本文關(guān)鍵詞： Hsp27 多囊卵巢綜合征 細(xì)胞凋亡 卵母細(xì)胞成熟 胚胎發(fā)育　出處：《南京醫(yī)科大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：研究背景 多囊卵巢綜合征(Polycystic Ovary Syndrome, PCOS)是育齡期婦女最常見的內(nèi)分泌和代謝紊亂性疾病,育齡婦女中發(fā)病率為5%-10%,主要特征為排卵障礙、高雄激素血癥、胰島素抵抗以及卵巢多囊樣改變。PCOS的病理生理變化涉及多種神經(jīng)內(nèi)分泌及代謝系統(tǒng)通路和多種卵巢局部的蛋白質(zhì)調(diào)控因子等,某種調(diào)節(jié)機(jī)制的失常可以導(dǎo)致多種反饋失衡和連鎖反應(yīng),產(chǎn)生臨床表型的多樣化和分類治療的復(fù)雜性。因此,更好的了解PCOS與卵巢外部和內(nèi)部的因子異常的關(guān)系,以及這些異常對(duì)顆粒細(xì)胞與卵母細(xì)胞之間的聯(lián)系,卵子成熟和胚胎發(fā)育潛力的影響等,對(duì)于需要進(jìn)行IVF治療的PCOS女性提高生育力、優(yōu)化臨床卵巢刺激方案和提高妊娠結(jié)局都是至關(guān)重要的。雖然PCOS患者的特點(diǎn)是在IVF促排卵過(guò)程中產(chǎn)生大量的卵子,但是通常卵子質(zhì)量較差,從而隨后的受精率、卵裂率和著床率都較低,流產(chǎn)率高。有證據(jù)表明卵子和胚胎質(zhì)量差可能是由于非整倍體率增加引起的,但最近的數(shù)據(jù)提示PCOS患者行體外受精(In Vitro Fertilization, ⅣF)治療產(chǎn)生大量的卵子,整倍體率卵子也較多,但總體妊娠率仍較低,流產(chǎn)率增高,提示這與胚胎非整倍體無(wú)關(guān)。因此,染色體之外還有其它因素導(dǎo)致PCOS患者妊娠丟失率增高。PCOS女性卵子成熟受損和胚胎發(fā)育潛能降低可能與患者的內(nèi)分泌／旁分因子異常、代謝異常和卵泡發(fā)育成熟過(guò)程中卵泡內(nèi)環(huán)境的改變都有關(guān)。 PCOS的病因和病理生理機(jī)制尚有很多未知的環(huán)節(jié)。大量研究提示,在PCOS患者卵巢中存在著凋亡／抗凋亡的失衡。本實(shí)驗(yàn)室在前期的研究工作中構(gòu)建了正常人與PCOS患者卵巢中差異蛋白質(zhì)表達(dá)譜系,其中54種蛋白質(zhì)在PCOS患者卵巢中表達(dá)上調(diào),15種蛋白質(zhì)表達(dá)下調(diào)。熱休克蛋白27(Heat Shock Protein27, Hsp27)是該差異表達(dá)譜系表達(dá)下調(diào)的蛋白質(zhì)之一。Hsp27是小分子量熱休克蛋白亞家族中的重要成員之一,Hsp27作為調(diào)控凋亡因子,通過(guò)與細(xì)胞色素c結(jié)合,防止Caspase9激活,阻礙Fas、TNF介導(dǎo)的外源性凋亡途徑產(chǎn)生抗凋亡作用。Hsp27可能通過(guò)抗凋亡機(jī)制的紊亂導(dǎo)致卵泡發(fā)育的停滯而不能正常周期性凋亡。我們前期研究了Hsp27對(duì)小鼠卵母細(xì)胞成熟的影響,研究證實(shí)Hsp27可以通過(guò)調(diào)節(jié)卵母細(xì)胞的早期凋亡從而調(diào)控其成熟,保證卵母細(xì)胞的正常生長(zhǎng)發(fā)育和成熟。Hsp27通過(guò)活化Caspase8介導(dǎo)的外源性凋亡信號(hào)通路調(diào)節(jié)卵母細(xì)胞的凋亡進(jìn)而調(diào)控卵母細(xì)胞的成熟；通過(guò)調(diào)節(jié)PKAc、PKCδ、PKOλ的表達(dá)影響卵母細(xì)胞的成熟。 有研究提示Hsp27在受精和早期胚胎發(fā)育過(guò)程中可能也起著重要作用。而對(duì)于Hsp27對(duì)卵母細(xì)胞的成熟受精和隨后胚胎發(fā)育的影響以及Hsp27在一系列過(guò)程中所起到的作用,尚未見報(bào)道。PCOS患者卵巢中存在著凋亡與抗凋亡的失衡,卵泡發(fā)育成熟障礙。而我們前期研究證實(shí),PCOS卵泡中Hsp27低表達(dá),可能引起抗凋亡機(jī)制的紊亂導(dǎo)致卵泡發(fā)育的停滯而不能正常周期性凋亡。卵泡發(fā)育障礙是否與此相關(guān)還不明確。而在臨床治療中發(fā)現(xiàn),PCOS患者進(jìn)行IVF促排卵治療時(shí),可以獲得大量的卵母細(xì)胞,但獲得的胚胎發(fā)育潛力和著床率較低。這是否與卵母細(xì)胞Hsp27的低表達(dá)有關(guān),還是胚胎發(fā)育過(guò)程中Hsp27低表達(dá)所致。這些問題都值得深入探討。因此,我們期望能研究PCOS患者小卵泡中HSP27的低表達(dá)對(duì)于卵母細(xì)胞成熟和隨后受精和胚胎發(fā)育的影響,進(jìn)一步探討凋亡在卵母細(xì)胞成熟和早期胚胎發(fā)育過(guò)程中起到的作用。 