大鼠卵巢组织冷冻保存和自体移植后形态与功能的研究

Study on the morphology and function of ovarian tissue in rats after cryopreservation and transplantation

目的:探讨大鼠卵巢组织冷冻、解冻和自体皮下移植后甾体激素分泌和卵泡生长发育的恢复情况。方法:将大鼠卵巢组织在程序性降温设备中缓慢冷冻,在液氮中保存14天后快速解冻。将新鲜和冻融后的大鼠卵巢组织分别自体异位移植于去势成年雌鼠的腹股沟皮下。术后每日行阴道细胞涂片检查,观察动情周期恢复情况,并分别于移植后4周和8周取移植卵巢进行组织学检查并测定血清雌孕激素水平。结果:新鲜组和冷冻组分别有80.6%和48.5%的大鼠恢复了动情周期,差异有统计学意义(P<0.01)。新鲜组和冷冻组首次恢复动情周期的天数分别为(8.72±1.69)天和(18.15±2.48)天,差异有统计学意义(P<0.01)。移植术后4周时冷冻组的E2和P水平与假手术组及新鲜组相比差异有统计学意义(P<0.05),而移植术后8周时3组激素水平差异已无统计学意义(P>0.05)。新鲜组和冷冻组移植术后4周和8周时移植物内均可见丰富的血管及不同发育阶段的卵泡和黄体。结论:大鼠的卵巢组织可以较好的耐受冷冻损伤,适宜条件下可以复苏恢复甾体激素的分泌,并且有卵泡的生长和发育。非血管吻合自体异位移植卵巢组织的方法简便易行且具有较高的成活率,是冻存卵巢组织较为安全有效的方法。

Objective： To explore the restoration of ovarian steroid secretion, follicular growth and development after autotransplantation of cryopreserved rat ovarian tissues. Methods： The rat ovarian tissues were slowly frozen in a programmable freezer, then the tissues were stored in liquid nitrogen for 2 weeks and fast thawed. Fresh and frozen - thawed ovarian tissues were autotransplanted （ in the right inguinal vascular plexus） to the bilaterally ovarieetomized adult female rats. Vaginal smears were taken daily after transplantation to observe the restoration of oestrous cycles. On the fourth and eighth week after transplantation, the ovarian tissues were removed and underwent histological examination. Serum estrogen and progesterone levels were detected. Results： 80. 6% of the rats in fresh group and 48.5% of the rats in frozenthawed group restored regular oestrous cyclic activity, there was significant difference between the two groups （P 〈 0. 01 ） . The vaginal cytology showed recovel7 times were （8.72 ± 1.69 ） days and （ 18. 15 ± 2. 48 ） days after transplantation in fresh and frozen -thawed groups, respectively （P 〈0. 01 ） . There was significant difference in estrogen and progesterone levels between frozen -thawed group and fresh group, sham operation group （P 〈 0. 05） on the fourth week, but there was no significant difference between the three groups on the eighth week （ P 〉 0. 05 ） . Lots of vessels, follicles in different developing stages and corpus luteum were observed on the fourth and eighth week after transplantation in both fresh group and frozen - thawed group according histological examination. Conclusion ： Cryopreserved rat ovarian tissues can be autotransplanted with subsequent restoration of hormone secretion and the follicular growth and development. Autotransplantation of cryopreserved ovarian tissues without vascular anastomosis is a practical and effective method.