冻融人卵巢组织腔前卵泡的分离及体外培养的研究

发布时间：2018-06-30 18:12

  本文选题：卵巢组织 + 分离卵泡　； 参考：《郑州大学》2011年硕士论文

【摘要】：目的 本研究采用直接覆盖玻璃化冷冻法冻存人类卵巢组织,解冻后采用酶加机械联合法与培养6天后机械法分别分离人卵巢组织的腔前卵泡,采用藻酸钙三维培养体系,在有、无血清两种培养系统中进行培养,通过比较分离后各级卵泡的存活率,培养前后卵泡直径和雌二醇水平的变化,探讨简捷有效地分离人腔前卵泡的方法及合适的腔前卵泡体外生长发育体系。 方法 研究对象为23例在我院行腹腔镜或手术切除卵巢良性肿瘤的患者。将收集到的正常人卵巢皮质切成0.5×0.5×1mm3的组织块,直接覆盖玻璃化冷冻,解冻后通过两种方法进行腔前卵泡的分离：一种是胶原酶加机械法联合分离,一种是培养6天后机械法分离,采用藻酸钙三维培养体系在10%胎牛血清(fetal calf serum, FBS)和0.3%牛血清蛋白(bovine serum albumin, BSA)两种培养体系中进行培养,隔天测量腔前卵泡的直径后半量换液,收集废弃的培养液保存于-20℃冰箱,采用电化学发光免疫分析法(electrochemiluminescence immunoassay, ECLIA)测定雌二醇的水平。采用SPSS17.0对实验数据进行分析。 结果 1.采用酶加机械联合法对人卵巢组织进行腔前卵泡分离。冷冻前后各级卵泡的存活率无统计学差异(P0.05),但始基卵泡的存活率较初、次级卵泡高,表明冷冻保存没有降低各级卵泡的存活率,始基卵泡更能耐受冷冻损伤。 2.采用无血清培养体系对腔前卵泡进行培养。组织培养6天后机械法分离腔前卵泡与直接联合法分离腔前卵泡培养6天相比,机械法分离得到的卵泡总数少,但次级卵泡的存活率较高,始基卵泡的存活率较低,差异有统计学意义(P0.05)。 3.解冻后的卵巢组织均采用联合法在有、无血清两种分离液中分离腔前卵泡。BSA组较FBS组回收到的卵泡多,但各级卵泡的存活率无统计学差异(P0.05)。 4.卵巢组织在无血清培养体系培养6天后,采用机械法分离得到腔前卵泡的直径及培养到第10天腔前卵泡的直径显著高于其他3组,差异有统计学意义(P0.05)。直径的增幅也较其它3组高,差异有统计学意义(P0.05)。 5.卵巢组织在无血清培养体系培养6天后,采用机械法分离得到腔前卵泡分泌的E2水平及培养到第8天、第10天的E2水平显著高于其他3组,差异有统计学意义(P0.05)。 结论 1.直接覆盖法玻璃化冷冻不影响人卵巢皮质中各级卵泡的存活率 2.培养6天后机械法分离回收到次级卵泡的存活率较高,有利于卵泡的后续培养； 3.采用无血清分离液可回收到较多的腔前卵泡,且不影响各级卵泡的存活率 4.无血清培养体系有助于人腔前卵泡的体外生长发育。
[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，

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