【作者】 李慧蓉；

【导师】 魏兆莲；

【作者基本信息】 安徽医科大学 ， 妇产科学， 2010， 硕士

【摘要】 多囊卵巢综合征( PCOS)是一种以内分泌紊乱为主、多种代谢异常导致的异质性临床综合征。PCOS患者双侧卵巢有大量窦卵泡存在,但是不能周期性产生成熟卵泡,其病理机制尚不清楚。目前认为原发性、内在性卵泡生长异常可能是引起PCOS不排卵及临床内分泌改变的病理基础。研究表明,内在性的卵泡发育异常可能与卵巢颗粒细胞的凋亡调控失常有关。内质网应激(ERS)启动的凋亡途径是近年才发现的一种新的凋亡途径。通过激活未折叠蛋白反应(UPR)以保护由ERS所引起的细胞损伤,恢复细胞功能,但是如果损伤太过严重,内环境稳定不能及时恢复, ERS则引起细胞凋亡。DNA受到损害的卵巢颗粒细胞存在内质网应激标志性蛋白CHOP表达。在上述研究的基础上,本文探讨了多囊卵巢综合征患者卵巢颗粒细胞凋亡与PCOS的发病可能的相关关系。目的:旨在研究多囊卵巢综合征患者卵巢组织切片各级卵泡数和其颗粒细胞的凋亡情况,以及凋亡调控蛋白Bcl-2、Caspase3和内质网应激蛋白Bip、CHOP蛋白的表达。方法:选择因多囊卵巢综合征行卵巢楔形切除的卵巢病理组织21例,同时选择年龄相匹配并于卵泡期手术的非多囊卵巢综合征正常卵巢组织15例为对照组。(1) HE染色,采用双盲读片,比较两组中各级卵泡的数目;(2) 3-末端原位标记法(TUNEL)检测各级卵泡颗粒细胞的凋亡情况,比较两组凋亡率差异;(3)免疫组化法检测凋亡调控蛋白Bcl-2、Caspase3和内质网应激蛋白Bip、CHOP蛋白在各级卵泡颗粒细胞表达情况,比较两组间的差异。结果:1.PCOS组患者卵巢生长卵泡数增加光镜下病理学观察:由两位病理科主治医师采取双盲法在光镜下观察其病理结构,随机挑选10个视野(10×10),记录各级卵泡的数量。两组卵巢组织切片始基卵泡、闭锁卵泡比较,差异均无统计学意义;PCOS组过渡型初级卵泡、典型初级卵泡、次级卵泡、窦卵泡较对照组增多,差异有统计学意义(P<0.05)。卵泡计数:PCOS组较对照组初级卵泡增加1.8倍,次级卵泡数和窦卵泡增加1.6倍,尤其典型初级卵泡数增加约3倍。2.PCOS患者窦卵泡颗粒细胞凋亡率增加TUNEL法:每张切片随机取10个视野,观察窦卵泡中TUNEL染色阳性颗粒细胞占颗粒细胞总数的百分比值,即凋亡指数AI。两组卵巢组织切片显示始基卵泡、初级卵泡、次级卵泡颗粒细胞的凋亡率比较,差异无统计学意义;窦卵泡颗粒细胞凋亡率比较,PCOS组的凋亡率高于对照组,差异有统计学意义(P<0.05)。3.PCOS患者窦卵泡颗粒细胞凋亡调控蛋白Bcl - 2蛋白表达减少,Caspase3表达增加免疫组化:Bcl-2、caspase3蛋白主要表达于颗粒细胞的胞浆和胞膜,呈棕黄色,PCOS组窦卵泡颗粒细胞Bcl - 2蛋白染色强度高于对照组, Caspase3蛋白染色强度低于对照组,两组比较,差异有统计学意义(P<0.05)。4.PCOS患者窦卵泡颗粒细胞内质网应激标志性蛋白CHOP蛋白表达增加免疫组化:内质网应激标志性蛋白Bip蛋白主要表达于颗粒细胞的胞浆和胞膜,呈棕黄色; CHOP蛋白主要表达于颗粒细胞的胞核,以胞核呈棕黄色颗粒为阳性。窦卵泡颗粒细胞CHOP蛋白染色强度高于对照组,两组比较,差异有统计学意义(P<0.05)。BiP蛋白在PCOS窦卵泡颗粒细胞的染色强度高于对照组,比较差异无显著性。结论:PCOS组患者卵巢切片早期生长卵泡及窦卵泡数增加,窦卵泡颗粒细胞凋亡增加,凋亡调控蛋白和内质网应激标志性蛋白表达异常,提示PCOS患者卵巢颗粒细胞凋亡异常,内质网应激可能参与PCOS的发病。更多还原

