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目的:系统地探索新生小鼠卵巢组织的玻璃化冻存建立卵巢库,移植和分离卵泡以及体外成熟培养的实验方法。方法:对1日龄小鼠卵巢组织进行冻存,解冻复苏和同种肾包膜下移植,从卵巢移植物种中进行卵泡分离和体外成熟培养。结果:①采用平衡液(ES)处理25min,玻璃化液(VS)处理3min方案冻存的卵巢组织具有更高比例的形态完整卵泡,其完整原始卵泡率均值达96.6%,显著性高于实验中其它四组方案(P<0.05);②在分别移植2周和4周后回收卵巢移植物,发现二者的卵巢回收率无显著差异(P>0.05),但移植4周组的卵泡回收数目要明显高于2周组(P<0.05);③在培养基中添加适量的自体血清(10%,V/V)能显著提高卵子的体外成熟率,培养12h后对照组中生发泡破裂(GVBD)发生率为(34.74±4.26)%,添加血清后提高至(54.60±3.37)%,成熟的MⅡ期卵子获得率从(43.17±1.31)%升高至(57.75±5.31)%,有显著性差异(P<0.05)。结论:通过该实验较好地建立了卵巢组织的玻璃化冻存、移植和卵泡分离以及体外成熟培养的实验方法。
OBJECTIVE: To systematically explore the vitrification of ovarian tissue in neonatal mice to establish ovarian bank, transplant and separate follicles and to develop in vitro maturation experiment. Methods: The 1-day-old mouse ovaries were cryopreserved, thawed and resuscitated and allogeneic subrenal transplantation was performed. Follicles were isolated from ovarian transplant recipients and cultured in vitro. Results: (1) The proportion of intact follicles in the frozen ovary tissue treated with VS (ES medium) for 25min and vitrification solution (VS) for 3min was higher than that of the experimental group (P <0.05). ② The ovarian transplantation was recovered after 2 weeks and 4 weeks of transplantation, respectively. There was no significant difference in ovary recovery between the two groups (P> 0.05) (P <0.05); ③Adding the appropriate amount of autologous serum (10%, V / V) to the medium can significantly increase the in vitro maturation rate of ovum, (34.74 ± 4.26)% of GVBD increased to (54.60 ± 3.37)% after addition of serum, and the rate of mature M Ⅱ ovary increased from (43.17 ± 1.31)% to (57.75 ± 5.31)% with Significant difference (P <0.05). Conclusion: The experimental method of vitrification, transplantation and follicular isolation and in vitro maturation culture of ovarian tissue is well established.