BMP-4在卵巢早衰小鼠卵巢中的变化

目的分析BMP-4在化疗后卵巢早衰(POF)小鼠卵巢中的表达,探索BMP-4与卵巢早衰卵巢中卵泡发育的关系,为卵巢早衰的发病机制及临床治疗的研究提供参考依据。方法选用48只4~5周龄具有正常动情周期的雌性昆明小鼠随机分为3组:POF组,腹腔注射环磷酰胺,皮下注射白消安建立卵巢早衰模型;对照组,腹腔注射0.9%的生理盐水;正常组。观察并记录小鼠的一般情况,应用HE染色、免疫组织化学技术和Western blot技术,观察卵巢形态学变化、各级卵泡数的变化及BMP-4的分布情况。结果 1POF组小鼠体重下降,卵巢体积缩小,HE染色显示卵泡数目减少,原始卵泡数目(15.44±2.16)与正常组(55.25±16.09)和对照组(53.37±18.11)相比明显减少(P0.05)。2免疫组化结果显示BMP-4在正常组卵巢和对照组卵巢中的表达比在POF组卵巢中的表达高。3免疫组化结果显示BMP-4在POF卵巢中的表达明显减少,其中原始卵泡(39.83±6.50)减少最为明显,与正常组(68.61±5.25)和对照组(66.72±6.26)相比差异有统计学意义(P0.05)。4Western blot结果显示POF卵巢中的BMP-4表达最低,约为正常组的0.6倍(P0.05)。结论 POF组的BMP-4表达降低明显,在原始卵泡中降低最为明显,推测其对原始卵泡的存活和发育及调控卵泡闭锁有促进作用。     

Htra1mRNA在PCOS大鼠卵巢颗粒细胞中的表达及意义

目的探讨Htra1 mRNA在PCOS大鼠卵巢颗粒细胞中的表达情况。方法皮下注射DHEA建立大鼠PCOS模型,并验证用RT-PCR方法及凝胶成像系统检测分析两组大鼠卵巢中Htra1的表达情况。结果与对照组比较,PCOS模型组血清雄激素(T),空腹血糖(FBG),空腹胰岛素(FINS)显著升高(P0.05)。卵巢切片HE染色显示:模型组卵巢有较多的囊状扩张卵泡,而发育阶段卵泡与颗粒细胞层数明显减少,未见黄体。对照组可见不同发育阶段的卵泡及多个黄体;模型组卵巢组织Htra1 mRNA的表达显著低于对照组(P0.05)。结论高雄激素血症和高胰岛素血症可能导致Htra1表达减弱,Htra1的低表达可能与多囊卵巢综合征(PCOS)卵泡发育成熟障碍及不排卵有关。     

环磷酰胺对大鼠睾丸C-Kit表达的影响

目的探讨抗肿瘤药物环磷酰胺对大鼠睾丸中分化型精原干细胞的特异性表面标志物c-kit表达的影响。方法 20只8周龄大鼠随机分为对照组和实验组,每组10只;实验组每天腹腔注射环磷酰胺50 mg·kg-1·d-1,注射3 d,对照组腹腔注射0.9%的生理盐水;用药4周后取双侧睾丸,应用HE染色、免疫组织化学技术和Western blot技术,观察睾丸组织学变化、c-kit在睾丸中的分布情况及表达变化。结果 1实验组大鼠睾丸中生精上皮呈有序性排列,但生精上皮层数减少,管腔中精子数量减少。2免疫组化结果显示c-kit在睾丸的生精细胞中均匀表达,但实验组的c-kit表达明显下降。结论环磷酰胺对大鼠生精功能的损害可能和通过下调c-kit的表达有关。     

AMH、FSH、E_2在卵巢早衰诊断中的价值

目的研究顺铂所致化疗损伤性卵巢早衰大鼠血清AMH、FSH、E2水平变化,探讨血清AMH、FSH、E2能否作为评估成年雌性大鼠卵巢储备的早期预测指标。方法通过给成熟雌性SD大鼠腹腔注射生理盐水、4.5mg/kg和6.0mg/kg顺铂,建立大鼠化疗损伤性卵巢早衰模型。检测血清AMH、FSH、E2水平。结果腹腔注射后,大鼠血清AMH水平均明显降低;血清E2水平与对照组相比,低剂量组明显升高,高剂量组无统计学意义,高剂量组与低剂量组相比明显降低;血清FSH水平：低剂量组与对照组相比无统计学意义,高剂量组与对照组、低剂量组相比均升高。腹腔注射前后,各顺铂组血清AMH水平注射后均较注射前明显降低;血清E2水平于注射低剂量顺铂后升高,而注射高剂量顺铂后差异无统计学意义。各顺铂组血清FSH水平注射后均较注射前明显升高。结论顺铂所致的卵巢损害早期血清E2代偿性升高,AMH明显降低,此两种激素的变化早于FSH的变化。血清E2的升高和AMH的降低可以作为暴露于化疗后的卵巢损害的早期预测指标,两者结合起来将有助于早期诊断卵巢早衰。     

