本文選題：卵巢組織 + 分離卵泡��； 參考：《鄭州大學(xué)》2011年碩士論文

【摘要】：目的 本研究采用直接覆蓋玻璃化冷凍法凍存人類卵巢組織,解凍后采用酶加機(jī)械聯(lián)合法與培養(yǎng)6天后機(jī)械法分別分離人卵巢組織的腔前卵泡,采用藻酸鈣三維培養(yǎng)體系,在有、無(wú)血清兩種培養(yǎng)系統(tǒng)中進(jìn)行培養(yǎng),通過(guò)比較分離后各級(jí)卵泡的存活率,培養(yǎng)前后卵泡直徑和雌二醇水平的變化,探討簡(jiǎn)捷有效地分離人腔前卵泡的方法及合適的腔前卵泡體外生長(zhǎng)發(fā)育體系。 方法 研究對(duì)象為23例在我院行腹腔鏡或手術(shù)切除卵巢良性腫瘤的患者。將收集到的正常人卵巢皮質(zhì)切成0.5×0.5×1mm3的組織塊,直接覆蓋玻璃化冷凍,解凍后通過(guò)兩種方法進(jìn)行腔前卵泡的分離：一種是膠原酶加機(jī)械法聯(lián)合分離,一種是培養(yǎng)6天后機(jī)械法分離,采用藻酸鈣三維培養(yǎng)體系在10%胎牛血清(fetal calf serum, FBS)和0.3%牛血清蛋白(bovine serum albumin, BSA)兩種培養(yǎng)體系中進(jìn)行培養(yǎng),隔天測(cè)量腔前卵泡的直徑后半量換液,收集廢棄的培養(yǎng)液保存于-20℃冰箱,采用電化學(xué)發(fā)光免疫分析法(electrochemiluminescence immunoassay, ECLIA)測(cè)定雌二醇的水平。采用SPSS17.0對(duì)實(shí)驗(yàn)數(shù)據(jù)進(jìn)行分析。 結(jié)果 1.采用酶加機(jī)械聯(lián)合法對(duì)人卵巢組織進(jìn)行腔前卵泡分離。冷凍前后各級(jí)卵泡的存活率無(wú)統(tǒng)計(jì)學(xué)差異(P0.05),但始基卵泡的存活率較初、次級(jí)卵泡高,表明冷凍保存沒(méi)有降低各級(jí)卵泡的存活率,始基卵泡更能耐受冷凍損傷。 2.采用無(wú)血清培養(yǎng)體系對(duì)腔前卵泡進(jìn)行培養(yǎng)。組織培養(yǎng)6天后機(jī)械法分離腔前卵泡與直接聯(lián)合法分離腔前卵泡培養(yǎng)6天相比,機(jī)械法分離得到的卵泡總數(shù)少,但次級(jí)卵泡的存活率較高,始基卵泡的存活率較低,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 3.解凍后的卵巢組織均采用聯(lián)合法在有、無(wú)血清兩種分離液中分離腔前卵泡。BSA組較FBS組回收到的卵泡多,但各級(jí)卵泡的存活率無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。 4.卵巢組織在無(wú)血清培養(yǎng)體系培養(yǎng)6天后,采用機(jī)械法分離得到腔前卵泡的直徑及培養(yǎng)到第10天腔前卵泡的直徑顯著高于其他3組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。直徑的增幅也較其它3組高,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 5.卵巢組織在無(wú)血清培養(yǎng)體系培養(yǎng)6天后,采用機(jī)械法分離得到腔前卵泡分泌的E2水平及培養(yǎng)到第8天、第10天的E2水平顯著高于其他3組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論 1.直接覆蓋法玻璃化冷凍不影響人卵巢皮質(zhì)中各級(jí)卵泡的存活率 2.培養(yǎng)6天后機(jī)械法分離回收到次級(jí)卵泡的存活率較高,有利于卵泡的后續(xù)培養(yǎng)； 3.采用無(wú)血清分離液可回收到較多的腔前卵泡,且不影響各級(jí)卵泡的存活率 4.無(wú)血清培養(yǎng)體系有助于人腔前卵泡的體外生長(zhǎng)發(fā)育。
[Abstract]:Objective to study the cryopreservation of human ovarian tissue by direct vitrification and cryopreservation, and to separate preantral follicles from human ovarian tissue by enzyme combined with mechanical method and mechanical method after 6 days of culture after thawing. Calcium alginate was used as a three-dimensional culture system. The follicle diameter and estradiol level before and after culture were compared by comparing the survival rate of follicles at all levels after separation and in serum-free culture system, and compared the changes of follicle diameter and estradiol level before and after culture. To explore a simple and effective method for the separation of human preantral follicles and a suitable system of preantral follicle growth and development in vitro. Methods 23 patients with benign ovarian tumors underwent laparoscopic or surgical resection in our hospital. The normal human ovarian cortex was cut into 0. 5 脳 0. 5 脳 1mm3 tissue mass and directly covered with vitrification and frozen. After thawing, preantral follicles were separated by two methods: one was collagenase combined with mechanical method. One was mechanical separation after 6 days of culture. Calcium alginate three-dimensional culture system was used in 10% fetal bovine serum (fetal calf serum,) and 0.3% bovine serum protein (bovine serum albumin,. The diameter of preantral follicles was measured the next day. The waste culture solution was collected and stored in the refrigerator at -20 鈩，

本文編號(hào)：2086403