双峰驼卵泡发育过程中卵泡液差异蛋白质组学

为了比较双峰驼卵泡发育过程中卵泡液中差异表达的蛋白质,将卵泡按照直径分为6 类,运用双向电泳技术构建双峰驼卵泡液蛋白质二维凝胶电泳图谱,凝胶经考马斯亮蓝染色后用PDQuest 8. 0 检测差异蛋白,结果表明在6 类大小不同的卵泡中共检测到13 个差异蛋白点,这些蛋白点经LC -MS/ MS 鉴定出7 种不同的蛋白质,它们分别是:血红蛋白、toll-like 受体9、抗凝血酶、聚泛素、γ 纤维蛋白原、重组活化蛋白1 和跨膜与卷曲螺旋域3。基于这些蛋白的功能和表达模式,结合实验结果讨论了这些蛋白质在生殖中的功能,发现toll-like 受体9、聚泛素、γ 纤维蛋白原和重组活化蛋白1 可能与卵泡发育或卵母细胞的成熟有关。这些蛋白质的发现为了解双峰驼卵泡发育和卵母细胞成熟的生理机制奠定基础。     

应用超高分辨双向凝胶电泳技术进行正常人类肝脏蛋白质组研究

肝脏是人体最大的实质器官, 承担着人体许多关键的生理功能, 在生命活动中占有重要地位. 根据人类肝脏蛋白质组计划, 本实验旨在构建人肝脏蛋白质双向凝胶电泳表达谱, 尽可能分离和鉴定更多的蛋白. 在双向凝胶电泳第一向等电聚焦水平上, 从上样方式、水化液配方、聚焦时间等方面优化了碱性蛋白的分离条件, 利用超放大胶分离技术搭建了高分辨的双向凝胶电泳分离平台, 构建了人类肝脏蛋白质2-DE参考谱, 检测到5481个蛋白质点. 这是目前国际上最为全面的人体器官蛋白质组2-DE参考谱, 为其他肝病的研究提供了较好的参照系. 成功鉴定了429个非冗余蛋白, 对pH 4.0~7.0, 5.0~6.0, 5.5~6.7, 6.0~9.0部分蛋白质点在胶上进行了注释, 由此构建了人肝2-DE蛋白质表达谱数据库. 对蛋白质的理化性质、功能及亚细胞定位进行了全面分析. 研究中构建的人类肝脏蛋白质2-DE图谱将为肝脏比较蛋白质组学研究提供参考.     

卵泡液蛋白质组学研究进展

蛋白质组的概念最初在1994年提出.蛋白质组学是以细胞内全部蛋白质的存在及其活动为研究对象,它极大地促进了我们对后基因组时代基因功能的理解.卵泡液作为卵母细胞生长和分化的培养基,直接或间接影响卵母细胞的生育力和发育潜能,其中的一些蛋白质可以作为卵母细胞成熟的标记.应用蛋白质组学方法可以筛选与卵母细胞成熟及一些与生殖疾病有关的标记蛋白.本文就卵泡液的蛋白质组学研究进展作简要综述.     

猪卵泡液样品制备方法对双向电泳图谱的影响

比较不同的蛋白制备方法,寻找适合猪卵泡液的双向电泳样品制备方法,提高2-DE图谱的分辨率和重复性。利用ProteoExtract Albumin/IgG Remove Kit去除卵泡液中的高峰度蛋白;然后分别用丙酮沉淀法、TCA/丙酮沉淀法和超滤法进行样品浓缩,比较分析对2-DE图谱的影响。结果表明,TCA/丙酮沉淀法制备的蛋白样品获得的电泳图谱质量较好,蛋白斑点数目较多,斑点较清晰、圆滑,分辨率较高,重复性较好。TCA-丙酮沉淀法更适合于制备猪卵泡液的双向电泳蛋白样品,为进一步对猪卵泡液2-DE图谱进行分析、鉴定及寻找生物标记物提供了保证。     

慢性应激大鼠海马的比较蛋白质组学研究

慢性应激可造成海马神经细胞丢失、树突萎缩等损伤,但有关其损伤机制仍有很多问题不甚明了.为了寻找应激致海马损伤相关的重要蛋白质、从蛋白质水平揭示应激致海马损伤的分子机制,应用双向凝胶电泳(2-DE)技术分离对照组和束缚应激组大鼠海马组织总蛋白质,图像分析检测差异表达的蛋白质点,基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MSS)和数据库检索对差异表达的蛋白质点进行鉴定,并采用半定量的RT-PCR在mRNA水平验证2-DE结果.得到了分辨率较高、重复性较好的对照和束缚应激大鼠海马2-DE图谱,质谱分析和数据库检索鉴定了14个差异表达蛋白质点中的11个蛋白质,大多数差异蛋白的功能涉及能量代谢、信号传递等过程.研究结果为揭示应激致海马损伤的机制、提高机体的应激适应能力提供了理论依据.     