持续光照对大鼠卵巢褪黑素受体表达的影响及与卵泡发育的相关性

目的:研究持续光照对青春期前雌性大鼠卵巢褪黑素受体的表达及与卵泡发育的相关性。方法:雌性大鼠随机分成对照组和24 h持续光照组,酶联免疫吸附法(ELISA)检测血清中褪黑素、雌二醇水平,免疫组织化学法观察卵巢组织褪黑素受体(MR)的表达,RT-PCR法检测卵巢组织中雌激素受体α、β(ERα、ERβ)的mRNA的表达量。结果:与对照组相比,24 h持续光照组阴门开启时间提前,动情期延长,血清褪黑素显著下降,雌二醇水平显著增高,卵巢组织中MR表达下降,ER表达增高。结论:持续光照可以抑制青春期前雌性大鼠褪黑素及其受体表达,并促进卵泡发育和卵巢雌激素受体的表达,这可能与女性性早熟的发病机制相关。     

Kisspeptin/kiss1r系统在多囊卵巢综合征大鼠卵巢中的表达及其对卵泡发育障碍的可能作用机制

目的:探讨多囊卵巢综合征(PCOS)大鼠卵巢中kisspeptin/kisslr系统的表达及其对PCOS大鼠卵泡发育障碍的可能作用机制。方法:构建PCOS大鼠模型;免疫组织化学检测实验大鼠卵巢中kisspeptin/kiss1r的表达;免疫印迹和qRTPCR分别检测kisspeptin/kisslr蛋白和mRNA水平。结果:与正常组相比,PCOS组的体质量及卵巢质量明显增加,血清雄激素、黄体生成素水平明显增加,卵泡刺激素水平变化无统计学差异。PCOS大鼠卵巢中含有较多发育早期的小卵泡及闭锁卵泡,囊状扩张卵泡明显增加,颗粒细胞层数减少至2~3层,黄体数量明显下降;kisspeptin/kisslr蛋白和mRNA水平明显降低。结论:卵巢表达的kisspeptin/kisslr在PCOS大鼠卵泡发育障碍中可能通过调节颗粒细胞发挥作用,本研究将为进一步探讨PCOS的发病机制提供一定的理论基础。     

CNTF基因在大鼠脊髓中的表达及生后发育的变化

目的 观察CNTF基因在大鼠脊髓中的表达以及生后发育过程中的变化。方法 以地高辛标记（dig）－CNTF cDNA为探针,采用原位杂交法,观察CNTF mRNA在大鼠脊髓中的分布;采用TR－PCR法,半定量分析大鼠生后发育过程中,脊髓CNTF mRNA表达水平的变化。结果 CNTF mRNA原位杂交阳性信号存在于正常大鼠脊髓白质的腹索、外侧索周边的部分胶质细胞中;灰质中未能发现阳性杂交信号。RT－PCR结果显示,大鼠生后1d脊髓细胞中即可见CNTF mRNA表达,但表达量较低;出生15d表达量迅速增加;30d时最高;60d时的表达量有下降的趋势。结论 大鼠脊髓白质的部分胶质细胞可表达CNTF mRNA;大鼠出生后1d CNTF mRNA即有表达,之后随脊髓的发育而呈动态变化的趋势。     

脐带间充质干细胞移植对卵巢早衰模型小鼠卵巢功能的影响

目的探讨人脐带间充质干细胞(hUCMSCs)移植小鼠卵巢修复化学治疗药物诱导的卵巢早衰(POF)。方法选择雌性ICR小鼠40只,鼠龄8周,体质量25～30 g。将ICR小鼠采用SPSS生成随机数字分为空白对照组、POF组、h UCMSCs组。空白对照组不做任何处理,POF组与hUCMSCs组每天腹腔注射环磷酰胺(CTX)70 mg/mL,连续15 d,建立POF模型。建模成功后hUCMSCs组小鼠经尾静脉注射hUCMSCs进行治疗,POF组注射同等剂量的0.9%氯化钠溶液(生理盐水)。观察小鼠一般情况及体质量,并在移植后的7 d、14 d测定血清中雌激素(E2)、卵泡刺激素(FSH)、抗苗勒管激素(AMH)含量及观察卵巢组织形态改变。结果建模完成后,与空白对照组比较,POF组和hUCMSCs组血清E2、AMH均都降低,血清FSH升高,其差异均有统计学意义(P <0.05);hUCMSCs移植后7 d,与POF组比较[(94.39±14.99) mIU/mL、(60.92±14.60) pg/mL、(2 870.00±801.74) pg/mL],hUCMSCs组血清FSH[(80...     