本研究以小鼠原核期胚胎和人PCOS患者未成熟的卵母細(xì)胞為對(duì)象,研究Hsp27在小鼠及PCOS患者卵母細(xì)胞成熟、受精、胚胎早期發(fā)育中的作用,觀察Hsp27對(duì)卵子成熟及胚胎體外發(fā)育能力的影響,進(jìn)一步探討PCOS患者卵母細(xì)胞Hsp27低表達(dá)對(duì)隨后胚胎發(fā)育潛力的影響。 研究?jī)?nèi)容和結(jié)果 1.Hsp27在小鼠早期胚胎體外發(fā)育過(guò)程中的表達(dá)與定位 6-8周齡ICR小鼠超排后,合籠。從輸卵管獲取受精卵。置于培養(yǎng)液中體外培養(yǎng),在培養(yǎng)的24h,48h,72h,96h后,受精卵分別發(fā)育到2細(xì)胞期,4-細(xì)胞期,8-16細(xì)胞期和囊胚期。收集后采用免疫熒光法對(duì)胚胎發(fā)育的各個(gè)時(shí)期Hsp27表達(dá)進(jìn)行定位。結(jié)果顯示,Hsp27蛋白定位于小鼠早期胚胎發(fā)育各時(shí)期的卵裂球除核仁外的細(xì)胞質(zhì)和細(xì)胞核中。 2.Hsp27在小鼠早期胚胎發(fā)育過(guò)程中的功能 應(yīng)用顯微注射方法分別將Hsp27抗體和已構(gòu)建成功的重組腺病毒Ad-H1-siRNA注射入小鼠原核期受精卵,分別從蛋白和RNA水平上阻斷受精卵的Hsp27的活性,觀察Hsp27下調(diào)對(duì)胚胎體外發(fā)育的影響。同時(shí)重組腺病毒Ad-CMV-Hsp27顯微注入受精卵胞漿中,上調(diào)受精卵的Hsp27表達(dá),觀察Hsp27上調(diào)后對(duì)胚胎發(fā)育的影響,將胚胎繼續(xù)體外培養(yǎng),觀察各組囊胚形成率和囊胚細(xì)胞總數(shù)的差異,通過(guò)TUNEL法檢測(cè)囊胚細(xì)胞的凋亡情況。結(jié)果發(fā)現(xiàn),Hsp27抗體組的囊胚形成比例為84.8%,對(duì)照組分別為81.8%和82.2%,P0.05。囊胚細(xì)胞數(shù)分別為：67.5、67.1和71.0,p0.05,沒有統(tǒng)計(jì)學(xué)差異。將重組腺病毒AdSiRNA-Hsp27采用顯微注射方法注射入小鼠原核期受精卵,注射AdSiRNA-GFP作為對(duì)照,空白對(duì)照不注射。結(jié)果發(fā)現(xiàn),三組之間囊胚形成率分別為,83.1%,86.3%87.7%,P0.05,沒有統(tǒng)計(jì)學(xué)差異。Hsp27表達(dá)上調(diào)結(jié)果顯示,顯微注射重組腺病毒Ad-CMV-Hsp27和Ad-CMV-GFP以及Control組,三組的囊胚形成率分別為：84.5%、83.8%和86.4%,P0.05,沒有統(tǒng)計(jì)學(xué)差異。以上試驗(yàn)結(jié)果表明,小鼠受精卵中Hsp27下調(diào)和上調(diào)后,對(duì)胚胎發(fā)育潛力均沒有明顯影響。通過(guò)總細(xì)胞數(shù)(total cell number,TCN)和細(xì)胞死亡率(cell death index,CDI)用來(lái)評(píng)估囊胚質(zhì)量,結(jié)果發(fā)現(xiàn)超標(biāo)達(dá)和干涉Hsp27表達(dá)后各組的囊胚質(zhì)量沒有差異。 3.Hsp27對(duì)人PCOS卵母細(xì)胞體外成熟的影響 3.1Hsp27表達(dá)上調(diào)對(duì)卵母細(xì)胞成熟的影響 通過(guò)小卵泡穿刺,獲得人PCOS患者的未成熟卵。將獲得的GV期卵母細(xì)胞隨機(jī)分為三組,利用顯微注射技術(shù),將重組的Hsp27質(zhì)粒載體(pAdTrack-CMV-Hsp27)注射到GV期卵子的生發(fā)泡中,從基因水平上調(diào)Hsp27的表達(dá)。實(shí)驗(yàn)組注射pAdTrack-CMV-Hsp27質(zhì)粒,一個(gè)對(duì)照組只注射空質(zhì)粒載體(pAdTrack-CMV),另一對(duì)照組不行注射。注射完成后,將GV期卵子轉(zhuǎn)移到體外成熟培養(yǎng)液中進(jìn)行成熟培養(yǎng),48h后觀察卵母細(xì)胞體外成熟情況。結(jié)果顯示,注射Hsp27超表達(dá)載體組卵母細(xì)胞的發(fā)育成熟率為33.5%,對(duì)照組分別為55.9%和61.4%,P0.05。表明,PCOS患者獲得的未成熟卵Hsp27表達(dá)上調(diào)后,抑制了卵母細(xì)胞的體外成熟。 3.2Real time RT-PCR驗(yàn)證卵母細(xì)胞特異的分泌因子的表達(dá) Hsp27超表達(dá)載體(pAdTrack-CMV-hHsp27)顯微注射到人PCOS卵母細(xì)胞后,體外培養(yǎng)48h后,收集卵母細(xì)胞,提取mRNA,檢測(cè)卵母細(xì)胞特異性的分泌因子Bmp15口Gdf9的表達(dá)變化,結(jié)果發(fā)現(xiàn),Hsp27表達(dá)增加后,Bmp15和Gdf9的mRNA表達(dá)下降,從另一方面驗(yàn)證Hsp27對(duì)卵母細(xì)胞成熟的影響。 