失血性休克大鼠肝脏蛋白质的差异分析

 应用蛋白质双向凝胶电泳 (two-dimensional polyacrylamide gel electrophoresis, 2-DE) 技术,分析在急性重度失血性休克 (refractory hemorrhagic shock, RHS) 条件下,大鼠肝脏蛋白质组表达的差异.16 只雄性 Wistar 大鼠随机分成正常对照组 (sham hemorrhage shock, SHS) 和 RHS 模型组,每组 8 只.采用股动脉放血的方法制备模型,在规定时间内处死大鼠并分离肝脏,提取肝脏总蛋白质后进行 2-DE.运用 Image Master 2D Platinum v 5.0 凝胶图像分析软件对 2-DE 凝胶图像进行差异表达分析.有意义的差异蛋白质点用基质辅助激光解析电离飞行时间质谱进行肽质量指纹图谱分析,借助 Swiss-prot 数据库进行蛋白质搜索和鉴定.SHS 组和 RHS 组肝脏的 2-DE 图谱,分别平均识别到 698±11 和 700±13 个蛋白质点,SHS 组和 RHS 组肝脏间平均匹配率达88%～92 %.共发现 10 个差异有意义的蛋白质点,鉴定出了肿瘤抑制性抗原gp96、葡萄糖调节蛋白58、过氧还蛋白Ⅰ、细胞色素b5、谷胱甘肽转移酶、ATP合酶β亚单位、二磷酸果糖酶 B、三磷酸甘油醛脱氢酶等8种蛋白质.结果表明,以 2-DE 技术得到重复性和分辨率都较好的 2-DE图谱,并初步鉴定急性重度失血性休克后大鼠肝脏的差异表达蛋白质,为深入研究失血性休克的生理病理机制及寻找失血性休克预防和治疗的生物标志物提供了依据.     

基于LC-MS/MS策略建立水牛卵母细胞蛋白质表达谱的方法研究

本研究采用了LC-MS/MS策略对如何建立水牛卵母细胞蛋白质表达谱进行了探讨,为后续研究不同时期的水牛卵母细胞和早期胚胎卵裂分化过程中蛋白质组变化建立研究平台。本实验收集水牛GV期和MⅡ期卵母细胞各500枚,去除透明带后将两个时期的细胞混合后提取蛋白质,蛋白质酶解后通过强阳离子交换色谱预分离多肽混合物,预分离后的馏分采用EASY-n LC结合LTQ-Orbitrap质谱鉴定蛋白质表达谱。通过SEQUEST检索,共鉴定出574种蛋白质,生物信息学分析表明,有明确分子功能相关的蛋白质有487种,与组成细胞成分相关的蛋白质有476种,与生物学进程相关的蛋白质有490种,初步建立了一套获得水牛卵母细胞蛋白质表达谱的方法。结果表明建立的LC-MS/MS策略方法适合于水牛卵母细胞蛋白质组学的研究,卵母细胞蛋白质表达谱数据为今后研究水牛不同时期的卵母细胞和早期胚胎卵裂分化过程中蛋白质组学奠定了方法与理论基础。     

番茄幼苗盐胁迫响应蛋白质组分析

采用营养液栽培,以盐敏感型番茄品种M82为试材,利用双向电泳(2-DE)研究盐胁迫处理下幼苗叶片蛋白质的表达谱,并采用基质辅助激光解析飞行时间串联质谱(MALDI-TOF/TOF-MS)技术进行差异蛋白质的分离及质谱鉴定。结果表明:(1)盐胁迫处理下,利用2-DE获得差异显著蛋白点20个,其中17个蛋白质点丰度上调表达,3个蛋白质点丰度下调表达。(2)通过质谱分析和蛋白质NCBInr数据库检索,共鉴定出19个差异蛋白,分别为果糖-二磷酸醛缩酶、S-腺苷甲硫氨酸合成酶、甘油醛-3-磷酸脱氢酶等及3个功能未知蛋白;这些鉴定出的差异蛋白质与能量代谢、光合作用、蛋白合成、氧化还原平衡等过程相关,暗示所分离鉴定的蛋白可能参与了番茄的盐胁迫响应,为进一步研究番茄抗逆机制奠定基础。     