去卵巢大鼠骨组织的变化

目的:观察去卵巢对大鼠骨组织的改变,以建立妇女绝经后骨质疏松的动物模型。方法:选用3月龄SD雌性大鼠16只,随机分为对照组和去卵巢组。去卵巢组大鼠的双侧卵巢被切除,对照组做假手术,持续90天。用骨矿测定仪测量大鼠股骨的骨密度及在半自动图像分析仪观测胫骨近端骨小梁的静、动态指标,并在扫描电镜下观察大鼠股骨松质骨结构的改变。结果:与对照正常组比较,去卵巢组大鼠股骨的远端的骨密度降低(P<0.05)。胫骨骨小梁的面积减少,骨小梁间隙增大。股骨的骨小梁变少,变细,断裂,连接不紧密,表面常见骨吸收形成的陷窝。结论:用切除卵巢的方法造成雌激素缺乏,导致骨质疏松,作为研究因绝经引起的原发性骨质疏松的可靠动物模型。     

Bcl-2/Bax蛋白在生后大鼠卵巢不同发育时期的表达

目的 探讨Bcl-2/Bax与卵泡发育和凋亡的关系. 方法 用免疫组织化学技术和图像分析系统,对95只大鼠生后不同发育时期卵巢中凋亡相关因子Bcl-2/Bax的表达情况进行研究. 结果 0～360 d卵巢各级卵泡中的卵母细胞均呈Bcl-2/Bax阳性染色.正常卵细胞中Bcl-2反应强于Bax,退化卵细胞中Bax的反应强于Bcl-2.Bcl-2/Bax在卵泡细胞中的表达模式有所不同,21d前 Bcl-2为阳性,21～150d之间为Bcl-2阴性;180d以后正常卵泡为阴性,闭锁卵泡为阳性;Bax的表达,180d前全为阴性,180d后的表达同Bcl-2.膜细胞中Bcl-2/Bax的表达与卵泡细胞存在相似之处.Bcl-2/Bax在黄体中的表达只见于180d后. 结论 Bcl-2/Bax对卵细胞的生长、发育、存活和凋亡可能有重要的调节作用,其对卵泡细胞、膜细胞及黄体细胞的存活和凋亡的调节作用不明显.     

The pathology of premature ovarian failure.

The monoclonal antibody Ki-67 reacts with a human nuclear cell proliferation-associated antigen that is expressed in all active parts of the cell cycle. Recently we have raised monoclonal antibodies, MIB 1-3, against recombinant parts of the Ki-67 antigen. These antibodies are true Ki-67 equivalents, as demonstrated by immunostaining of fresh specimens, biochemistry, and molecular biological techniques. Formalin-fixed, paraffin-embedded sections routinely processed for immunohistochemistry failed to stain for Ki-67 and MIB 2. Antibodies MIB 1 and MIB 3 labelled mitotic figures, while non-mitotic proliferating cells were negative under these conditions. However, when dewaxed microwave oven-processed paraffin sections of formalin-fixed tissues were used, MIB 1 and MIB 3 gave strong nuclear staining of those cells presumed to proliferate under a variety of normal and neoplastic conditions. Moreover, routine decalcification or depigmentation techniques did not alter the immunoreactivity of MIB 1 and MIB 3 with microwave-processed paraffin sections. This method is highly reproducible, easy to perform at low cost, and no additional technical skill is needed because after microwave treatment just routine immunohistochemical methods are used. Since we have successfully applied this new method to sections obtained from paraffin blocks stored for a long time (in one case more than 60 years), the assessment of cell kinetics through the detection of Ki-67 antigen is now possible on archival material collected in histopathology departments all over the world.     

