脑源性神经营养因子在生殖领域的研究进展

脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)是广泛分布于神经系统的一种神经营养因子。近年来研究发现,BDNF在生殖系统也有表达,参与生殖系统各项生理功能的调节,包括调节性激素的分泌、促进两性生殖细胞的形成等,并与女性多囊卵巢综合征(polycystic ovary syndrome,PCOS)、卵巢储备功能减退(diminished ovary reserve,DOR)、子宫内膜异位症(endometriosis,EMS)及男性少、弱精子症等不孕症相关疾病密切相关。     

脑源性神经营养因子在人卵泡及早期胚胎发育中的作用

脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)是广泛分布于神经系统的一种神经营养因子,人BDNF基因位于11q13。近年来研究发现,BDNF及其受体在人卵巢中也有表达,它经由旁分泌和自分泌方式,通过与促性腺激素、卵巢甾体激素的相互作用,影响卵泡生成和生长、卵子成熟、排卵和早期胚胎的发育等过程。多囊卵巢综合征和卵巢储备功能减退女性卵泡液中的BDNF分泌异常,这可能与卵泡内活性氧增加和过度的氧化应激有关,对BDNF与多囊卵巢综合征、卵巢储备功能减退关系的研究可能有助于揭示排卵异常性不孕的部分发病机制。     

取卵日血清脑源性神经营养因子与辅助生育结局的相关性

目的:探讨取卵日血中脑源性神经营养因子(BDNF)能否预测卵母细胞质量和其后的卵胞质内单精子注射(ICSI)的妊娠结局。方法:总共纳入57例进行ICSI患者,在取卵日清晨抽取空腹静脉血,测量BDNF,E2和P的浓度。比较妊娠组与非妊娠组的相关数据,并分析取卵日BDNF水平与E2、P、年龄和IVF相关数据之间的相关性。结果:妊娠组与非妊娠组的BNDF浓度无明显差异;取卵日BDNF浓度与E2和P呈明显正相关。BDNF与获卵数和成熟卵母细胞数成正相关。结论:尚不能用取卵日BDNF来预测ICSI的妊娠结局,但BDNF可能在卵母细胞发育成熟过程中发挥着重要作用。     

单纯性肥胖儿童血清脑源性神经营养因子表达的变化及意义

目的观察单纯性肥胖儿童和健康正常体重儿童血清脑源性神经营养因子(BDNF)质量浓度的差异,探讨BDNF与儿童肥胖及瘦素抵抗、胰岛素抵抗的关系。 方法南京军区福州总医院儿科等于2004年5月至2005年5月应用酶联免疫法检测单纯性肥胖儿童(37例)和健康儿童(31例)血清BDNF质量浓度与胰岛素(INS)浓度，应用放射免疫法检测血清瘦素(LEP)质量浓度。比较两组儿童血清BDNF、INS、LEP的差异，分析血清BDNF质量浓度与血清LEP质量浓度和INS浓度的关系。 结果(1)两组儿童的体重指数(BMI)、BDNF、INS及LEP均差异显著(BMI：F=175.05，P<0.01；BDNF：F=12.35，P<0.01；INS：F=21.71，P<0.01；LEP：F=48.89，P<0.01)，肥胖组BMI、INS及LEP均明显高于健康组，而肥胖组BDNF明显低于健康组。(2)影响LEP的因素依次为BMI、丙氨酸转氨酶(ALT)、BDNF(R2=0.5946，F=0.31，P<0.01)；影响INS的因素依次为BMI、BDNF(R2=0.2647，F=11.34，P<0.01)。去除BMI、ALT影响后，BDNF与LEP、INS负相关(BDNF与LEP：r=-0.2455，P<0.05；BDNF与INS：r=-0.2878，P<0.05)。 结论(1)肥胖儿童血清BDNF缺乏，未发现“BDNF抵抗”的特点。(2)学龄前儿童血清LEP、INS受BMI影响最大，还受血清BDNF影响。BDNF是二者独立的负相关因素。     

脑源性神经营养因子在子宫内膜异位症及其相关疼痛中的研究进展

子宫内膜异位症(EMs)是一种常见且影响广大育龄期女性的妇科良性疾病,该病的早期诊断和治疗是目前临床工作中亟需解决的难题.EMs有3种公认的病理类型,分别是腹膜型、卵巢型及深部浸润型.然而不论哪种病理类型,疼痛都是EMs患者最为突出的临床表现,包括经期腹痛、慢性盆腔痛及性交痛等不适,严重影响女性的身心健康,并增加经济负...     

