本文選題：慢速冷凍 + 玻璃化冷凍�。� 參考：《北京協(xié)和醫(yī)學(xué)院》2015年博士論文

【摘要】：研究背景腫瘤患者經(jīng)過手術(shù)或放化療后生殖功能會受到明顯影響,已經(jīng)有多種方法能夠?qū)@些患者的性腺功能進行保護,而在卵巢功能受損前進行卵巢組織的冷凍更適合于青春期前卵泡尚未發(fā)育成熟的患者、不能延誤腫瘤治療、激素治療或促排卵有禁忌以及有切除雙側(cè)附件風(fēng)險的女性患者,因此具有很大的潛在意義。卵巢皮質(zhì)內(nèi)具有大量的卵泡儲備,原始卵泡因體積小、代謝率低等特征能夠耐受低溫損傷,在皮質(zhì)冷凍中可以相對多地完好保存。目前的卵巢組織冷凍保存尚處于研究階段。從冷凍方法上講,可以分為慢速冷凍和玻璃化冷凍。慢速冷凍是傳統(tǒng)的冷凍方法,采用低濃度的冷凍保護劑從而降低細胞毒性,玻璃化冷凍因為降溫迅速,組織或細胞與冷凍劑接觸時間短,則需要更高濃度的冷凍保護劑和更快的降溫速率。目前卵巢組織的冷凍方案尚無統(tǒng)一意見,但多數(shù)取得臨床活產(chǎn)的研究采用慢速冷凍。而玻璃化冷凍因其較好的胚胎/卵母細胞存活率、操作耗時短、無需特殊儀器而備受青睞,也逐步應(yīng)用至卵巢組織冷凍的研究中。目的比較不同冷凍方案對小鼠卵巢組織的保存效果。方法25只六周齡ICR雌鼠,取卵巢皮質(zhì)切塊后,隨機分成5組：新鮮(對照)組、二甲基亞砜慢速冷凍(A)組、丙二醇慢速冷凍(B)組、冷凍管玻璃化冷凍5min(C)組及冷凍管玻璃化冷凍8min(D)組。分別進行卵巢組織冷凍復(fù)蘇、復(fù)蘇后體外培養(yǎng)兩周以及復(fù)蘇后進行同種體內(nèi)移植,并飼養(yǎng)兩周。比較三種情況下各組的原始卵泡形態(tài)正常率；卵巢組織中的原始卵泡凋亡情況；比較各體外培養(yǎng)組培養(yǎng)液中以及各復(fù)蘇移植后小鼠靜脈血中雌二醇(E2)水平。結(jié)果復(fù)蘇后,與對照組相比, C、D組原始卵泡形態(tài)正常率顯著降低(P0.001),A、B組無顯著性差異。體外培養(yǎng)兩周后及復(fù)蘇移植飼養(yǎng)兩周后,各組原始卵泡形態(tài)正常率無顯著差異(P0.1)。細胞凋亡檢測顯示,三種情況下卵泡凋亡率均無顯著性差異。體外培養(yǎng)過程中,各組培養(yǎng)液E2水平均不斷增加(P0.001),A、B兩組的E2平均濃度顯著高于對照組及C、D兩組(P0.05),對照組與C組及D組差異無顯著性(P0.05)。移植飼養(yǎng)兩周后,小鼠血清E2水A、B組顯著高于C、D組及對照組(P0.05)。結(jié)論慢速冷凍與冷凍管玻璃化冷凍均能較好地保存小鼠卵巢組織,經(jīng)過體外培養(yǎng),能保持一定的生殖內(nèi)分泌功能,復(fù)蘇移植的卵巢組織生長良好。本研究條件下,慢速冷凍對卵巢組織的保存效果優(yōu)于玻璃化冷凍,仍需進一步摸索更合適的冷凍方法和冷凍條件。
[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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R713.6

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本文編號：1893896