【摘要】 背景:胚胎卵巢组织免疫原性低,不易引起宿主的免疫排斥反应,是卵巢异体移植的理想供体。但由于获取胎儿卵巢的不随意性和来源限制,标本的冷冻显得至关重要,成功的冷冻保存和复苏技术是建立胎儿卵巢库的关键。目的:通过对大鼠新鲜或冻融胚胎卵巢组织异体无血管移植后的组织形态和功能的观察,探讨胚胎卵巢组织异体移植和超速冷冻保存的可行性。设计、时间及地点:随机对照动物实验,于2006-03/05在云南省第一人民医院生殖遗传科进行课题设计,于2006-05/2007-02在云南大学单克隆抗体研究中心完成实验。材料:卵巢来源于孕龄为18～20dSD大鼠胚胎。取阴道脱落细胞涂片证实有正常动情周期未交配过的SD成年雌性大鼠作为移植对象,分4组:正常对照组(n=10)、去势对照组(n=10)、新鲜胚胎卵巢移植组(n=25)、冷冻胚胎卵巢移植组(n=25)。方法:正常对照组仅行皮肤筋膜肌肉切开缝合术,去势对照组切除双侧卵巢组织。将孕龄为18～20d的新鲜或经超速冷冻法冷冻复苏的大鼠胚胎卵巢植入去势后4周的大鼠颈部皮下作为实验组,分别为新鲜移植组和冷冻移植组。主要观察指标:通过阴道脱落细胞的观察和血清雌二醇、孕酮的测定了解大鼠内分泌恢复情况,并对移植卵巢进行组织学观察。结果:新鲜移植组和冷冻移植组分别有52.17%和38.01%大鼠恢复了动情周期,两组血清雌二醇和孕酮在分别在35和49d恢复到正常,且动情周期天数与正常对照组比较,差异无显著性意义。冷冻移植组大鼠恢复动情周期虽稍短于新鲜移植组,但差异并无统计学意义。63d后取出移植物观察,见移植物色泽红润,表面有丰富的血管形成,镜下见卵巢内有大量的小动脉和小静脉;移植物组织学检查发现卵泡的发育,光镜下可见不同发育阶段的卵泡,有成熟卵泡和黄体形成,形态与正常卵巢无明显差别。结论:大鼠胚胎卵巢异体异位无血管皮下移植后能存活、生长发育并具有与正常卵巢相似的内分泌功能;超速冷冻法能较好的保存胚胎卵巢的活性,复苏后与新鲜胚胎卵巢组织具有相同的生理活性。更多还原

【Abstract】 BACKGROUND: Fetal ovarian tissue with low immunogenicity is an ideal donor for ovarian allograft transplantation. However,it is often difficult to be obtained,so cryopreservation and resuscitation technique for fetal ovaries is a key for the building of fetal ovary bank. OBJECTIVE: To observe the tissue morphology and functional recovery of fresh or freeze-thaw fetal ovaries following avascular subcutaneous allotransplantation,and to explore the feasibility of ovarian tissue allograft and ultra rapid freezing. DESIGN,TIME AND SETTING: This study was a randomized control animal experiment and completed in Yunnan University of Monoclonal Antibody Research Centre from May 2006 to February 2007 after designed in the Department of Reproduction and Genetics,the First People’s Hospital of Yunnan Province from March to May in 2006. MATERIALS: Sprague-Dawley (SD) rat embryos with gestational age between 18 to 20 days were obtained. Adult female SD rats in normal estrous cycle without mating were chosen as transplantation objects and divided into 4 groups: normal control group (n=10),castrated control group (n=10),fresh embryo transfer group (n=25) and frozen embryo transfer group (n=25). METHODS: After anesthesia,rats in the castrated group were treated with bilateral ovariectomy,and those in the normal control group were only given fasciotomy. 18-20 days fresh and frozen-thawed rat ovaries were allotransplanted subcutaneously to adult female SD rats that had been castrated for 4 weeks as experimental group,named fresh embryo transfer group and fozen embryo transfer group. MAIN OUTCOME MEASURES: The restoration of estrous cycle was evaluated by observing vaginal smears and measuring serum oestradiol and progesterone levels. Histological observation was carried on transplanted ovaries. RESULTS: 52.17% of the rats in fresh embryo transfer group and 38.01% in frozen embryo transfer group resulted in restoration of regular estrous cyclic activity. Serum oestradiol and progesterone levels became normal on day 35 and day 49 after transplantation. Though it was slower in the frozen-thawed embryo transfer group than that of the fresh embryo transfer group,there were no significant differences in estrous cycle between experimental group and normal control group. The grafts were taken out and observed on day 63 after transplantation,rich blood vessels were formed on the cover of the graft,arterioles and venules could be seen under microscope. Histological examination of graft revealed that transplanted fetal ovary contained follicles in different developing stages,vesicula graafianae and corpus luteum were formed,and the morphous of graft was no obvious differences to normal ovaries. CONCLUSION: Subcutaneously allotransplanted fetal ovaries can survive,develop and restore the endocrine function as nomal ovaries. The activity of frozen fetal ovaries can be conserved by ultra rapid freezing method. Frozen-thawed ovaries possess identical physiological activity as fresh ovaries after being allotransplanted.更多还原