促性腺激素对人卵巢脑源性神经营养因子及其受体表达的影响

目的 :探讨促性腺激素 ( Gn)对脑源性神经营养因子 ( BDNF)及其受体——酪氨酸蛋白激酶受体 B( Trk B)在人卵巢中表达的影响。方法 :体外培养来自体外受精 -胚胎移植 ( IVF- ET)周期的黄体化卵丘颗粒细胞与壁层颗粒细胞 ,ELISA方法检测不同浓度人绝经期促性腺激素 ( h MG)对卵丘颗粒细胞分泌 BDNF的影响 ,Western免疫印迹方法检测不同浓度 h MG对壁层颗粒细胞表达 Trk B的影响。结果 :h MG促进卵丘颗粒细胞 BDNF的分泌 ,增加壁层颗粒细胞 Trk B的表达 ;不同浓度h MG作用的实验组之间表达差异无统计学意义。结论 :Gn促进 BDNF及其受体 Trk B在人卵巢的表达。     

脑组织S100B蛋白及脑源性神经营养因子对胎鼠宫内脑损伤的预测价值

目的:寻找胎鼠宫内脑损伤的有效预测指标。方法:比较正常对照组和缺氧组胎鼠脑组织形态学变化、脑组织中S100B蛋白和脑源性神经营养因子(BDNF)的表达及含量。结果:(1)脑组织形态学结果显示:缺氧组胎鼠大脑皮质和海马区细胞排列紊乱,细胞数目减少,结构模糊或消失,组织间质疏松水肿。(2)免疫组织化学染色结果显示:S100B蛋白主要在海马区的齿状回部位表达,BDNF主要在海马区和皮质区表达。缺氧组S100B蛋白及BDNF阳性细胞平均灰度值较正常对照组明显增强(189.90±31.66 vs 84.66±12.90,P=0.000;139.04±15.83 vs 74.86±14.08,P=0.000)。(3)酶联免疫吸附试验检测结果显示:缺氧组胎鼠脑组织中S100B蛋白、BDNF含量均明显高于正常对照组(3.17±1.07 ng/ml vs 2.25±1.20 ng/ml,P=0.006;152.08±36.29 pg/ml vs 128.21±43.43 pg/ml,P=0.039)。结论:孕鼠缺氧后可导致胎鼠脑损伤,脑组织中S100B蛋白、BDNF的测定可作为预测胎鼠宫内脑损伤的指标。     

脑源性神经营养因子在宫内缺氧环境中对胎盘及血脑屏障通透性的实验研究

目的 探讨外源性脑源性神经营养因子(brain—derived neurotrophic factor，BDNF)在官内缺氧环境下能否通过胎盘屏障进入胎鼠体内，再通过胎鼠血脑屏障进入胎脑而发挥其生物功能。方法 钳夹孕17d(孕中期)鼠子官动脉30min后，经孕鼠尾静脉注射不同剂量的^125I标记的BDNF(^125I-BDNF)。24、48及72h后取孕鼠胎盘、羊水及胎鼠脑、心、肺、肝、肾，检测和比较各组织中BDNF的放射活性。结果 (1)BDNF能够部分通过胎盘屏障，胎鼠体内检测到^125I-BDNF的分布。(2)^125I-BDNF在胎盘屏障通透性及在各组织中分布的量随外源性^125I—BDNF注射剂量增加而升高。(3)BDNF在胚胎缺血缺氧时，能通过血脑屏障到达胚鼠大脑。(4)缺氧缺血时BDNF对胎盘屏障和血脑屏障的通透性增加。结论外源性BDNF在官内缺血缺氧时能够部分通过胎盘屏障和血脑屏障。     

转化生长因子β受体Ⅱ在大鼠实验性隐睾中的表达及与生精细胞凋亡的关系

目的:探讨转化生长因子β受体Ⅱ(TGFβ-RⅡ)与实验性隐睾诱导的生精细胞凋亡的关系。方法:建立大鼠单侧隐睾动物模型,SABC法检测TGFβ-RⅡ在隐睾及对照侧睾丸组织内的分布及表达,TUNEL法检测凋亡生精细胞。结果:术后不同时间组隐睾侧曲细精管TGFβ-RⅡ表达均低于对照侧(P<0.01)。术后隐睾侧细胞凋亡数量较对照侧明显增加(P<0.01)。结论:TGFβ可能通过TGFβ-RⅡ介导的信号转导过程在睾丸生精细胞凋亡过程中发挥重要的调控作用。     

