【作者】 王丹丹；

【导师】 王兴玲；

【作者基本信息】 郑州大学 ， 妇产科学， 2010， 硕士

【摘要】 早期诊断、有效地放化疗以及骨髓移植的应用,明显改善了肿瘤的治疗效果,大大提高了肿瘤患者的生存率。但对于年轻的女性患者而言,生存率的提高并不意味着生活质量的提高。因为卵巢组织对这些细胞毒性治疗尤其是烷化剂及放疗特别敏感,有可能导致女性生殖内分泌功能的丧失。多方面的研究已经证实对这些患者实施卵巢组织冷冻保存是有效的。卵巢组织中存在大量的卵泡,如果能够有效地将其冷冻保存,需要时解冻并加以利用,必将为生命科学的进一步发展提供丰富的资源和强有力的研究平台。近年卵巢组织冷冻研究发展迅速,并逐渐成为生命科学研究中的热点问题。玻璃化冷冻技术是20世纪90年代发展起来的冷冻保存技术,目前已在胚胎冷冻保存方面得到广泛的应用,相继有学者报道了玻璃化冷冻成功保存鼠、兔、羊、猴等卵巢组织的研究,并证实玻璃化冷冻可以成为保存卵巢组织功能较为有效的手段。目的当前卵巢组织玻璃化冷冻保存还没有统一的冷冻方案,包括冷冻保护剂和冷冻载体。国内的卵巢组织玻璃化冷冻尚处于探索阶段,本次研究通过观察新鲜和冻融后的卵巢组织的某些生理特点,着重探讨不同玻璃化冷冻方案对人卵巢组织冷冻的效果,初步比较不同浓度玻璃化冷冻保护剂以及不同的冷冻载体对人卵巢组织玻璃化冷冻效果,初步建立一种人卵巢组织玻璃化冷冻方案。材料与方法1材料2008年8月至2009年3月间,本研究募集了20例因卵巢囊肿需要手术的患者,年龄为24-38岁(29.79±4.14)。活检卵巢组织均通过腹腔镜下获得,在进行囊肿剥离术后剪下附于囊肿壁上且无明显血栓的卵巢组织片进行实验。2方法2.1卵巢组织的处理将取得的卵巢组织片置于装有生理盐水液的试管内并放入冰桶中保存,30分钟内送至实验室,在磷酸盐缓冲液中反复漂洗、去除髓质,将卵巢皮质片切割成2*2*1mm3大小的组织块,放在磷酸盐缓冲液中备用。随机选取部分组织块置于中性福尔马林液中固定作新鲜对照组。2.2卵巢组织玻璃化冷冻保存采用低浓度比二甲基亚砜(DMSO)和乙二醇(EG)为玻璃化Ⅰ组,高浓度比为玻璃化Ⅱ组。将部分人卵巢组织块在平衡液和两种玻璃化冷冻液中暴露一定的时间后分别经微滴法、麦管法和坚硬表面玻璃化法(SSV)投入液氮中进行玻璃化冷冻保存。2.3卵巢组织冷冻复苏及体外培养采用三步复苏法将冷冻保存的组织颗粒逐个复苏,随机选取部分复苏后的卵巢组织块置于CO2培养箱中培养14天,每48小时收集培养上清液,离心、等待测激素水平。将剩余组织块及培养结束后的组织块中性固定后等待组织学检查。2.4组织学检查及激素水平测量每四个组织块为一待检平面行石蜡包埋,每个石蜡块以4μm厚度切片,行HE染色。以RIA测定上清液中雌二醇(estradiol, E2)水平,以评定冻融后卵巢组织的内分泌功能。3统计学处理利用SSPS13.0对数据进行统计学分析。组间原始卵泡形态正常率行χ2检验分析,组间凋亡率比较行单因素方差分析,体外培养两周各组间激素分泌量行随机配对方差分析,以a=0.05检验水准。1冷冻对人卵巢组织间质及各级卵泡比例和形态学的影响1.1冷冻对人卵巢组织间质的影响新鲜人卵巢组织间质细胞排列紧密且规律,将各级卵泡紧密的包裹在卵巢皮质内。与新鲜对照组相比,两种玻璃化冷冻方案冷冻保存的卵巢组织,复苏后均可见卵巢组织内部分间质细胞分离,但原始卵泡变化不明显。体外培养后卵巢组织间质细胞间连接疏松、甚至断裂形成裂隙,细胞排列紊乱。1.2冷冻对人卵巢组织内各级卵泡比例和形态学的影响与新鲜组相比,两冷冻组原始卵泡的数目和正常形态率都有不同程度的下降,但玻璃化Ⅰ组原始卵泡正常形态率差异无统计学意义(P>0.05),玻璃化Ⅱ组差异有统计学意义(P<0.05),两冷冻组间差异有统计学意义(P<0.05)。新鲜组与各冷冻组初级卵泡和次级卵泡数目均较少,本次研究中不再对其进行统计学分析。2冷冻对人卵巢组织中各类细胞凋亡情况的影响卵巢组织内原始卵泡、初级卵泡的卵母细胞以及颗粒细胞内均未见凋亡细胞的阳性表达；而卵巢组织内部分间质细胞的细胞核内可见阳性表达的凋亡细胞,呈棕色或棕褐色。与新鲜对照组相比,玻璃化冷冻各组卵巢组织内间质细胞凋亡率差异无统计学意义(P>0.05)。3冷冻对复苏后卵巢组织分泌E2的影响组织体外培养期间,各冻融组分泌E2不断增高,两冷冻组间同种载体间差异有统计学意义(P<0.05),两冷冻组内各载体间差异无统计学意义(P>0.05)。结论1各冷冻组中SSV法明显优于微滴法及麦管法,较适合卵巢组织冻存。2较低浓度冷冻保护剂及SSV玻璃化冷冻方法保存卵巢组织的效果更佳。更多还原

【Abstract】 Early diagnosis and effective chemotherapy, as well as the application of bone marrow transplantation has significantly improved the therapeutic effect of cancer and greatly enhanced the survival rate of cancer patients. But for young female patients, raising the survival rate does not mean that the quality of life improved. Because ovarian tissue toxicity of the treatment of these cells, in particular, alkylating agent and radiotherapy are particularly sensitive, it may lead to the loss of female reproductive endocrine function. Various studies have confirmed that the implementation of these patients with ovarian tissue cryopreservation is effective. There are a lot ovarian follicles, if we can effectively save their frozen, thawed and be used when necessary, is bound to the further development of life sciences provides a wealth of resources and a strong research platform.In recent years, research and development ovarian tissue frozen rapidly, and has become a hot issue in the life sciences. Vitrification