3.3Hsp27上調(diào)后凋亡調(diào)控因子的表達(dá) 為了檢測(cè)Hsp27上調(diào)后激活的凋亡通路,顯微注射pAdTrack-CMV-hHsp27后,卵母細(xì)胞繼續(xù)體外成熟培養(yǎng),培養(yǎng)48h后,收集各組卵母細(xì)胞,提取mRNA,RNA反轉(zhuǎn)錄為cDNA,運(yùn)用Real time RT-PCR檢測(cè)Caspase3、Caspase8、Caspase9、Cytc的表達(dá)。結(jié)果顯示,Track-CMV-hHsp27顯微注射組中Caspase8、Caspase9和Caspase3的表達(dá)明顯低于pAdTrack-CMV注射組(P0.05),而Cyt c的表達(dá)未有明顯的差異。說(shuō)明,HSP27在人PCOS卵母細(xì)胞中通過(guò)capase-8介導(dǎo)的外源性及capase-9介導(dǎo)的內(nèi)源性凋亡通路發(fā)揮作用。 4.Hsp27表達(dá)上調(diào)對(duì)人PCOS卵母細(xì)胞體外受精胚胎發(fā)育能力的影響。 獲取的PCOS患者的GV期卵母細(xì)胞,顯微注射Hsp27超表達(dá)載體后,繼續(xù)體外成熟培養(yǎng),將培養(yǎng)成熟的MII期卵母細(xì)胞行卵胞漿內(nèi)單精子注射(ICSI)法受精。注射完成后,將卵子移入卵裂期胚胎培養(yǎng)液中培養(yǎng),ICSI后16～-18h觀察卵子受精情況。培養(yǎng)的第三天,將胚胎移入囊胚培養(yǎng)液中繼續(xù)培養(yǎng)。在第五天和第六天觀察囊胚形成情況,統(tǒng)計(jì)囊胚形成率。結(jié)果提示,Hsp27超表達(dá)后,卵子成熟率顯著低于對(duì)照組(33.5%vs55.9%和61.4%,P0.05),Hsp27上調(diào)后成熟卵子的受精率、day3優(yōu)質(zhì)胚胎率與對(duì)照組沒有顯著差異,而Hsp27上調(diào)組的囊胚形成率顯著高于對(duì)照組(41.30%vs23.53%,P0.05)。說(shuō)明HSP27表達(dá)上調(diào)導(dǎo)致PCOS卵母細(xì)胞成熟發(fā)生不同程度的阻滯,然而HSP27卻顯著提高卵母細(xì)胞發(fā)育潛能,說(shuō)明HSP27在卵母細(xì)胞進(jìn)一步發(fā)育潛能起著重要的調(diào)節(jié)作用。 數(shù)據(jù)分析 運(yùn)用SPSS17.0進(jìn)行數(shù)據(jù)統(tǒng)計(jì)分析,采用單向方差分析和對(duì)數(shù)線性模型來(lái)比較mRNA和蛋白水平,而對(duì)于卵母細(xì)胞成熟率,受精率和胚胎發(fā)育數(shù)據(jù),采用卡方分析進(jìn)行比較,P0.05認(rèn)為是具有統(tǒng)計(jì)學(xué)差異。 結(jié)論 1.在小鼠早期胚胎發(fā)育過(guò)程中,Hsp27蛋白定位于早期胚胎發(fā)育各時(shí)期的卵裂球除核仁外的細(xì)胞質(zhì)和細(xì)胞核中。Hsp27的上調(diào)或下調(diào)對(duì)體外培養(yǎng)胚胎發(fā)育潛能沒有顯著影響。 2.Hsp27通過(guò)Caspase8介導(dǎo)的外源性及capase-9介導(dǎo)的內(nèi)源性凋亡信號(hào)通路調(diào)節(jié)卵母細(xì)胞的凋亡而影響其成熟,Hsp27的表達(dá)上調(diào),使得caspase8凋亡相關(guān)因子表達(dá)降低,降低卵母細(xì)胞體外成熟率。 3.Hsp27作為抗凋亡活性因子,參與了PCOS卵母細(xì)胞凋亡的失衡。由于PCOS的卵巢凋亡的失衡狀態(tài)導(dǎo)致了Hsp27的代償性下降。
[Abstract]:Research background
Polycystic ovary syndrome (Polycystic Ovary, Syndrome, PCOS) is a disease of endocrine and metabolic disorders are most common in women of childbearing age, the incidence rate of 5%-10% in women of childbearing age, the main features of anovulation, Kaohsiung hormones, insulin resistance and polycystic ovary.PCOS involves a variety of pathophysiological