本文選題：結(jié)締組織生長因子　切入點：基質(zhì)細胞衍生因子-1　出處：《內(nèi)蒙古大學(xué)》2017年碩士論文

【摘要】：綿羊是非常重要的經(jīng)濟型家畜,為人們提供肉、奶、皮毛等產(chǎn)品。運用卵母細胞體外成熟(In Vitro Maturation,IVM)、體外受精(In Vito Fertilization,IVF)和胚胎移植(Embryo Transfer,ET)技術(shù)對優(yōu)質(zhì)家畜進行良種擴繁,能有效提高家畜生產(chǎn)力。卵母細胞的發(fā)育與成熟是這些技術(shù)的基礎(chǔ),因此,卵母細胞成熟的品質(zhì)好壞直接影響子代能否成活及存活后代的健康狀況。從卵母細胞成熟到早期胚胎發(fā)育的過程中,除內(nèi)源性基因的表達調(diào)控作用之外,培養(yǎng)環(huán)境中的信號分子作用也影響其能否正常發(fā)育。通過旁分泌因子發(fā)揮調(diào)控作用是這種調(diào)控最常見、最直接的方式之一。結(jié)締組織生長因子(Connective tissue growth factor,CTGF)、基質(zhì)細胞衍生因子-1(Stromal cell-derived factor-1,SDF-1)、神經(jīng)生長因子(Nerve growth factor,NGF)、肝細胞生長因子(Hepatocyte growth factor,HGF)是卵巢旁分泌因子。研究發(fā)現(xiàn)CTGF能促進小鼠、大鼠卵泡發(fā)育,SDF-1能增加顆粒細胞存活和提高胚胎質(zhì)量,NGF能以劑量依賴的方式增強山羊初級卵泡的體外生長,HGF能誘導(dǎo)牛的顆粒細胞增殖。但這四種旁分泌因子是否能夠作用于綿羊卵母細胞,并提高卵母細胞體外成熟率、受精率和早期胚胎發(fā)育率,尚未做過充分研究。因此,我們利用綿羊卵母細胞作為實驗材料,研究CTGF、SDF-1、NGF和HGF單獨添加和組合添加對綿羊卵母細胞成熟和早期胚胎發(fā)育的作用。我們在綿羊卵母細胞成熟液中分別單獨添加不同濃度梯度的CTGF、SDF-1、NGF和HGF因子,統(tǒng)計綿羊卵母細胞成熟率,找到單獨添加CTGF、SDF-1、NGF和HGF因子的最佳濃度。接著將這四種因子組合添加到綿羊卵母細胞成熟液和早期胚胎發(fā)育液中,統(tǒng)計綿羊卵母細胞成熟率、卵子激活率和早期胚胎發(fā)育率。以卵丘擴散指數(shù)(cumulus expansion index,CEI)、細胞膜表面早期細胞凋亡信號和胚胎的囊胚細胞數(shù)作為評價卵母細胞成熟和早期胚胎發(fā)育潛能的指標,并且檢測了凋亡基因Bax、Bcl-2、P53和PTGS2的基因表達量。實驗結(jié)果發(fā)現(xiàn):(1)在四種旁分泌因子單獨使用的實驗中,添加30.0 ng/mL CTGF 因子,10.0ng/mLSDF-1 因子,3.0ng/mLNGF 因子,100.0ng/mLHGF因子能促進綿羊卵母細胞成熟和早期胚胎發(fā)育;(2)在四種旁分泌因子組合使用的實驗中,添加30.0 ng/mLCTGF + 10.0 ng/mL SDF-1+3.0 ng/mLNGF+100.0ng/mL.HGF 時,卵母細胞成熟率、激活率以及早期胚胎各個時期的發(fā)育率都優(yōu)于單獨添加單一因子組和不添加因子組;(3)組合因子對CEI、抑制卵子凋亡率和提高囊胚細胞數(shù)有顯著作用;(4)組合因子的使用能降低卵丘細胞、卵母細胞和囊胚細胞中凋亡基因的表達量。實驗結(jié)果表明,旁分泌因子CTGF、SDF-1、NGF和HGF能促進卵母細胞成熟;四種因子組合使用能促進綿羊卵母細胞成熟和早期胚胎的發(fā)育。
[Abstract]:Sheep are very important economic livestock, providing people with meat, milk, fur and other products. Using in Vitro maturation in vitro maturation of oocytes, in Vito fertilization IVF) and embryo transfer transfer et) technology to improve the breeding of high quality domestic animals. The development and maturation of oocytes are the basis of these techniques. The quality of oocyte maturation directly affects the survival of offspring and the health status of surviving offspring. During the process from oocyte maturation to early embryonic development, the expression and regulation of endogenous genes are involved in the process of oocyte maturation. Signaling molecules in the culture environment also affect their normal development. Regulation through paracrine factors is the most common regulation. One of the most direct ways is that connective tissue growth factor-CTGFs, stromal cell-derived factor-1, nerve growth factor (NGF) and hepatocyte growth factor (HGFs) are the paracrine factors of ovary. SDF-1 can increase granulosa cell survival and improve embryo quality. NGF can enhance the growth of goat primary follicles in vitro in a dose-dependent manner. HGF can induce the proliferation of bovine granulosa cells. But whether these four paracrine factors can promote the proliferation of bovine granulosa cells? Can act on ovine oocytes, The rate of oocyte maturation in vitro, fertilization rate and early embryo development rate have not been fully studied. Therefore, we use sheep oocytes as experimental materials. To study the effects of HGF and CTGFF-1NGF on ovine oocyte maturation and early embryo development, we added CTGF- SDF-1NGF and HGF factor in ovine oocyte maturation fluid separately. The maturation rate of ovine oocytes was calculated, and the optimum concentration of HGF factor and SDF-1NGF were found. Then the four factors were added to ovine oocyte maturation fluid and early embryonic development fluid to calculate the ovine oocyte maturation rate. Oocyte activation rate and early embryo development rate. The cumulus expansion index CEI, early cell apoptosis signal on cell membrane surface and blastocyst number of embryos were used as indicators to evaluate oocyte maturation and early embryo development potential. The expression of apoptotic gene Bax-Bcl-2P53 and PTGS2 was detected. The results showed that the expression of Bax-Bcl-2P53 and PTGS2 was detected in four paracrine factors. Adding 30.0 ng/mL / mLSDF-1 factor and 3.0 ng / mLNGF factor 100.0ng / mLHGF can promote ovine oocyte maturation and early embryonic development. In the experiment of four paracrine factors, the oocyte maturation rate was increased by adding 30.0 ng/mLCTGF 10.0 ng/mL SDF-1 3.0 ng/mLNGF 100.0ng/mL.HGF. The activation rate and the development rate of each stage of early embryo were better than those of single factor group and no factor group. The combination factor had significant effect on CEI, inhibiting egg apoptosis rate and increasing blastocyst cell number. Use can reduce the number of cumulus cells, The expression of apoptotic genes in oocytes and blastocysts. The results showed that the paracrine factor CTGFF- SDF-1NGF and HGF could promote oocyte maturation, and the combination of four factors could promote ovine oocyte maturation and early embryo development.
【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S826
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本文編號：1677659