【作者】 郑华；

【导师】 邓晓惠；

【作者基本信息】 山东大学 ， 妇产科学， 2009， 硕士

【摘要】 目的:评估成年雌性Wistar大鼠冻存卵巢组织自体异位移植后的长期生殖、内分泌功能。方法:将78只健康、存在规律动情周期的性成熟雌性Wistar大鼠,按随机数字表法分为假手术组(1组,15只)、新鲜组织移植组(2组,24只)、冻存组织移植组(3组,24只)和去势组(4组,15只)。1组:仅行假手术,常规麻醉后切开大鼠皮肤、肌肉,保留卵巢,依次缝合肌肉、皮肤;2组:切除大鼠双侧卵巢,将卵巢用生理盐水冲洗干净后平均切为8块,每块组织块体积约为1mm×2mm×2mm。随机取6块新鲜卵巢组织自体异位多点移植于每侧肾被膜下;3组:切除大鼠双侧卵巢,将每侧卵巢用生理盐水冲洗干净后平均切为8块,采用玻璃化冷冻方法冷冻保存,做好标记。2～4周后将冻存卵巢组织复苏,随机取6块自体卵巢组织块多点移植于每侧肾被膜下;4组:切除大鼠双侧卵巢,作为去势对照组。术后定期行阴道冲洗术,检查阴道脱落细胞直至有典型角化细胞出现。分别于移植后5、8、10个月随机抽取各处理组1/3数量动物,结合阴道细胞学检查在大鼠动情期从其颈静脉抽血监测血清雌、孕激素水平,评估移植后卵巢组织的生殖内分泌功能。大鼠处死后取其卵巢及子宫,称量子宫湿重、做卵巢及子宫组织切片。通过光镜下行组织学观察,计数各级卵泡数目,反映移植后卵巢组织存活及卵泡生长发育情况。结果:所有动物模型中,新鲜及冻存卵巢组织异位移植后都可观察到存活组织块,组织块表面肉眼可见有新生血管覆盖。2、3组大鼠卵巢移植术后阴道细胞学检查均有典型角化细胞出现。2、3组大鼠动情期血清雌、孕激素水平与假手术组无显著性差异(P＞0.05),且均明显高于去势组(P＜0.01)。移植后冻存卵巢组织与新鲜卵巢组织光镜下均可观察到各级卵泡及黄体存在。冷冻复苏后组织与新鲜组织各级卵泡构成比无显著性差异(P＞0.05)。从2、3组取得的移植后卵巢组织中均存在各级卵泡,其各级卵泡构成比与同期1组大鼠卵巢组织相比均无显著性差异(P＞0.05)。与冷冻复苏后组织相比,3组大鼠分别在移植后5、8、10个月取得的卵巢组织中各级卵泡构成比均有显著性差异(P＜0.05),成熟卵泡数明显增加(P＜0.05)。各组内不同时间取得的卵巢组织各级卵泡构成比相比较均无显著性差异(P＞0.05)。移植后5个月,2、3组卵巢组织中原始卵泡数明显减少,分别为假手术组的59.5%和55.1%。结论:小块卵巢组织可耐受冻融过程;冻存卵巢组织自体肾被膜下移植后,卵巢组织内有各级卵泡及黄体存在,原始卵泡总数虽然明显减少,但有成熟卵泡发育并排卵,并能长期维持生殖内分泌功能。更多还原

【Abstract】 OBJECTIVE To assess the long-term efficacy of cryopreserved ovarian tissues after heterotopic autologous transplanted in rats.METHODS Seventy-eight Wistar adult female rats were divided into four groups randomly as follows:sham-operated group（group 1,n=15）, fresh-transplanted group（group 2,n=24）,frozen-thawed-transplanted group（group 3,n=24） and ovariectomized group（group 4,n=15）.Group 1,underwent a sham operation;Group 2,underwent bilateral ovariectomy and their each ovarian were cut into 8 blocks（about 1mm×2mm×2mm）,selected randomly 6 blocks of fresh ovarian tissues and autografted immediately to their each renal capsule;Group 3, underwent bilateral ovariectomy followed by ovarian tissue cryopreservation,then transplanted the ovarian tissues autologously（6 blocks of frozen-thawed ovarian tissues per capsule） under the renal capsules 2-4 weeks later;Group 4,underwent bilateral ovariectomy,and served as castration group.Vaginal irrigation samples were collected daily every two weeks until typical cornified epithelial cells were observed.One third of the animals in each group were killed and their uteri were weighted,and the ovaries and parts of the uteri were removed for morphologic analysis at the 5^th,8^th,or 10^th months after transplantation respectively.Before being killed,the serum concentrations of estradiol-17β（E₂） and progesterone（P） were measured during the period of estrus-like cytological variation according to completely cornified epithelial cells shown by the samples of vaginal irrigation.The follicles of each stage of folliculogenesis were counted under the light microscope. RESULTS Both fresh and frozen ovarian grafts survived in all the animal models.Blood vessels surrounding the grafts were visible.Daily vaginal irrigation showed completely comified epithelial cells in groups 2 and 3.In groups 2 and 3, the serum E₂ and progesterone concentrations during estrus remained comparable to the sham-operated rats’ hormone levels（groupl）,and significantly higher than the concentrations in group 4 at the 5^th,8^th,10^th months respectively（P＜0.01）. Morphologically,there were each stage of follicles and lutea.No significant differences were observed in the proportion of each stage of follicles between frozen-thawed tissues and fresh ovarian tissues（P＞0.05）.And the proportion of each stage follicles in both types of grafts was comparable to that in group 1 at the same time（P＞0.05）.The number of primordial follicles in fresh grafts and frozen grafts were significantly lower than that in group 1.Grafts in group 2 contained 59.5%of the primordial follicles in sham-operated controls while frozen grafts contained 55.1%at the 5^th month after implantation.CONCLUSIONS Cryopreservation of small pieces of ovarian tissues is feasible.After autologous implanted under the renal capsule,the cryopreserved ovarian tissues contain each stages of follicles and lutea.The follicles survive and develop well,though the number of primordial follicles decreased.The secretary function of ovarian tissues could be preserved in a long term.更多还原