changes of neuroendocrine and metabolic pathways and systems a variety of ovarian local regulatory protein factors, some regulatory mechanisms of arrhythmias can lead to a variety of complex feedback imbalance and chain reaction, clinical phenotype and diverse classification treatment. Therefore, a better understanding of the relationship between abnormal factor PCOS and ovarian internal and external, and these abnormalities of cumulus cells and oocytes contact oocyte maturation and embryo development potential, and the effects of the need for IVF treatment PCOS women improve fertility, optimize clinical Ovarian stimulation protocols and improve the outcome of pregnancy is essential. Although the characteristics of PCOS patients in IVF induced ovulation process produces a large number of eggs, but eggs usually of poor quality, and then the fertilization rate, cleavage rate and implantation rate were lower, the abortion rate is high. There is evidence that the quality of oocytes and embryos may be poor the aneuploidy rate increased, but recent data suggest that patients with PCOS in vitro fertilization (In Vitro Fertilization F, IV) treatment produced a large number of eggs, aneuploid rate of eggs is more, but the overall pregnancy rate is still low, the abortion rate increased, suggesting that the embryo aneuploidy has nothing to do therefore, other factors lead to the loss of chromosome.PCOS increased female egg maturation and impaired embryo development potential decrease may be associated with endocrine / paracrine factor PCOS in patients with abnormal pregnancy, abnormal metabolism and follicle development The changes in the follicular environment in the process of ripening are all related.
The etiology and pathophysiology of PCOS still has many unknown links. A large number of studies suggest that, in patients with ovarian PCOS exist in apoptosis / anti apoptosis imbalance. The previous study was constructed between normal persons and patients with ovarian PCOS in protein expression spectrum, upregulation of the expression of 54 proteins in patients with ovarian PCOS in 15 kinds of protein expression. Heat shock protein 27 (Heat Shock, Protein27, Hsp27) is