technology was developed in the 20th century,90 years of cold-storage technology has been in the embryo cryopreservation in a wide range of applications, some scholars have reported the successful vitrification preservation rats, rabbits, sheep, monkey, etc. Study of ovarian tissue and confirmed that vitrification can be stored ovarian tissue function in a more effective means.ObjectiveCurrent ovarian tissue by vitrification freezing still there is no uniform program, including cryoprotectants and frozen carrier. Domestic ovarian tissue vitrification is still at the exploratory stage, this study by observing the fresh and frozen-thawed ovarian tissue after certain physiological characteristics, focusing on the programs of different vitrification of ovarian tissue freezing effect, a prliminary comparison of different concentrations of Vitrification protecting agent and the different carriers of human ovarian tissue freezing vitrification effect initially established a human ovarian tissue vitrification program. Materials and methods1 MaterialsFrom August 2008 to March 2009, the present study because of raised 20 cases of ovarian cysts need surgery patients, aged 24-38 years (29.79±4.14). Laparoscopic ovarian tissue biopsies were obtained by carrying out spin-off after cyst cyst attached to the cut with no obvious thrombosis of ovarian tissue piece 1-2cm2 experiment.2 Methods2.1 The treatment of ovarian tissueWill achieve the ovarian tissue pieces placed in saline solution and placed in ice bucket can be preserved, sent to the laboratory within 30 minutes, the rinsing in PBS to remove the medulla, the ovarian cortical pieces cut into the size 2*2*1mm3 The tissue block, placed in PBS solution standby. Randomly selected part of the tissue placed in neutral formalin fixed as fresh control group.2.2 Ovarian tissues by vitrificationLow-concentration ratio of dimethyl sulfoxide (DMSO) and ethylene glycol (EG) for vitrification groupⅠ, a high concentration ratio of glass groupⅡ. Part of human ovarian tissue fluid balance and two vitrification solution in a certain time after exposure to each of the two micro-drop method, wheat-tube method and a hard surface vitrification (SSV) into liquid nitrogen for vitrification.2.3 Ovarian tissue cryopreservation and in vitro cultureUsing three-step recovery method will be the organization of cryopreservation recovery of particles one by one randomly selected part of the revival of the ovarian tissue pieces placed in CO2 incubator for 14 days every 48 hours to collect the supernatant centrifuged under test hormone levels. The remaining tissue and cultured tissue after the end of neutral fixation until after the histological examination.2.4 Histological examination and measurement of hormone levelsEach block of four organizations to be seized for a flat-line embedded in paraffin wax block in order to 4μm thickness of each slice, line HE staining. Supernatant measured by RIA of estradiol (estradiol, E2) levels, in order to assess ovarian tissue after freezing and thawing of the endocrine function.3 StatisticalUse SSPS 13.0 the date were analyzed statistically.Between the two groups the rate of morphologically normal primordial follicle useχ2 tests and analysis, comparing the rate of apoptosis between the two groups line single-factor analysis of variance, two weeks in vitro hormone secretion in each group were randomly paired line analysis of variance, withα=0.05 significance level.Results1 Freezing of human ovarian follicles, interstitial and all levels of the proportion and morphology.1.1 Frozen human ovarian tissue stromaFresh human ovarian mesenchymal cells arranged closely and the law will be close at all levels, wrapped in ovarian follicles within the cortex. Two kinds of vitrification solution, as well as three kinds of human ovarian tissue cryopreservation carrier recovery is seen after ovarian tissue in parts of mesenchymal cell separation, but the primordial follicle did not change significantly. After the ovarian tissue cultured in vitro stromal cell connections loose, or even fracture the formation of fissures, and disordered cell arrangement.1.2 Human ovarian tissue before and after freezing at all levels of follicle ratio and morphology Compared with the fresh, two frozen groups the number of primordial follicles and the normal form of the rate of decline in varying degrees, but the glass of the number of primordial follicles in groupⅠand the normal form of the rate of no statistically significant difference (P>0.05), glass transitionⅡgroup differences were statistically significant (P<0.05), two freezing difference between the groups was statistically significant (P<0.05). Frozen fresh group and the group the number of primary follicles and secondary follicles are small, this study and not subject to statistical analysis.2 Frozen human ovarian tissue of various types of cells in apoptosisHuman ovarian tissue in the primordial follicle, primary follicle oocytes and granulosa cells no apoptotic cells in both the positive expression, and ovarian stromal cells within the visible expression of the nucleus of apoptotic cells, brown, or brown color. With the fresh control group, each group vitrification of ovarian tissue mesenchymal cells was no significant difference in apoptosis rate (P>0.05).3 The frozen ovarian tissue after resuscitation group of E2 secretionDuring in vitro tissue culture, the constant increase in E2 secretion of freeze-thaw group, two freezing difference between the groups was statistically significant (P<0.05), the carrier was no significant difference between (P>0.05). Conlusions1 Solid-surface vitrification method is superior to micro-drop method and Mai-tube method, more suitable for primordial follicles in ovarian tissue cryopreservation.2 It is a better effect for the conservation of ovarian tissue low concentration of cryoprotective agents and solid-surface vitrification method.更多还原