差异蛋白质组学的研究进展

差异蛋白质组是蛋白质组学研究的一个主要内容,其核心在于寻找某种特定臣寸素引起样本之间蛋白质组的差异,揭示并验证蛋白质组在生理或病理过程中的变化。进一步对蛋白质组差异信息分析后,理论上可以推断造成这种变化的原因。因此,对于临床上肿瘤预诊、药物靶标寻找、细胞调控分子的鉴别等有着极大的实际意义。差异蛋白质组研究要求可靠性和可重复性。因此,对于样本处理要求较高,激光微切割技术和高丰度蛋白去除技术的应用优化了样本处理方法。目前差异蛋白质组的主要研究方法仍是2-DE分离和MS鉴定联合应用,基于2-DE的2-DDIGE方法弥补了2-DE的弱点,更适用于差异蛋白质组研究。除2-DE技术外的其他几种技术手段,如多维液相色谱分离技术、ICAT技术、蛋白芯片技术等差异蛋白质组学研究技术可以作为2-DE技术的补充,甚至或替代技术。     

应用LCM和蛋白质组学技术筛选肺腺癌的差异表达蛋白质

为筛选肺腺癌(lung adenocarcinoma,AdC)发病相关蛋白质,首先采用激光捕获显微切割技术(laser capture microdissection,LCM)分别从AdC组织和正常支气管上皮(normal bronchial epithelium,NBE)组织中切割并收集AdC细胞和NBE细胞,再应用双向凝胶电泳技术(two-dimensional gel electrophoresis,2-DE)分离经LCM收集的细胞蛋白质,通过PDQuest软件分析差异表达的蛋白质点,基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)鉴定差异表达蛋白质,组织芯片免疫组化方法检测差异蛋白质膜联蛋白A4(annexin A4)在30例AdC组织、配对的癌旁组织和淋巴结转移癌组织中的表达水平。研究结果显示,通过蛋白质组学方法建立了LCM收集的AdC和NBE细胞的2-DE图谱,质谱鉴定得到了33个差异表达蛋白质,其中21个蛋白质在AdC细胞中表达上调,12个蛋白质在AdC细胞中表达下调。组织芯片免疫组化结果显示,与癌旁肺组织相比,annexin A4在AdC组织中的表达水平显著上调,且在AdC淋巴结转移癌组织中的表达明显高于其原发癌组织。上述结果提示annexin A4与AdC的发病及淋巴结转移相关,有望成为诊断AdC及预测AdC转移的分子标志物。     

Immunoglobulins from human follicular fluid induce the acrosome reaction in human sperm

The ability of human follicular fluid (hFF), retrieved from women undergoing IVF to induce the acrosome reaction (AR) in human sperm has been documented by several laboratories. However, the nature of the active factors in the hFF and the physiological meaning of the AR induction are highly controversial. We performed a three step purification scheme for hFF and all the fractions were screened for the AR-inducing activity. AR activity was associated with a protein fraction of M_r > 180 kD that on further analysis under PAGE was found to be composed by subunits of apparent M_r 50,000 and 29,000. The N-terminal sequences of these bands showed a 100% homology with the heavy and light chains of human lgG. A polyclonal antibody raised against the purified protein and anti-human lgG were both able to suppress the acrosome reactioninducing activity of crude hFF. However, neither normal human serum nor a purified preparation of human lgG were able to mimic the AR-inducing activity of hFF. We concluded that the AR-inducing activity of hFF is, at least in part, due to the presence of antisperm antibodies. © 1994 Wiley-Liss, Inc.     

Steroidogenesis-stimulating activity in the gonads: comparison of rat testicular fluid with bovine and human ovarian follicular fluids.

The steroidogenesis-stimulating activities of ovarian follicular fluid from bovine (bFF) and human (hFF) sources, were compared with those of adult rat testicular fluid (rTF) using an in vitro bioassay system based on stimulation of testosterone production by purified adult rat Leydig cells during a 20-h incubation. Rat TF and bFF were charcoal-treated to remove steroids prior to assay, and the major active fraction of hFF was collected after gel-permeation chromatography. All three fluid samples stimulated both basal and maximal hCG-stimulated testosterone production, although the resulting log dose-response lines of bFF and the hFF extracts were not parallel with those of rTF. Both rTF and bFF were active over a similar dose range (5.2-150 microliters and 9.7-150 microliters, respectively) and both had a more than additive interaction with hCG on testosterone production. The stimulatory activity of the hFF extract was considerably greater than that of either rTF or bFF in the absence of hCG, but hFF extract had only an additive effect with hCG in stimulating testosterone production. Moreover, unlike rTF activity, which was inhibited by co-incubation with the protein synthesis inhibitor, cycloheximide, the activity of the hFF extract was not affected by cycloheximide. The factors responsible for activity in all three fluids were of a large molecular size (greater than 30 kDa), as determined by ultrafiltration or gel-permeation chromatography. However, in contrast to both rTF and bFF, hFF extract activity was removed by charcoal extraction. Human FF extract was inactivated by heat (100 degrees C, 30 min), whereas rTF activity was partially (70%) heat-labile and bFF was not affected by heart.(ABSTRACT TRUNCATED AT 250 WORDS)     