one of the pedigree of the down regulated protein.Hsp27 is an important member of the small heat shock protein subfamily of the Hsp27 as a differential expression, regulation of apoptosis factor by binding to cytochrome c, preventing Caspase9 activation block Fas, exogenous apoptosis mediated by TNF may have anti apoptosis effect of.Hsp27 through anti apoptosis mechanism of follicular development disorder leads to stagnation and not the normal cycle of apoptosis. We Preliminary study of the effects of Hsp27 on mouse oocyte maturation, the study confirmed that Hsp27 can regulate the apoptosis of oocytes to regulate its mature through the development and maturation of.Hsp27, ensure the apoptosis of activated extrinsic apoptosis pathway of Caspase8 mediated regulation of oocyte and control oocytes through normal growth of oocytes. Mature; by adjusting the PKAc, PKC 8, PKO lambda expression affects oocyte maturation.
Studies have shown that Hsp27 may also play an important role in fertilization and early embryonic development process. But for Hsp27 on oocyte maturation and fertilization affect subsequent embryo development and Hsp27 in a series of process function, has not been reported there and anti apoptotic ovarian imbalance in.PCOS patients. Follicular maturation disorder. Our preliminary studies confirmed that PCOS follicles in the low expression of Hsp27, may cause the anti apoptosis mechanism of follicular development disorder leads to stagnation and not the normal cycle. The apoptosis of follicular development disorder is associated with this is not clear. But in clinical treatment, PCOS patients with IVF in ovulation induction treatment. You can get a lot of oocytes, but the embryo developmental potential and implantation rate is relatively low. This is the low expression of Hsp27 on oocytes, or during embryonic development of Hsp27 low expression . these problems are worth further discussion. Therefore, we can expect the low expression of HSP27 on PCOS patients with small follicle in effect on oocyte maturation and subsequent fertilization and embryo development, further discussion to apoptosis in oocyte maturation and early embryonic development process.