Genetic disorders in premature ovarian failure

This review presents the genetic disorders associated with premature ovarian failure (POF), obtained by Medline, the Cochrane Library and hand searches of pertinent references of English literature on POF and genetic determinants cited between the year 1966 and February 2002. X monosomy or X deletions and translocations are known to be responsible for POF. Turner's syndrome, as a phenotype associated with complete or partial monosomy X, is linked to ovarian failure. Among heterozygous carriers of the fragile X mutation, POF was noted as an unexpected phenotype in the early 1990s. Autosomal disorders such as mutations of the phosphomannomutase 2 (PMM2) gene, the galactose-1-phosphate uridyltransferase (GALT) gene, the FSH receptor (FSHR) gene, chromosome 3q containing the Blepharophimosis gene and the autoimmune regulator (AIRE) gene, responsible for polyendocrinopathy-candidiasis-ectodermal dystrophy, have been identified in patients with POF. In conclusion, the relationship between genetic disorders and POF is clearly demonstrated in this review. Therefore, in the case of families affected by POF a thorough screening, including cytogenetic analysis, should be performed.     

Antiovarian antibody in premature ovarian failure

Premature ovarian failure is a syndrome consisting of primary or secondary amenorrhoea, hypergonodotropiremia and hypoestrogenemia in women under the age of 40. An autoimmune mechanism was suggested as possible etiology when Vallolton and Forbes in 1966-67 found antibodies to the cytoplasm of rabbit ova in 29 of 232 tested sera. Immune mechanism in the pathogenesis of premature ovarian failure (POF) is suggested by association of autoimmune phenomenon with POF in some cases and demonstration of circulating antibodies to ovary in serum samples from women with POF. The incidence of presence of antiovarian antibody of POF patients has been reported earlier. Evidence of autoimmunity is present in 18-92% of patients with POF. In the present study we have studied 18 cases of POF without any overt manifestation of autoimmune disorder but the antiovarian antibody was detected, with the idea that this autoantibody might be the cause of ovarian dysfunction which is evident in POF. Presence of antiovarian antibody in 16.67% cases with POF in our study that ovarian antibodies may play a role in or reflect an autoimmune process responsible for the development of POF.     

Incipient ovarian failure and premature ovarian failure show the same immunological profile

PROBLEM: Incipient ovarian failure (IOF) is characterized by regular menstrual cycles, infertility and a raised early-follicular FSH in women under 40. IOF might be a precursor or a mitigated form of premature ovarian failure (POF). Disturbances in the immune system may play a role in ovarian failure. METHOD OF STUDY: Autoantibodies and lymphocyte subsets were determined in 63 POF patients, 50 IOF patients, and 27 controls. RESULTS: The prevalence of autoantibodies did not differ between the groups. There was a statistically significant difference in lymphocyte subsets between the control group and the POF group, with the IOF group taking an intermediate position. We found a decrease in percentage of T-suppressor cells with a rise in T-helper/T-suppressor cell ratio, a decrease in natural killer cells, and an increase in B lymphocytes and HLA-DR positive T cells. CONCLUSIONS: These data support the concept that IOF is a mitigated form of POF. The question remains whether these changes are the cause or the consequence of the ovarian failure.     

Treatment of autoimmune premature ovarian failure.

There is no known immunosuppressive therapy for autoimmune premature ovarian failure that has been proven safe and effective by prospective randomized placebo-controlled study. Nevertheless, immunosuppression using corticosteroids has been used on an empirical basis for this condition. Here we present two cases of young women with premature ovarian failure who were treated with glucocorticoids in the hopes of restoring fertility. The first case illustrates the potential benefit of such therapy, and the second case illustrates a potential risk. The first patient with histologically proven autoimmune oophoritis was treated with alternate day glucocorticoid treatment. She had return of menstrual bleeding six times and ovulatory progesterone concentrations four times over a 16 week period. The second patient with presumed but unconfirmed autoimmune ovarian failure was referred to us after having been treated with a 9 month course of corticosteroids. During that treatment her menses did not resume. The corticosteroid treatment was complicated by iatrogenic Cushing syndrome and osteonecrosis of the knee. Identifying patients with autoimmune premature ovarian failure presents the opportunity to restore ovarian function by treating these patients with the proper immune modulation therapy. On the other hand, potent immune modulation therapy can have major complications. Corticosteroid therapy for autoimmune premature ovarian failure should be limited to use in placebo-controlled trials designed to evaluate the safety and efficacy of such treatment.     

FRAXA premutation associated with premature ovarian failure

A family is described in which six females in three generations experienced premature ovarian failure (POF). In three of them a FRAXA premutation was documented and the carrier status of a fourth female could be inferred, because her son had the fragile X syndrome. These findings provide further evidence for a nonrandom association between POF and the FRAXA premutation. © 1996 Wiley-Liss, Inc.