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目的:研究细胞筛网作为载体运用于玻璃化冷冻人卵巢组织的效果,并寻找合适的冷冻剂浓度。方法:选择来自卵巢切除、子宫内膜异位、卵巢浆液性囊肿患者的卵巢组织共10例,以细胞筛网为载体,以二甲基亚砜、乙二醇和蔗糖为冷冻保护剂进行玻璃化冷冻。分别以二甲基亚砜和乙二醇等浓度混配(12%、15%、18%和20%)和0.5 mol/L的蔗糖配成4组不同的冷冻液,并以新鲜组织及常规程序化冷冻组织为对照,观察不同冷冻液组合对卵巢组织始基卵泡的形态学、凋亡以及体外培养后雌激素、孕酮和乳酸脱氢酶的影响,评价冷冻效果。结果:与新鲜组相比,细胞网筛玻璃化冷冻的卵巢组织冻融后,始基卵泡正常率虽有所降低,但优于常规程序冷冻组,其中18%组和15%组的正常率显著增高。组织细胞的凋亡率比新鲜组有所增加,但低于常规程序冷冻组,其中15%组和12%组明显降低。因此,15%组的冷冻效果最好。比较了该浓度组合对卵巢组织培养后的雌激素、孕酮、乳酸脱氢酶的影响,结果发现,与新鲜组和程序化冷冻组相比,培养液中雌激素水平无明显差异,孕酮水平略好于程序组,但乳酸脱氢酶与新鲜组的变化完全一样,都显著优于程序化组。结论:细胞筛网玻璃化冷冻法优于程序化冷冻法,可作为临床卵巢组织冷冻的一种方法,15%的二甲基亚砜和乙二醇等浓度组合效果最佳。
OBJECTIVE: To study the effect of cell sieve as a carrier on vitrification of human ovarian tissue and to find a suitable concentration of cryogen. Methods: A total of 10 cases of ovarian tissue from ovarian resection, endometriosis and ovarian serous cyst were selected and vitrified by using cell sieve as carrier and dimethyl sulfoxide, ethylene glycol and sucrose as cryoprotectant freezing. Four different freezing solutions were prepared by mixing with dimethyl sulfoxide and ethylene glycol (12%, 15%, 18% and 20% respectively) and 0.5 mol / L sucrose. Fresh tissues and routine The frozen tissue was used as a control to observe the effect of different freezing fluid combinations on the morphology and apoptosis of primordial follicles in ovarian tissue and estrogen, progesterone and lactate dehydrogenase after in vitro culture to evaluate the freezing effect. Results: Compared with the fresh group, the normal rate of primordial follicles decreased after freeze-thaw in vitrified vitrified cells, but it was better than that in normal group (18% vs 15%) Significantly increased. The apoptotic rate of tissue cells increased compared with fresh group, but it was lower than that of routine frozen group, of which 15% and 12% groups were significantly decreased. Therefore, 15% of the best group of freezing effect. The effect of this concentration combination on estrogen, progesterone and lactate dehydrogenase after ovarian tissue culture was compared. Compared with fresh group and programmed freezing group, there was no significant difference in estrogen level between the two groups. Progesterone Level slightly better than the program group, but lactate dehydrogenase and fresh group exactly the same, are significantly better than the programmed group. CONCLUSION: Cell-cell vitrification is superior to programmed freezing in freezing freezing process, which can be used as a freezing method for clinical ovarian tissue. The combination of 15% dimethyl sulfoxide and ethylene glycol has the best effect.