In this study, mouse pronuclear embryos and PCOS were immature oocytes as the object of study of Hsp27 in mice and PCOS patients with oocyte maturation, fertilization and early development of embryos, the effects of Hsp27 on oocyte maturation and developmental ability of embryos in vitro, to further explore the oocytes with PCOS low expression of Hsp27 influence on subsequent embryonic development potential.
Research content and results
Expression and localization of 1.Hsp27 in the development of early mouse embryos in vitro
6-8 week old ICR mice after superovulation, cage. Obtaining fertilized eggs from the fallopian tube. In cultured medium, in cultured 24h, 48h, 72h, 96h, respectively. The fertilized eggs develop into 2 cell stage, 4- cell stage, 8-16 cell and blastocyst stage. After collected by immunofluorescence the embryonic development of each period the expression of Hsp27 positioning. The results showed that Hsp27 protein was located in the blastomeres of mouse early embryo development in the nucleolus in the cytoplasm and the nucleus.
Function of 2.Hsp27 in the development of early mouse embryos
Application of micro injection methods respectively, Hsp27 antibody and has successfully constructed the recombinant adenovirus Ad-H1-siRNA was injected into mouse pronuclear zygote, blocking the activity of Hsp27 from the fertilized egg protein and the level of RNA, observe the Hsp27 downward effect on embryo development in vitro. The recombinant adenovirus Ad-CMV-Hsp27 were injected into a fertilized egg in the cytoplasm. Expression of Hsp27 of zygotes, influence on embryonic development observation of Hsp27 increases, will continue to observe the in vitro embryo, blastocyst formation rate and blastocyst cell number differences, the apoptosis of blastocyst cells was detected by TUNEL. The results showed that Hsp27 antibody group of the blastocyst formation ratio was 84.8%, the control group were 81.8% and 82.2%, P0.05. cell number of blastocyst were 67.5,67.1 and 71, P0.05, the difference was not statistically significant. The recombinant adenovirus AdSiRNA-Hsp27 by microinjection method was injected into mouse primary The nuclear stage fertilized eggs, AdSiRNA-GFP injection as control, blank control without injection. The results showed that the blastocyst formation rates between the three groups respectively, 83.1%, 86.3%87.7%, P0.05, no statistical difference between the.Hsp27 expression results showed that microinjection of recombinant adenovirus Ad-CMV-Hsp27 and Ad-CMV-GFP and Control group, three groups of blastocyst formation rates were: 84.5% 83.8%, and 86.4%, P0.05, the difference was not statistically significant. The above results show that the downregulation of Hsp27 and upregulation of mouse fertilized eggs, had no obvious effect on embryonic development potential. The total cell number (total cell, number, TCN) and cell death rate (cell death index, CDI) were used to assess the quality of blastocysts, the results found to exceed the standard of quality and interference blastocysts of Hsp27 expression after no difference.
Effect of 3.Hsp27 on the in vitro maturation of human PCOS oocytes
The effect of up-regulated expression of 3.1Hsp27 on oocyte maturation
Through the small follicle puncture, human immature oocytes retrieved from PCOS patients. GV stage oocytes were randomly divided into three groups obtained, using microinjection technique, the recombinant plasmid vector Hsp27 (pAdTrack-CMV-Hsp27) injection into germinal vesicle GV oocytes in gene expression level from the upregulation of Hsp27. The experimental group was injected with pAdTrack-CMV-Hsp27 plasmid, a control group was injected with empty plasmid vector (pAdTrack-CMV), a control group without injection. After the injection, the GV oocytes transferred to culture medium on in vitro maturation of mature oocytes in vitro, observe the maturation of 48h. The results showed that the injection of Hsp27 over expression vector group of oocytes maturation rate was 33.5%, the control group were 55.9% and 61.4%, P0.05. showed that the expression of PCOS in patients with immature oocytes after Hsp27 inhibited oocyte maturation in vitro.