亮丙瑞林可促进供体精原干细胞(SSCs)在受体小鼠睾丸的归巢效率

目的:研究亮丙瑞林(GnRH-激动剂)处理受体小鼠促进精原干细胞(SSCs)移植后克隆率的作用时间、方式。方法:使用亮丙瑞林(7.6 mg/kg)或其载体剂(生理盐水)皮下注射处理受体小鼠,然后将供体SSCs(具有GFP标记)通过睾丸输出管注射法移植到经白消胺处理过的受体睾丸曲细精管内,分别于移植后1周和4周采样,在荧光显微镜下观察供体细胞在受体睾丸曲细精管中的归巢数量,供体细胞来源的睾丸数量及曲细精管数量;反转录PCR检测移植后1周和4周睾丸组织中β1-整合素(β1-integrin)mRNA表达水平。结果:在SSCs移植1周后,显微镜下可见亮丙瑞林处理组(实验组)曲细精管中SSCs的归巢数量为20.4±4.3个/曲细精管,显著高于对照组(11.5±3.8个/曲细精管)(P<0.05);在SSCs移植后4周,处理组显示绿色荧光的曲细精管比率(24.1±4.5%)也显著高于对照组(13.4±5.3%,P<0.05)。用反转录PCR检测mRNA表达量,在移植后1周时实验组小鼠睾丸内β1-整合素明显增加(P<0.05),而在移植后4周时β1-整合素与对照组相比无显著差异(P>0.05)。结论:GnRH-激动剂对SSCs的归巢具有重要促进作用,并且提示其归巢作用的发生可能是通过β1-整合素来完成的,从而使SSCs在受体小鼠曲细精管的数量一直(到移植后4周)保持着高于对照组的数量优势。     

Proliferation of spermatogonial stem cells and spermatogenesis in vitro

Detection of spermatogonial stem cells (SSC) became possible 10 years ago, with the transplantation of germ cells into the seminiferous tubules of mice. Using this assay system, attempts to maintain and expand SSC in vitro finally bore fruit. Gonocytes from neonatal mice and spermatogonial stem cells from adult mice were plated on feeder cells in a medium supplemented with Glial cell line-derived neurotrophic factor (GDNF) along with certain other factors. The germ cells that emerged under such conditions, named germline stem (GS) cells, proliferated exponentially through self-renewing division. GS cells in vitro show features of differentiation as well. Some expressed c-kit, which is a cell surface marker of differentiating spermatogonia. Therefore, it seems that GS cells undergo both self-renewing and differentiating cell divisions in vitro . There is a century of history behind attempts to reproduce spermatogenesis in vitro and significant progress has been made. Nonetheless, there are few established culture-based protocols for recreating spermatogenesis in vitro . GS cells would be an ideal starting material in this regard. (Reprod Med Biol 2006; 5 : 169–174)     

Expression of stem cell-related genes in the endometrium and endometriotic lesions: a pilot study

Objective: To compare the expression of stem cell-related genes in the endometrium (END), superficial endometriosis (SE), and deep infiltrating endometriosis (DIE).

Study design: We performed a prospective pilot study of six women suffering from SE and DIE who gave consent for laparoscopy surgery, endometrial biopsies, and participation in this study. Quantitative RT-PCR analysis of 84 stem cell-related genes was performed in 18 biopsy samples.

Results: A total of 40 of 84 genes were expressed in SE and DIE, but were different from END as follows. Seven genes were over-expressed in SE and 33 genes were under-expressed in DIE compared with END. Two genes were only over-expressed in SE and three genes were only over-expressed in DIE. Six under-expressed genes were exclusively located in SE and one was only located in DIE. The remaining 31 genes were not different among the groups. There was no significant difference in gene expression between SE and DIE samples.

Conclusion: Tissue of DIE and SE appears to have similar stem cell-related genes. Nevertheless, there are differences in gene expression between SE and DIE.     

小鼠胚胎干细胞体外诱导分化为精原干细胞的研究

目的:初步探讨在体外条件下诱导小鼠胚胎干细胞(EGFP-mESC)分化为精原干细胞(SSC)的条件,建立有效的技术平台。方法:采用部分模拟胚胎早期发育的方法,将EGFP-mESC(XY)经体外悬滴培养,制备拟胚体(EB);使用免疫磁珠分选法从EB中分离出表达原始生殖细胞(PGC)表面标志SSEA-1的细胞;获得的细胞经2μmol/L维甲酸(RA)诱导增殖,使之向雄性生殖细胞分化。对诱导的细胞采用RT-PCR及免疫荧光检测特异性基因及蛋白的碱性磷酸酶。结果:在诱导EGFP-mESC向雄性生殖细胞分化的过程中,采用RT-PCR方法检测到了雄性生殖细胞特异表达基因Sry、fragilis、stella、Tex14等mRNA的表达;利用激光共聚焦方法检测到SSC表面标志蛋白Stra8、integrin-α6及Hsp90α的表达。结论:采用RA体外诱导EGFP-mESC定向分化为SSC是可行的。     