Further indications of the multicomponent nature of the acrosome reaction-inducing substance of human follicular fluid

Human follicular fluid (hFF), which has been treated with either unspecific proteases or dextran-coated charcoal (DCC) to remove proteins and/or steroids, cannot successfully induce the acrosome reaction (AR). After the removal of steroids, AR-inducing activity can be restored to hFF by supplementation with exogenous progesterone, but only in the presence of intact protein. Gel filtration experiments with ³H-progesterone-labelled hFF showed elution of the radioactive signal in the high molecular weight range, corresponding to bound progesterone. AR-inducing activity was seen in exactly the same fraction. Based on these results, the acrosome reaction-inducing substance (ARIS) appears to be a complex of progesterone and a progesterone-binding protein, which was shown to be identical with the plasma protein corticosteroid-binding globulin (CBG) by immunological techniques. AR induction was only observed in the presence of both CBG and progesterone, suggesting a combined effect of the two components. © 1995 wiley-Liss, Inc.     

Demonstration of a nonsteroidal factor in human follicular fluid that attenuates the self-priming action of gonadotropin-releasing hormone on pituitary gonadotropes

The self-priming effect of gonadotropin-releasing hormone (GnRH) on luteinizing hormone (LH) release can be demonstrated in vitro by perfusing pituitary tissue with a continuous GnRH stimulus. A characteristic biphasic response is produced. We have used this system to investigate whether or not human follicular fluid (hFF) contains a nonsteroidal substance that can attenuate the GnRH-induced LH secretion in perfused rat pituitary glands. Steroid-extracted hFF, added to the perfusing medium, attenuated the self-priming action of GnRH in a dose-dependent manner. This was not abolished by selectively depleting the inhibin content of hFF by 97% on an immunoaffinity column. Furthermore, the biological activity of the substance was resistant to heating and was removed by dialysis. It is concluded that hFF contains a nonsteroidal factor, distinct from inhibin, that can attenuate the self-priming action of GnRH on pituitary gonadotropes.     

Strontium supports human sperm capacitation but not follicular fluid-induced acrosome reaction.

The ability of strontium (Sr(2+)) to replace calcium (Ca(2+)) in maintaining human sperm function has still not been completely characterized. In the present study, acrosome reaction (AR) inducibility in response to human follicular fluid (hFF) was compared in spermatozoa incubated in either Ca(2+)- or Sr(2+)-containing media. Other events related to sperm capacitation, such as protein tyrosine phosphorylation and hyperactivation as well as zona pellucida (ZP) recognition under both conditions, were also analyzed. Spermatozoa incubated overnight in the presence of Sr(2+) were unable to undergo the AR when exposed to hFF. Nevertheless, when spermatozoa were incubated under this condition and then transferred to medium with Ca(2+), sperm response to hFF was similar to that of cells incubated throughout in the presence of Ca(2+). The sperm protein tyrosine phosphorylation patterns and the percentages of sperm motility and hyperactivation were similar after incubation in Ca(2+)- or Sr(2+)-containing media. Under both conditions, the same binding capacity to homologous ZP was observed. Similar results were obtained when EGTA was added in order to chelate traces of Ca(2+) present in Sr(2+) medium. From these results, it can be concluded that Sr(2+) can replace Ca(2+) in supporting capacitation-related events and ZP binding, but not hFF-induced AR of human spermatozoa.     

