發(fā)布時間：2018-03-19 09:13

  本文選題：水牛卵母細(xì)胞　切入點：卵丘細(xì)胞　出處：《廣西大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：卵母細(xì)胞體外成熟(In Vitro Maturation, IVM)和胚胎體外培養(yǎng)(In Vitro Culture, IVC)是胚胎體外生產(chǎn)的關(guān)鍵技術(shù)環(huán)節(jié)。近年來,雖然二者均已取得了長足進(jìn)展,但體外生產(chǎn)的胚胎發(fā)育潛力仍較體內(nèi)胚胎低。本研究旨在研究低氧分壓對水牛卵母細(xì)胞體外成熟與早期胚胎體外發(fā)育的影響,同時初步探討低氧誘導(dǎo)因子(Hypoxia Inducible Factor, HIF)通過糖代謝途徑對水牛卵母細(xì)胞體外成熟和早期胚胎體外發(fā)育影響的分子機制,以期通過模擬體內(nèi)卵母細(xì)胞成熟和胚胎發(fā)育的理化環(huán)境來優(yōu)化卵母細(xì)胞和早期胚胎體外培養(yǎng)體系中的氣相環(huán)境,從而提高水牛卵母細(xì)胞體外成熟和胚胎體外培養(yǎng)的效率。首先研究了低氧分壓對水牛卵母細(xì)胞體外成熟的影響,并初步探討了低氧分壓通過HIF調(diào)節(jié)的低氧通路及糖代謝影響水牛卵母細(xì)胞體外成熟的作用機制。結(jié)果顯示：水牛卵冠丘復(fù)合物(Cumulus-Oocyte Complexes, COCs)在不同低氧分壓(2%O2、3.5%O2、5%o2、6.5%O2)環(huán)境中體外成熟培養(yǎng)時,相對于常氧(20%02)組,5%02和6.5%02組對卵母細(xì)胞的成熟率無顯著影響(50.73% vs 55.3 1%,49.58% vs 55.3 1%,P0.05),而2%02和3.5%02組卵母細(xì)胞的成熟率均顯著降低(21.22% vs 55.31%,29.71% vs55.31%,P0.05)；與常氧(20%02)組相比,2%02、3.5%02、5%02組COCs卵丘細(xì)胞擴展指數(shù)顯著增高(2.84 vs 2.70,2.88 vs 2.70,2.89 vs 2.70,P0.05),而6.5%02組對COCs卵丘細(xì)胞擴展指數(shù)無顯著影響(2.74 vs 2.70,P0.05)。水牛COCs在5%02和20%O2條件下體外成熟培養(yǎng)后進(jìn)行體外受精(In Vitro Fertilization, IVF),獲得的胚胎在20%02中體外培養(yǎng),結(jié)果發(fā)現(xiàn)：與常氧(20%02)組相比,COCs在低氧(5%02)條件下IVM組能顯著提高其IVF早期胚胎的8-ce11分裂率和囊胚發(fā)育率(44.88% vs38.47%,26.81%vs 15.60%,P0.05)。對HIF1α和HIF2α進(jìn)行免疫熒光檢測結(jié)果發(fā)現(xiàn)：在IVM前水牛COCs的卵丘細(xì)胞及在不同氧分壓(5%O2、20%02)條件下IVM后的卵丘細(xì)胞上均有HIF1α和HIF2α的表達(dá)；而在IVM后的卵母細(xì)胞中HIF1α均有表達(dá),HIF2α幾乎不表達(dá)。進(jìn)一步進(jìn)行實時熒光定量PCR (Real-time Quantitative PCR, Q-PCR)分析,結(jié)果顯示：在低氧(5%02)環(huán)境中體外成熟培養(yǎng)水牛COCs,顯著上調(diào)了COCs中卵丘細(xì)胞HIF1α、HIF2α、FSH的受體(FSHR)、IGF1的受體(IGF1R)、FOXO1、 VEGF及其受體(VEGFR1、VEGFR2)、糖代謝相關(guān)基因(GLUT1、LDHA、 G6DP)、熱休克因子(HSP70、HSP90)和五聚環(huán)蛋白-3(Ptx3)的mRNA水平(P0.05),且顯著下調(diào)了前列腺素內(nèi)過氧化物合酶-2(Ptgs2)及凋亡相關(guān)基因Bax和Bc12的mRNA水平(P0.05)。TUNEL檢測結(jié)果亦發(fā)現(xiàn),水牛COCs在低氧(5%02)中體外成熟培養(yǎng),能顯著降低其卵丘細(xì)胞的凋亡率(21.84% vs 30.87%,P0.05)。隨后,探討了低氧分壓(5%02)體外培養(yǎng)水牛體外早期胚胎對其發(fā)育能力的影響,并對低氧通路中HIF的調(diào)控路徑及其通過無氧糖酵解影響水牛早期胚胎發(fā)育的作用機制進(jìn)行了初步研究。水牛COCs在5%O2中IVM后進(jìn)行IVF,受精卵分別在不同氧分壓(5%02、20%02)條件下進(jìn)行IVC，并收集囊胚,結(jié)果發(fā)現(xiàn)：低氧(5%O2)IVC(M5C5)組的胚胎發(fā)育到2-cell、 4-cell和8-cell的比率均顯著高于常氧(20%O2) IVC (M5C20)組(72.38%vs 68.98%,62.32%vs 57.18%,49.65% vs 45.23%,P0.05),但兩組的囊胚發(fā)育率差異不顯著(27.24% vs 26.11%,P0.05)；免疫熒光分別檢測兩組囊胚中HIF1α和HIF2α的表達(dá)情況,結(jié)果表明：HIF1α有表達(dá),而HIF2α幾乎不表達(dá)；經(jīng)Q-PCR分析發(fā)現(xiàn)：相對于M5C20組,M5C5組囊胚中HIF1α及調(diào)控其表達(dá)的相關(guān)基因(IGF1R、FOXO1)、糖代謝相關(guān)基因(GLU1、 LDHA、G6PD)、熱休克因子(HSP70/90)和抗凋亡基因(Bcl2)的mRNA水平顯著上調(diào)(P0.05),而促凋亡基因(Bax)的mRNA水平顯著下調(diào)(P0.05)。TUNEL檢測結(jié)果亦發(fā)現(xiàn),M5C5組囊胚細(xì)胞的凋亡率顯著低于M5C20組(3.89% vs 6.90%,P0.05)。上述結(jié)果表明：(1) HIF1α在水牛COCs的卵丘細(xì)胞和卵母細(xì)胞以及囊胚中均有表達(dá),而HIF2α僅在水牛COCs的卵丘細(xì)胞中有表達(dá)； (2)低氧分壓(5%02)條件有利于水牛COCs的體外成熟及其IVF早期胚胎的體外發(fā)育；(3)在低氧分壓(5%02)條件下,FSH和IGF1可能通過PI3K/AKT途徑調(diào)節(jié)低氧通路的核轉(zhuǎn)錄因子HIF1α穩(wěn)定表達(dá),穩(wěn)定表達(dá)的HIF1α可能又通過促進(jìn)無氧糖酵解有利于水牛卵丘細(xì)胞和早期胚胎適應(yīng)低氧環(huán)境,促進(jìn)水牛卵丘細(xì)胞的擴展與增殖、上調(diào)發(fā)育相關(guān)基因的表達(dá)、抑制細(xì)胞的凋亡,進(jìn)而促進(jìn)水牛卵母細(xì)胞的成熟,并提高其早期胚胎的發(fā)育潛力。