TrkB及其配体BDNF在卵巢上皮癌中的表达及其临床意义

目的检测酪氨酸激酶B（tyrosine kinase,TrkB）及其配体脑源性神经营养因子（brainderived neurotrophic factor,BDNF）在卵巢上皮癌（EOC）中的表达,并探讨其临床意义。方法选择中山大学附属肿瘤医院病理存档的石蜡标本108例,其中10例正常卵巢、17例良性卵巢肿瘤、21例卵巢交界性肿瘤和60例卵巢上皮癌,采用免疫组化链霉素抗生物素蛋白-过氧化物酶法（SP法）检测TrkB及其配体BDNF蛋白的表达水平,分析其与临床病理因素之间的关系。结果 TrkB和BDNF蛋白在正常卵巢组织和良性卵巢上皮性肿瘤中无表达;在交界性和卵巢上皮性癌中的阳性表达率分别为19.0%、71.7%和14.0%、51.7%（P〈0.05）。TrkB和BDNF蛋白在EOC组中的表达水平与其FIGO分期及病理分级有关,在Ⅲ期＋Ⅳ期、低分化组（G3）比Ⅰ期＋Ⅱ期、高中分化（G1、G2）组表达率高（P〈0.05）。TrkB和BDNF在卵巢上皮癌中的表达水平呈正相关（r=0.428,P〈0.05）。结论卵巢上皮癌中存在TrkB和BDNF的异常高表达。TrkB和BDNF可能协同作用促进了卵巢上皮癌的发生、发展,并有望成为卵巢上皮癌基因治疗的新靶点。     

SEPT11基因在小鼠睾丸精子发生过程中的表达特征

目的初步探讨Septin11(SEPT11)基因在精子发生中的作用。方法通过实时定量PCR、蛋白免疫印迹及免疫荧光等方法检测SEPT11在不同周龄野生型小鼠以及成年睾丸支持细胞激素受体(Ar)特异性敲除小鼠(SCARKO)和Ar全敲除(ARKO)小鼠睾丸中的表达特征。结果 SEPT11基因小鼠出生2周内高表达,随后表达水平降低,出生6周后出现并与未释放的成熟精子共定位且高表达。与野生型小鼠相比,SEPTIN11在SCARKO小鼠和ARKO小鼠睾丸中表达降低(P0.01),m RNA水平显著升高(P0.01);SEPTIN11在成熟精子中定位于尾部。结论 SEPT11基因在小鼠出生时表达最高;SEPTIN11在小鼠成熟精子中定位于尾部;Ar的敲减提高了SEPT11的表达。     

Recent advances of in vitro culture systems for spermatogonial stem cells in mammals

Background

Spermatogonial stem cells (SSCs) in the mammalian testis are unipotent stem cells for spermatozoa. They show unique cell characteristics as stem cells and germ cells after being isolated from the testis and cultured in vitro. This review introduces recent progress in the development of culture systems for the establishment of SSC lines in mammalian species, including humans.

Methods

Based on the published reports, the isolation and purification of SSCs, identification and characteristics of SSCs, and culture system for mice, humans, and domestic animals have been summarized.

Results

In mice, cell lines from SSCs are established and can be reprogrammed to show pluripotent stem cell potency that is similar to embryonic stem cells. However, it is difficult to establish cell lines for animals other than mice because of the dearth of understanding about species‐specific requirements for growth factors and mechanisms supporting the self‐renewal of cultured SSCs. Among the factors that are associated with the development of culture systems, the enrichment of SSCs that are isolated from the testis and the combination of growth factors are essential.

Conclusion

Providing an example of SSC culture in cattle, a rational consideration was made about how it can be possible to establish cell lines from neonatal and immature testes.     

A specific and quick gene expression study in mouse ES cells

Purpose To study gene expression at single embryonic stem cell colony levels with a new RT-PCR protocol. Methods Forty-five mouse ES cell colonies were retrieved at the 5th, 10th, 15th, 20th and 25th passages. The pluripotent state was analyzed for OCT-4 and Nanog, and β-actin as a control for the presence of templates. RT-PCR was done using the SuperScript™ III CellsDirect cDNA Synthesis System. Every 2 or 3 days just before passage, a single colony was loaded into a 0.5 ml PCR tube containing 10 μl of resuspension buffer using a pulled glass pipette. Results The RT-PCR protocol was completed in less then 150 min. All colonies were positive for OCT-4 and β-actin and 42 out of 45 were positive for Nanog. Conclusions This protocol requires as little as 10 pg of total RNA starting material and is therefore useful for low cell number tissues, such as single stem cell colonies or preimplantation embryonic materials. A gene expression study at the single ES cell colony level is described which may improve previous procedures that use materials from pooled colonies.