Inhibition of LHRH-induced LH and FSH release by gonadotrophin surge-attenuating factor (GnSAF) from human follicular fluid

It has been suggested that in superovulated women the endogenous LH surge is attenuated by a non-steroidal factor, called gonadotrophin surge-attenuating factor (GnSAF), which reduces gonadotrophin secretion in response to LHRH. To determine whether human follicular fluid (hFF) from superovulated women contains GnSAF activity, the secretion of LH and FSH by cultured sheep pituitaries was studied. After charcoal extraction of steroids, hFF was treated by heparin/Sepharose chromatography, which reversibly binds inhibin. The effects of whole hFF and the bound and unbound fractions on basal and LHRH-induced gonadotrophin secretion were then assessed. Steroid-free hFF significantly reduced basal FSH, but not basal LH, secretion, and significantly attenuated the LH and FSH responses to LHRH. The bound (inhibin) fraction significantly decreased both basal and LHRH-induced FSH secretion but did not affect LH release. The unbound fraction had no effect on basal LH or FSH secretion, but significantly attenuated LHRH-induced secretion of both LH and FSH. We conclude that the unbound fraction of hFF from superovulated women contains GnSAF. It has been demonstrated that GnSAF is a non-steroidal factor and its activity is distinct from that of inhibin.     

Proteomic analysis of human follicular fluid using an alternative bottom-up approach

Human follicular fluid (hFF) is the in vivo environment of oocytes during follicular maturation in the ovaries. It contains a huge variety of compounds such as, e.g., proteins that might play an important role in follicular development and oocyte growth. Previous proteomic studies on follicular fluid have isolated and already identified a certain number of proteins. Nevertheless, only a small part of proteins present in follicular fluid have been covered so far and a large number have still not been identified. Therefore, the need for new, more resolving, and sensitive approaches in proteome research is evident. We utilized a proteomic setup based on in solution isoelectric focusing (IEF) and reversed-phase nanoliquid chromatography coupled to matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (nano-LC MALDI TOF/TOF MS) for in depth protein analysis of human follicular fluid samples of patients undergoing controlled ovarian hyper stimulation (COH) for in vitro fertilization therapy (IVF). This approach led to the significant identification of 69 proteins, where 32 have not been reported before to be found in human follicular fluid with proteomic methods. Among these findings, at least two relevant compounds essentially involved in hormone secretion regulation during the folliculogenetic process were identified: sex hormone binding globulin (SHBG) and inhibin A (INHA). To confirm these results, both proteins were further validated by immunoassays.     

Regulation of prostaglandin biosynthesis by human ovarian follicular fluid: a mechanism for ovulation?

We have studied the effect of human ovarian follicular fluid on PG production by bovine seminal vesicles in vitro and found that hFF1 contains a factor of high molecular weight (Mr greater than 30,000) which inhibits PG synthase in a dose-dependent manner. Exposure of this substance to protease activity produced a factor of lower molecular weight (Mr less than 1000) which stimulated PG synthase activity. If this is true of ovarian follicles in vivo, it is possible that increased follicular protease activity stimulates PG synthesis at the time of ovulation.     

Regulation of prostaglandin biosynthesis by human ovarian follicular fluid: A mechanism for ovulation?

We have studied the effect of human ovarian follicular fluid on PG production by bovine seminal vesicles and found that hFF contains a factor of high molecular weight (Mr > 30,000) which inhibits PG synthase in a dose-dependent manner. Exposure of this substance to protease activity produced a factor of lower molecular weight (Mr < 1000) which stimulated PG synthase activity. If this is true of ovarian follicles , it is possible that increased follicular protease activity stimulates PG synthesis at the time of ovulation.     

Proteomic analysis of recurrent spontaneous abortion: Identification of an inadequately expressed set of proteins in human follicular fluid

Recurrent spontaneous abortion (RSA), defined as the loss of three or more consecutive pregnancies prior to the 20th week of gestation, affects up to 5% of the child-bearing population. To investigate the proteins associated with RSA, the protein expression in human follicular fluid was analyzed using 2-DE. Follicular fluid contains a variety of biologically important proteins for oocyte fertilization and follicle maturation in the mammalian reproductive process. Therefore, it can be used as a provisional source for identifying proteins involved in RSA. In this study, we identified five aberrantly expressed proteins (complement component C3c chain E, fibrinogen gamma, antithrombin, angiotensinogen, and hemopexin precursor) in follicular fluid from RSA patients with MALDI-TOF-MS and nano-LC MS/MS. Western blot analysis confirmed that the protein expression level of fibrinogen gamma and antithrombin was less in follicular fluid from RSA patients than those from normal controls. Semiquantitative RT-PCR and real-time PCR analyses revealed that mRNA level of these coagulation factors was also decreased significantly in chorionic villi of RSA patients compared with normal samples. Taken all together, it is likely that coagulation factors (fibrinogen gamma and antithrombin) play an important role in maintaining the normal pregnancy.