荷人卵巢上皮癌裸鼠冻融卵巢组织移植的效果评价

Evaluation of the effect of ovarian cryopreservation and transplantation in nude mice bearing human epithelial ovarian cancer

目的:获取人卵巢上皮癌裸鼠原位移植瘤癌旁正常卵巢组织,经安全筛查并冻融后移植至去势裸鼠体内,探讨移植后效果,为临床应用提供依据。方法:将人卵巢上皮癌OVCAR3细胞种植于裸鼠皮下以获取瘤源,并进行卵巢原位移植,建立卵巢癌原位移植瘤模型,解剖裸鼠获取癌旁正常卵巢组织。癌旁组:取筛选后的癌旁卵巢组织进行玻璃化冷冻,复苏后分别进行皮下及原位移植,各20例;对照组:同龄正常裸鼠卵巢组织,皮下移植及原位移植各20例;去势裸鼠组:20只同龄去势裸鼠;正常卵巢组:20只同龄正常裸鼠。移植12周后,分析各组裸鼠卵巢组织内卵泡形态及激素分泌功能。结果:40只人卵巢上皮癌裸鼠原位移植瘤模型中共获取35份活检正常的癌旁卵巢组织,获取率87.5%(35/40)。玻璃化冷冻前后卵巢组织中卵泡形态及各级卵泡比例均无显著差异(P>0.05),癌旁冷冻组织和癌旁新鲜组织的异常卵泡比例差异无统计学意义(P>0.05),冷冻组织以初级卵泡及次级卵泡等窦前卵泡为主。癌旁组皮下移植组织存活率80%(16/20),对照组皮下移植组织存活率90%(18/20),癌旁组原位移植组织存活率90%(18/20),对照组原位移植组织存活率95%(19/20)。移植后组织内卵泡以次级卵泡及窦状卵泡为主,各级卵泡的形态及构成比和未移植的同龄正常裸鼠卵巢相似(P>0.05);癌旁组卵泡数明显低于对照组(P<0.05)及正常卵巢组(P<0.01);皮下移植和原位移植组织内的各级卵泡数差异无统计学意义(P>0.05)。癌旁组卵泡刺激素水平明显低于去势裸鼠组,而雌二醇水平明显高于去势裸鼠组(P<0.01);癌旁组卵泡刺激素水平明显高于对照组(P<0.05)及正常卵巢组(P<0.01),而雌二醇水平明显低于对照组(P<0.05)及正常卵巢组(P<0.01);皮下移植和原位移植的卵泡刺激素水平和雌二醇水平差异无统计学意义(P>0.05)。结论:癌旁卵巢组织冻融移植后有正常卵泡发育及激素分泌功能;皮下移植和原位移植均可取得较好的效果;癌旁卵巢组织冻融移植有望作为卵巢上皮癌患者治疗后恢复卵巢内分泌功能的有效手段。

Objective：Normal ovarian tissues after security screening from the paraneoplastic ovarian tissues with orthotopic transplantation of human ovarian epithelial cancer were got to be frozen and transplanted in castrated nude mice.To investigate the effect of transplantation and provide a basis for clinical application.Methods：Human ovarian epithelial cancer cell line OVCAR3 were grown in subcutaneous of nude mice in order to fetch tumor source and the tumor tissues were orthotopic implanted.Orthotopic transplantation tumor model were established and the normal ovarian tissues were got from the paraneoplastic ovarian tissues.Paraneoplastic group：Screened paraneoplastic ovarian tissues were vitrified cryopreservation.Tissues after anabiosis were subcutaneously and orthotopic transplanted in 20 cases nude mice respectively.Control Group：Tissues from 20 cases normal ovarian tissue of the same age nude mice were subcutaneously and orthotopic transplanted respectively.Castrated nude mice group：20 cases.Normal ovarian group：20 cases.The follicular morphology and hormone secretion of all groups were analyzed in 12 weeks after transplantation.Results：35 samples ovarian tissues with normal biopsy were gained from 40 cases of human epithelial ovarian cancer of orthotopic implantation model in nude mice.There were no differences in follicular morphology and constituent ratio between ovarian tissues before and after vitrification（P0.05）.No difference was found in the proportion of abnormal follicles between the frozen tissues and fresh tissues of paraneoplastic ovarian tissues（P0.05）.There was mainly based on primordial follicles and primary follicles in the frozen tissues.Survival rate of the tissues adjacent to tumor with subcutaneous implantation was 80%（16/20）while those of control group was 90%（18/20）.Survival rate of the tissues adjacent to tumor with orthotopic implantation was 90%（18/20）while those of control group was 95%（19/20）.There was mainly based on secondary follicles and antral follicles in the tissues after implantation.There was no difference in follicular morphology and constituent ratio between implanted tissues and the ovarian tissues of normal same age nude mice without implantation（P0.05）.The follicular numbers of the paraneoplastic group were lower than those of control group（P0.05）,and also lower than those of normal ovarian group（P0.01）.No difference was found in follicular numbers between the tissues with subcutaneous and orthotopic transplantation（P0.05）.The FSH level of the paraneoplastic group was lower and the E2 level was higher than those of castrated nude mice obviously（P0.01）.The FSH level of paraneoplastic group was higher than those of control group（P0.05）and normal ovarian group（P0.01）,while the E2 level was lower than those of control group（P0.05）and normal ovarian group signifcantly（P0.01）.No difference was found in FSH and E2 levels between the groups of subcutaneous and orthotopic transplantation（P0.05）.Conclusions：Normal follicular development and hormone secretion were found after cryopreservation and transplantation of ovarian tissues adjacent to tumor.Better effect can be obtained both in subcutaneous and orthotopic transplantation.Cryopreservation and transplantation of paraneoplastic ovarian tissues expected to be an effective way for recovery of ovarian function in patients with epithelial ovarian cancer.