不同玻璃液冻存卵巢组织移植于鸡胚前后的氧化应激和细胞凋亡研究

Oxidative stress reaction and apoptosis of human ovarian tissue pre/post transplanted into chicken embryos after cryopreservation with different vitrification solutions

目的 研究不同玻璃液（VS）保护下冻存的卵巢组织移植于鸡胚前后的氧化应激和细胞凋亡情况,以评估不同玻璃液的冷冻保护效果。方法 将卵巢组织分割成1-2mm×1mm×1mm的小块分别放入VS1、VS2、VS3、VS4 4种不同的玻璃液中,经过4种玻璃液平衡后冻存并复苏的卵巢组织称为冻存卵巢组织（FOT）,分别将经4种玻璃液冻存的FOT移植到胚龄10d的鸡卵绒毛尿囊膜（CAM）上培养,于鸡卵胚龄16d取出移植于CAM的卵巢组织（TOT）,以未经玻璃液平衡的新鲜卵巢组织为对照（COT）,分析4种玻璃液保存的FOT、TOT、COT中线粒体活性、细胞内活性氧自由基（ROS）水平和间质细胞凋亡情况。结果 线粒体活性：经过VS1、VS3和VS4冻存的FOT和TOT中的线粒体活性低于COT,差异有统计学意义（P〈0.05）;经VS2冻存的TOT与COT中线粒体活性无显著性差异（P〉0.05）。细胞内ROS水平：经过VS1、VS3和VS4冻存的FOT和TOT细胞内ROS水平低于COT,有统计学差异（P〈0.05）;经VS2冻存的FOT细胞内ROS水平与COT组比较无显著性差异（P〉0.05）。间质细胞凋亡情况：经过VS1、VS2、VS3和VS4平衡的FOT间质细胞凋亡率和COT组之间的差异无统计学意义（P〉0.05）。结论 由玻璃液VS2保护冻存卵巢组织的冷冻损伤小、移植后缺血损伤轻,是本研究优化的卵巢冷冻保护玻璃液;玻璃化冷冻对间质细胞损伤小,适于人卵巢组织的冷冻保存。

Objective： To evaluate the effect of cryopreservation with different stress reaction and apoptosis of human ovarian tissues before and after chorioallantoic membrane （CAM）. vitrification solutions on oxidative transplantation into chick embryo Methods： Human ovarian tissues were dissected to 1-2 mm× 1 mm× 1 mm size,and then put into in four kinds of vitrification solutions （VS1, VS2, VS3, VS4） for cryopreservation. Part of the frozen ovarian tissues（FOT）was grafted into CAM. The transplanted ovarian tissues（TOT）were collected 5 days after transplantation. The tissue without treatment was as control ovarian tissue （COT）. The mitochondrial activity,reactive oxygen species （ROS）and apoptosis of stromal cells in COT, FOT and TOT after cryopreservation with four different vitrification solutions were evaluated and compared. Results： Mitochondrial activity of tissue after cryopreservation with VS1, VS3 or VS4 was significantly lower than COT （P 〈 0.05） . TOT after cryopreservation with VS2 was not significant different with COT（P〉0.05）. ROS levels in FOT and TOT after cryopreservation with VS1 ,VS3 or VS4 were significantly lower than COT（P〈0.05）. ROS levels in FOT with VS2 were not significantly different with COT（P〉0.05）. There was no significant difference in apoptosis between FOT and COT among the four VS groups（P〉0.05）. Conclusions： VS2 results in a higher level of mitochondrial activity and ROS,therefore,it is an optimal solution. Vitrification cryopreservation shows little damage to stromal cells, which is an alternative way to protect human ovarian tissue.