小鼠卵巢组织冷冻保存方法的初步研究

发布时间：2018-05-15 20:59

本文选题：慢速冷冻 + 玻璃化冷冻　；参考：《北京协和医学院》2015年博士论文

【摘要】：研究背景肿瘤患者经过手术或放化疗后生殖功能会受到明显影响,已经有多种方法能够对这些患者的性腺功能进行保护,而在卵巢功能受损前进行卵巢组织的冷冻更适合于青春期前卵泡尚未发育成熟的患者、不能延误肿瘤治疗、激素治疗或促排卵有禁忌以及有切除双侧附件风险的女性患者,因此具有很大的潜在意义。卵巢皮质内具有大量的卵泡储备,原始卵泡因体积小、代谢率低等特征能够耐受低温损伤,在皮质冷冻中可以相对多地完好保存。目前的卵巢组织冷冻保存尚处于研究阶段。从冷冻方法上讲,可以分为慢速冷冻和玻璃化冷冻。慢速冷冻是传统的冷冻方法,采用低浓度的冷冻保护剂从而降低细胞毒性,玻璃化冷冻因为降温迅速,组织或细胞与冷冻剂接触时间短,则需要更高浓度的冷冻保护剂和更快的降温速率。目前卵巢组织的冷冻方案尚无统一意见,但多数取得临床活产的研究采用慢速冷冻。而玻璃化冷冻因其较好的胚胎/卵母细胞存活率、操作耗时短、无需特殊仪器而备受青睐,也逐步应用至卵巢组织冷冻的研究中。目的比较不同冷冻方案对小鼠卵巢组织的保存效果。方法25只六周龄ICR雌鼠,取卵巢皮质切块后,随机分成5组：新鲜(对照)组、二甲基亚砜慢速冷冻(A)组、丙二醇慢速冷冻(B)组、冷冻管玻璃化冷冻5min(C)组及冷冻管玻璃化冷冻8min(D)组。分别进行卵巢组织冷冻复苏、复苏后体外培养两周以及复苏后进行同种体内移植,并饲养两周。比较三种情况下各组的原始卵泡形态正常率；卵巢组织中的原始卵泡凋亡情况；比较各体外培养组培养液中以及各复苏移植后小鼠静脉血中雌二醇(E2)水平。结果复苏后,与对照组相比, C、D组原始卵泡形态正常率显著降低(P0.001),A、B组无显著性差异。体外培养两周后及复苏移植饲养两周后,各组原始卵泡形态正常率无显著差异(P0.1)。细胞凋亡检测显示,三种情况下卵泡凋亡率均无显著性差异。体外培养过程中,各组培养液E2水平均不断增加(P0.001),A、B两组的E2平均浓度显著高于对照组及C、D两组(P0.05),对照组与C组及D组差异无显著性(P0.05)。移植饲养两周后,小鼠血清E2水A、B组显著高于C、D组及对照组(P0.05)。结论慢速冷冻与冷冻管玻璃化冷冻均能较好地保存小鼠卵巢组织,经过体外培养,能保持一定的生殖内分泌功能,复苏移植的卵巢组织生长良好。本研究条件下,慢速冷冻对卵巢组织的保存效果优于玻璃化冷冻,仍需进一步摸索更合适的冷冻方法和冷冻条件。
[Abstract]:Background the reproductive function of cancer patients is significantly affected after surgery or radiotherapy and chemotherapy, and there are many ways to protect the gonadal function of these patients. However, cryopreservation of ovarian tissue prior to impaired ovarian function is more suitable for women with immature prepubertal follicles who do not delay tumor therapy, have taboos in hormone therapy or ovulation promotion, and are at risk of excision of bilateral appendages. Therefore, it has great potential significance. There are a lot of follicular reserves in the ovarian cortex. The primary follicles can withstand low temperature damage due to their small size and low metabolic rate, and can be preserved relatively well in the cryopreservation of the cortex. The cryopreservation of ovarian tissue is still in the research stage. In terms of freezing method, it can be divided into slow freezing and vitrification. Slow freezing is a traditional method of freezing. Low concentration of cryopreservation protectant is used to reduce cytotoxicity. Vitrification is due to rapid cooling, and the contact time between tissue or cell and refrigerant is short. Higher concentrations of cryoprotectants and faster cooling rates are needed. At present, there is no uniform opinion on cryopreservation of ovarian tissue, but slow freezing is used in most studies of obtaining clinical live delivery. Vitrified cryopreservation has been widely used in the research of ovarian tissue cryopreservation because of its good survival rate of embryo / oocyte, short operation time and no need of special instruments. Objective to compare the effects of different cryopreservation protocols on mouse ovarian tissue preservation. Methods Twenty-five six-week-old ICR female rats were randomly divided into five groups: fresh (control) group, dimethyl sulfoxide slow freezing group (DMSO) group and propanediol slow cryopreservation group (B) group. Group C (5 min) and group D (8 min). Ovarian tissue was resuscitated by cryopreservation, cultured in vitro for two weeks after resuscitation, and transplanted in vivo after resuscitation, and fed for two weeks. The normal rate of primordial follicle morphology in each group, the apoptosis of primordial follicle in ovarian tissue and the level of estradiol E _ 2 in culture medium of each culture group in vitro and in vein blood of mice after resuscitation transplantation were compared. Results after resuscitation, there was no significant difference in the normal rate of primordial follicle morphology between Con D group and control group. After two weeks of culture in vitro and two weeks after resuscitation transplantation, there was no significant difference in the normal rate of primordial follicles in each group. Apoptosis detection showed that there was no significant difference in the rate of follicular apoptosis in the three conditions. During in vitro culture, the level of E _ 2 in culture medium of each group was significantly higher than that of control group and Con D group (P 0.05), but there was no significant difference between control group and C group and D group. After transplantation for two weeks, the serum E _ 2 of Agna B group was significantly higher than that of C _ (2) D group and control group (P _ (0.05). Conclusion both slow freezing and vitrification with cryopreservation tube can preserve the ovarian tissue of mice. After culture in vitro, the reproductive endocrine function can be maintained, and the ovarian tissue of resuscitation and transplantation can grow well. In this study, the cryopreservation effect of slow cryopreservation on ovarian tissue was better than that of vitrification.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R713.6

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