orthotopic transplantation tumor model and metastasis mo del in nude mice,which made up for the lack of poor metastatic pote ntial of subcutaneous graft,and retaining the invasion and metastasis c haracteristics of primary tumors.They are ideal in vivo models for stu dying the progression of human tumor metastasis,and have laid the fo undation for further exploration of the pathophysiology of choriocarcino ma.Based on the construction of human choriocarcinoma nude mice metastasis model and previous in vitro experimental studies,this study further explored the effect of dihydromyricetin on the growth of choriocarcinoma lung metastases and its possible molecular mechanism.The experimental results found that dihydromyricetin can significantly inhibit the formation of choriocarcinoma lung metastases,and may play regulatory role through the PI3K/Akt signaling pathway.This study provides experimental and theoretical basis for drug treatment of lung metastasis from choriocarcinoma.Part I Establishment of orthotopic transplantation model of human chor iocarcinoma in nude mice.Objective:To establish orthotopic transplantation model of human choriocarci noma JAR cells in nude mice and explore its biological characteristics.Methods:1.5 nude mice were injected subcutaneously with human choriocarcinoma JAR cells to construct a subcutaneous transplanted tumor model.2.The subcutaneous tumor tissue was removed and surgically imp lanted into the uterus of 10 female nude mice to establish a human ch oriocarcinoma orthotopic transplanted nude mice model.3.We then performed survival and anatomical observations to draw weight changes and survival curves for nude mice.4.HE staining was used to identify the histopathological characteristics of subcutaneous xenografts,orthotopic xenografts and peritoneal metastases.5.Immunohistochemical staining was used to detect the expression of?-HCG in choriocarcinoma tissues.Results:1.Survival and anatomical observation of choriocarcinoma orthotopic transplantation tumor model Beginning on the 10 th day after orthotopic transplantation surgery,tumor-forming nude mice showed symptoms such as body wasting and weight loss.The abdomen gradually swelled and cachexia appeared.The overall survival time was 26-82 days,which was significantly lower than that of non-tumor nude mice 58-82 days(P=0.009).Anatomical findings showed that 7 nude mice formed a lump in the uterus and the success rate of orthotopic transplantation was 70%.Among them,2 orthotopic uterine transplanted tumors and peritoneal metastases were observed at the same time.2.Pathomorphological examination under HE staining light microscope Human choriocarcinoma pathological structure was shown by ortho topic transplantation tumor,peritoneal metastasis tumor,and subcutaneo us transplantation tumor under light microscope.The tumor tissue is c omposed of a large number of proliferating trophoblasts.The cytoplas m is rich,the nucleus is large and deep,the chromatin is thick,the n uclear atypia is obvious,the mitotic image can be seen,and a sheet o f bleeding can be seen around the tumor cells.3.?-HCG expression in choriocarcinoma Immunohistochemical results showed that ?-HCG was positively expressed in uterine orthotopic transplantation tumors,peritoneal transplantation tumors and subcutaneous transplantation tumor tissues,and mainly expressed in the cytoplasm.Conclusion:The orthotopic transplantation model of human choriocarcinoma JAR cells in nude mice were successfully established,which retained the pathological characteristics of the primary tumor,and provided a better animal model for the research of choriocarcinoma disease progression.Part ? Establishment of metastasis model of human choriocarcinoma in nude mice.Objective:To establish metastasis model of human choriocarcinoma JAR cells in nude mice and explore its biological characteristics.Methods:1.Human choriocarcinoma JAR cells were injected through the tail vein to establish a human choriocarcinoma nude mice model of metastasis.2.Survival and anatomical observations were performed in order to draw a survival curve for nude mice with metastatic tumors.3.HE staining was used to identify the histopathological characteristics of lung metastases.4.The expression of ?-HCG in lung metastases was detected by immunohistochemistry.Results:1.Survival and anatomical observation of choriocarcinoma metastasis model Observation found that the tumor-forming nude mice developed cachexia symptoms at the fourth week after after injecting JAR cells into the tail vein,with a survival time of 27 to 51 days and an average survival time of 32.5 ± 6.75 d,which was significantly lower than that of non-tumor-bearing nude mice at 64 to 82 days(mean 73.00±12.73 days,P= 0.018).Anatomical findings showed that lung metastases from choriocarcinoma showed invasive growth,and some metastases fused into tumor nodules.2.Histopathological characteristics of lung metastases from chorioc arcinoma The results of HE staining microscopy showed that lung metastase s from choriocarcinoma were the same as orthotopic transplanted tumor s,and they all showed the pathological structure of human choriocarci noma.3.?-HCG expression in choriocarcinoma lung metastases Immunohistochemistry showed that ?-HCG was positively expressed in choriocarcinoma lung metastases,mainly in the cytoplasm.Conclusion:The model of human choriocarcinoma JAR cell metastasis in nude mice was successfully constructed,which retained the original biological characteristics of human choriocarcinoma,and may provide experimental basis for the study of the advanced lung metastasis of choriocarcinoma.Part ? The effects of dihydromyricetin on the growth of metastases in nude mice with choriocarcinoma stablishment of metastasis model of human choriocarcinoma in nude mice.Objective:Based on the successful establishment of a nude mice metastasis model of human choriocarcinoma JAR cells,we explored the effects of dihydromyricetin on the growth of choriocarcinoma metastases and related molecular mechanisms.Methods:1.Human choriocarcinoma JAR cells were injected through the tail vein to establish a human choriocarcinoma nude mice model of metastasis.2.25 human choriocarcinoma metastases nude mice were divided into five groups: negative control group(Nacl),DMY(50mg / kg)group,DMY(100mg / kg)group,DMY(150mg / kg)group,and positive control group(methotrexate).Dosing for 30 days.3.Real-time quantitative PCR was used to detect the m RNA expression of PI3 K and Akt in lung metastases from different experimental groups.4.Western blotting was used to detect the expression of PI3 K and Akt protein in lung metastases of each experimental group.5.Immunohistochemistry was performed to detect the expression of PI3 K and Akt in lung metastases of each experimental group.Results:1.Effect of Dihydromyricetin on the weight of nude mice with choriocarcinoma metastasis The weight change curve of nude mice showed that the weight decrease of nude mice was significantly reduced after administration of dihydromyricetin,and the concentration was drug-dependent(P <0.05).2.Effect of Dihydromyricetin on the growth of lung metastases in nude mice The experimental results showed that dihydromyricetin can significantly inhibit the formation and growth of lung metastases in nude mice with choriocarcinoma,and the greater the concentration of dihydromyricetin,the fewer the number of lung metastases(P<0.05).3.Effect of Dihydromyricetin on the expression of PI3 K and Akt in lung metastases from nude mice with choriocarcinoma Real-time quantitative PCR,western blotting experiments and Immunohistochemistry showed that dihydromyricetin significantly inhibited the expression of PI3 K and Akt in choriocarcinoma lung metastases,and the expression levels of PI3 K and Akt were concentration-dependent(P<0.05).Conclusion:Dihydromyricetin can significantly inhibit the growth of metastatic tumor in choriocarcinoma nude mice,which may be related to the influence of PI3 K / Akt signaling pathway. |
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