3.2Real time RT-PCR verifies the expression of specific secretory factors in oocytes
Hsp27 over expression vector (pAdTrack-CMV-hHsp27) microinjection into human PCOS oocytes after 48h cultured in vitro after extraction of mRNA oocytes were collected, and the expression of secreted Bmp15 Gdf9 in detection of oocyte specific results showed that increased expression of Hsp27, Bmp15 and Gdf9 decreased expression of mRNA, from the impact of on the other hand, verification of Hsp27 on oocyte maturation.
Expression of regulation factor of apoptosis after up regulation of 3.3Hsp27
In order to detect the apoptosis pathway upregulation of Hsp27 after activation, micro pAdTrack-CMV-hHsp27 after injection of oocytes to mature in vitro, cultured 48h, collected oocytes, mRNA extraction, RNA reverse transcription into cDNA, detected by Caspase3, Real time RT-PCR Caspase8, Caspase9, Cytc expression. The results showed that Caspase8 Track-CMV-hHsp27 microinjection in the group, the expression of Caspase9 and Caspase3 were significantly lower than that of pAdTrack-CMV group (P0.05), and the expression of Cyt C. There is no obvious explanation, PCOS HSP27 in human oocytes by exogenous and endogenous apoptosis pathway mediated by capase-9 capase-8 mediated by play a role.
The effect of up-regulated expression of 4.Hsp27 on the development ability of human PCOS oocyte in vitro fertilization embryo.
Gets the PCOS of patients with GV stage oocytes microinjected with Hsp27 over expression vector, continue to mature in vitro, cultured mature MII oocytes intracytoplasmic sperm injection (ICSI) method of fertilization. After injection, the eggs in cleavage stage embryos cultured in ICSI 16 ~ -18h observation of fertilization. Third days of culture, the embryo into blastocyst culture medium to culture. Observation on the fifth day and the sixth day of blastocyst formation and blastocyst formation rate. The statistical results suggest that over expression of Hsp27, egg maturation rate was significantly lower than that of control group (33.5%vs55.9% and 61.4%, P0.05), mature the fertilization rate of Hsp27 increases, day3 high-quality embryo rate no significant difference between the two groups, Hsp27 increased group blastocyst formation rate was significantly higher than the control group (41.30%vs23.53%, P0.05). The results showed that HSP27 up-regulated the expression of PCOS in oocyte maturation occurs However, HSP27 significantly improves the developmental potential of oocyte, indicating that HSP27 plays an important role in the further developmental potential of oocyte.
Data analysis
SPSS17.0 was used for data analysis. One-way ANOVA and log linear models were used to compare mRNA and protein levels. For oocyte maturation rate, fertilization rate and embryo development data, chi square analysis was used for comparison. P0.05 thought there was statistical difference.
conclusion
1. in the process of early embryonic development in mice, the Hsp27 protein was located in the period of early embryonic development of blastomere.Hsp27 except outside the nucleolus in the cytoplasm and the nucleus upregulation or downregulation of embryo development potential has no significant effect on in vitro.
2.Hsp27 regulates the apoptosis of oocytes by Caspase8 mediated exogenous and capase-9 mediated endogenous apoptotic signaling pathways, and affects the maturation. Hsp27 expression is upregulated, which reduces the expression of apoptosis related factors and reduces the in vitro maturation rate of oocytes.
As an anti apoptotic active factor, 3.Hsp27 is involved in the imbalance of apoptosis in PCOS oocytes. The imbalance of PCOS's ovarian apoptosis leads to a compensatory decline in Hsp27.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R711.75

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