【Abstract】 Polycystic ovary syndrome (PCOS) is a heterogeneity of clinical syndrome, which is caused by a wide range of metabolic abnormalities, mainly endocrine disorders. Both ovaries of PCOS patients contain a large number of antral follicle, however they are not able to produce mature follicle periodically. The pathological mechanism of PCOS has not yet been made clear. Presently the primary, intrinsic abnormalities of follicle growth are considered to be the possible pathological mechanism basis which causes PCOS non-ovulation and clinical endocrine abnormality. Studies show that the intrinsic follicular dysplasia may be associated with the ovarian granulosa cell apoptosis regulation disorders. Recent studies point to the role of the endoplasmic reticulum (ER) in the sensing and transduction of apoptotic signals. Studies have established that expression of mutant, folding incompetent proteins causes ER stress and elicits an ER stress response, called the unfolded protein response (UPR). The initial intent of the UPR is adaptation and restoration of the normal ER function. The failure of such adaptative mechanisms leads to alarm signaling and finally to cell suicide, usually in the form of apoptosis, as a last resort to do away with dysfunctional cells.CHOP expressed in DNA-damaged ovarian granulosa cells. On the basic of these results, here we investigated test our hypothesis that differences in granulosa cell apoptosis may underlie abnormalities that affect follicular development, and to explore the pathogenesis of PCOS.OBJECTIVE:The purposes of this study were to score the composition of follicles in normal and PCOS ovaries, and to investigate apoptosis and the expressions of Bcl - 2 , Caspase3 , Bip and CHOP in granulosa cells of PCOS .Based on these experiments, we tried to make some exploration on the pathogenesis of PCOS.METHODS:We took 21 cases of ovarian wedge resection of ovarian pathology due to PCOS, and 15 cases of normal (non-PCOS) ovarian tissue from age-matched patients at early stage of the menstrual cycle as control group. Ovarian follicles of two groups were counted by morphometric analysis.Granulosa cells apoptosis in ovaries was observed by TdT-mediated dUTP-biotin nick end-labeling (TUNEL).The expression of apoptosis regulatory protein caspase3, Bcl-2 and endoplasmic reticulum stress protein Bip, CHOP protein was analysed with immunohistochemical staining.RESULTS:1.Stockpiling of growing follicles in ovaries of women with PCOSTwo pathology physicians observed the pathological structure in the optical microscope double-blindly. 10 visual fields (10*10) were randomly selected, and recorded the number of follicles at all levels. The total number of growing follicles was significantly greater in PCOS ovaries than normal, but the number of nongrowing primordial follicles did not differ. The transitional primary follicles, typical primary follicles, secondary follicles and antral follicle are more than control group, the differences are statistically significant. (P<0.05).Differential counts showed that the number of growing follicles at each stage of development was significantly greater: PCOS had 1.8-fold more primary, 1.6-fold more secondary, and 1.6-fold more Graafian follicles than normal. The greatest effect was on the classic primary follicles where the number was almost 3-fold greater in PCOS ovaries.2. Granulosa cells from PCOS patients have higher apoptotic ratesTen visual fields were randomly chosen for each slice, and observed the percentage of TUNEL-positive granulosa cells of the total granulosa cell number, that is, apoptotic index (AI). The comparison of ovarian tissue biopsy revealed that the apoptosis rate of primordial follicle, primary follicle and secondary follicle granulosa cells of the two groups are not significantly different. The antral follicular granulosa cell apoptosis rate of PCOS group is higher than control group. The difference is statistically significant. (P<0.05)3. Expression of apoptotic regulators in granulosa cellsCaspase3 and Bcl-2 protein was mainly expressed in the cytoplasm and membrane of granulosa cells, showing brown yellow. Significantly higher apoptotic rates were associated with stronger intensities immunostaining of the apoptotic effector caspase-3 (P<0.05) and weaker intensities immunostaining of the anti-apoptotic survival factor cellular Bcl -2 in the PCOS group (P<0.05).4. Expression of endoplasmic reticulum stress protein in granulosa cellsHallmark of endoplasmic reticulum stress protein CHOP protein was mainly expressed in the nuclei of granulosa cells.BIP protein was mainly expressed in the cytoplasm and membrane of granulosa cells, showing brown yellow. The intensities of CHOP immunostaining in PCOS ovaries were significantantly stronger compared to human normal ovaries.No significant difference of Bip expression was found between follicle of PCOS and the normal control group.CONCLUSIONS:PCOS patients have higher numbers of primary follicles, secondary follicles and antral follicle in ovarian compared with normal women. Significantly higher apoptotic rates were found in granulosa cells of patients with PCOS, compared with women with regular ovulatory cycles.The intrinsic follicular dysplasia may be associated with the ovarian granulosa cell apoptosis regulation disorders. Endoplasmic reticulum stress might be involved in the pathogenesis of PCOS.更多还原