[Abstract]:In vitro maturation of oocytes (In Vitro, Maturation, IVM) and cultured embryos in vitro (In Vitro Culture, IVC) is the key technology of embryo production in vitro. In recent years, although the two have made considerable progress, but the production of in vitro embryo developmental potential is still relatively low body endodermal. This study was aimed to study fetal hypoxia the partial pressure and influence on early embryonic development in vitro maturation of oocytes in vitro and buffalo, preliminary study of hypoxia inducible factor (Hypoxia Inducible, Factor, HIF) through the metabolic pathways of buffalo oocyte maturation and early embryo development in vitro molecular mechanism, in order to simulate in vivo oocyte maturation and embryo development the physicochemical environment to optimize the oocytes and early embryos in vitro culture system in the gas phase, so as to improve the efficiency of buffalo culture in vitro maturation of oocyte and embryo in vitro. The first study The oxygen partial pressure on the impact of buffalo oocyte maturation in vitro, and to explore the low oxygen partial pressure regulated by HIF pathway and effects of glucose metabolism in hypoxia buffalo oocyte in vitro maturation mechanism. Results: Buffalo OCCC (Cumulus-Oocyte Complexes, COCs) at different oxygen partial pressure (2%O2,3.5%O2,5%o2,6.5%O2) in vitro maturation, compared with normoxia (20%02) group, 5%02 group and 6.5%02 on the maturation rate of oocytes had no significant effect (50.73% vs 55.31%, 49.58% vs 55.31%, P0.05), and the maturation rate of oocytes from group 3.5%02 and 2%02 were significantly decreased (21.22% vs 55.31%, vs55.31% 29.71%, P0.05); and normoxia (20%02) group, 2%02,3.5%02,5%02 group, COCs cumulus cell expansion index increased significantly (2.84 vs 2.70,2.88 vs 2.70,2.89 vs 2.70, P0.05), and 6.5%02 group of COCs had no significant effect on cumulus expansion index (2.74 vs 2.70, P0.05). The buffalo COCs in 5%02 and 20%O2 during maturation after in vitro fertilization (In Vitro, Fertilization, IVF) of embryos in 20%02 cultured in vitro. The results showed that: compared with normoxia (20%02) group, COCs (5%02) under the condition of hypoxia in IVM group significantly to improve the IVF 8-ce11 early embryo cleavage rate and blastocyst rate (44.88% vs38.47%, 26.81%vs 15.60%, P0.05). The HIF1 alpha and HIF2 alpha immunofluorescence assay results showed that: in the former IVM of buffalo COCs and cumulus cells at different oxygen partial pressure (5%O2,20%02) expression of IVM after cumulus cells were HIF1 and alpha HIF2 a condition; while in IVM after oocyte expressed HIF1 alpha, alpha HIF2 almost no expression. Further real-time fluorescence quantitative PCR (Real-time Quantitative PCR, Q-PCR) analysis, results showed that: (5% 02) in the hypoxic environment in vitro maturation of water Cattle COCs, increased COCs in cumulus cells HIF1 alpha, alpha HIF2, FSH receptor (FSHR), IGF1 receptor (IGF1R), FOXO1, VEGF and its receptors (VEGFR1, VEGFR2), glucose metabolism related genes (GLUT1, LDHA, G6DP), heat shock factor (HSP70, HSP90) and five poly ring -3 (Ptx3) mRNA protein level (P0.05), and significantly reduced the prostaglandin endoperoxide synthase -2 (Ptgs2) and apoptosis related gene Bax and Bc12 mRNA (P0.05).TUNEL test results also found that Buffalo COCs in hypoxia (5%02) maturation in vitro, can significantly reduce the apoptosis of cumulus cells the rate (21.84% vs 30.87%, P0.05). Then, discusses the low oxygen partial pressure (5%02) on the impact of buffalo early embryos in vitro development ability of cultured in vitro, and regulatory pathways to hypoxia and HIF pathway via anaerobic glycolysis mechanism of early embryonic development of buffalo were studied. In the buffalo COCs 5% IVF IVM O2, the fertilized eggs respectively at different oxygen partial pressure (5%02,20%02) of IVC, and collecting blastocysts, results showed that hypoxia (5%O2) IVC (M5C5) group of embryonic development to 2-cell, 4-cell and 8-cell ratio were significantly higher than those in normoxia (20%O2) group (IVC (M5C20) 72.38%vs 68.98%, 62.32%vs 57.18%, 49.65% vs 45.23%, P0.05 two), but the blastocyst development rate had no significant difference (27.24% vs 26.11%, P0.05); the results showed that immunofluorescence was used to detect the expression situation, two groups of blastocysts of HIF1 alpha and HIF2 alpha HIF1 alpha HIF2 alpha expression, while almost no expression by; Q-PCR analysis showed that: compared with M5C20 group, the related gene HIF1 and regulation of M5C5 expression in the blastocyst group (IGF1R, FOXO1), glucose metabolism related genes (GLU1, LDHA, G6PD), heat shock factor (HSP70/90) and anti apoptosis gene (Bcl2) of mRNA was significantly increased (P0.05), and apoptosis (Bax) mRNA gene Levels were significantly reduced (P0.05).TUNEL test results also found that the rate of blastocyst cell apoptosis in M5C5 group was significantly lower than that of M5C20 group (3.89% vs 6.90%, P0.05). The results showed that: (1) HIF1 in Buffalo COCs cumulus cells and oocytes and blastocysts were expressed, and HIF2 alpha only in Buffalo COCs cumulus cells; (2) low oxygen pressure (5%02) growth conditions in favor of buffalo COCs IVF in vitro maturation and early embryo in vitro; (3) in the low oxygen partial pressure (5%02) conditions, the expression of nuclear transcription factor HIF1 alpha stable FSH and IGF1 pathway regulated by PI3K/AKT pathway of hypoxia, stability the expression of HIF1 may be through promoting anaerobic glycolysis in favor of buffalo cumulus cells and early embryos adapt to the hypoxic environment, promote the expansion and proliferation of buffalo cumulus cells, up-regulated expression of genes related to growth, inhibit cell apoptosis, and promote buffalo eggs The maturation of the mother cell and the development potential of its early embryos.

【學(xué)位授予單位】：廣西大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S823.83
，

本文編號：1633612