發(fā)布時(shí)間：2018-04-23 21:47

  本文選題：牦牛 + 生長(zhǎng)因子��； 參考：《甘肅農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：牦牛作為青藏高原地區(qū)重要物種資源,通過(guò)輔助繁殖技術(shù)提高其繁殖性能對(duì)我國(guó)“絲綢之路經(jīng)濟(jì)帶”畜牧業(yè)的發(fā)展至關(guān)重要。為了提高體外生產(chǎn)牦牛胚胎的質(zhì)量,闡明生長(zhǎng)因子對(duì)牦牛體外胚胎發(fā)育的調(diào)控作用,及其對(duì)細(xì)胞凋亡的影響,并試圖通過(guò)生長(zhǎng)因子改善牦牛卵母細(xì)胞和胚胎冷凍技術(shù),本研究進(jìn)行了以下實(shí)驗(yàn)并取得了一定成果。(1)采用Real-time PCR和間接免疫熒光方法檢測(cè)COCs成熟及體外受精胚胎發(fā)育過(guò)程中EGF及EGFR表達(dá)動(dòng)態(tài),分析EGF對(duì)牦牛卵母細(xì)胞成熟率、胚胎發(fā)育能力、囊胚質(zhì)量的影響,并檢測(cè)細(xì)胞凋亡相關(guān)基因的表達(dá)變化。研究發(fā)現(xiàn)卵母細(xì)胞成熟和胚胎發(fā)育各個(gè)時(shí)期均可表達(dá)EGF和EGFR,且成熟卵母細(xì)胞的表達(dá)水平高于未成熟卵母細(xì)胞,囊胚的表達(dá)水平高于其他發(fā)育時(shí)期的胚胎,EGFR的表達(dá)水平高于EGF。加入EGF可以顯著提高卵母細(xì)胞的成熟率、胚胎的發(fā)育能力和囊胚質(zhì)量,最佳的作用濃度為100 ng·mL-1。EGF作用后成熟卵母細(xì)胞中促凋亡基因Bax表達(dá)顯著降低(p?0.05),而抗凋亡基因Baxi的表達(dá)水平顯著增加(p?0.05),囊胚中HSP70和Survivin的表達(dá)也可在100 ng·mL-1 EGF處理組中顯著提高。結(jié)果表明牦牛卵母細(xì)胞成熟和胚胎發(fā)育過(guò)程中EGF和EGFR作為重要的自分泌或旁分泌因子,可通過(guò)調(diào)控Bax、Baxi、HSP70和Survivin等凋亡相關(guān)基因的表達(dá)提高牦牛卵母細(xì)胞和體外受精胚胎的發(fā)育能力。(2)運(yùn)用Real-time PCR、WB和間接免疫熒光方法從基因和蛋白水平檢測(cè)COCs成熟及體外受精胚胎發(fā)育過(guò)程中IGF-1及IGF-1R表達(dá)動(dòng)態(tài),同時(shí)在牦牛卵丘細(xì)胞體外培養(yǎng)、體外胚胎發(fā)育中加入不同濃度的IGF-1,分析卵丘細(xì)胞的凋亡率及凋亡相關(guān)基因的表達(dá)變化,及其對(duì)胚胎發(fā)育能力的影響。研究發(fā)現(xiàn)卵母細(xì)胞成熟和胚胎發(fā)育各個(gè)時(shí)期均可表達(dá)IGF-1和IGF-1R,成熟卵母細(xì)胞的表達(dá)水平高于未成熟卵母細(xì)胞,囊胚的表達(dá)水平高于其他發(fā)育時(shí)期的胚胎,IGF-1R的表達(dá)水平高于IGF-1、外源的IGF-1可以顯著提高卵母細(xì)胞的成熟率、胚胎的發(fā)育能力和囊胚質(zhì)量,濃度為100 ng·mL-1時(shí),作用效果最佳。體細(xì)胞培養(yǎng)過(guò)程中IGF-1可以顯著降低細(xì)胞凋亡率(p?0.05),在IGF-1濃度為100 ng·mL-1處理組中卵丘細(xì)胞的凋亡率最低,且Bax的表達(dá)水平最低,HSP70和Bcl-2的表達(dá)水平最高。研究結(jié)果表明,igf-1可調(diào)節(jié)卵丘細(xì)胞的hsp70、bax和bcl-2表達(dá),降低細(xì)胞凋亡率,且igf-1在牦牛移植前胚胎發(fā)育過(guò)程中作為重要的生長(zhǎng)因子,可以提高囊胚的形成和細(xì)胞增殖。(3)采集6月齡和24月齡牦牛睪丸組織,real-timepcr、wb和ihc方法從基因和蛋白水平分析牦牛睪丸發(fā)育過(guò)程中egf和egfr的表達(dá)變化;在牦牛凍精體外獲能過(guò)程中加入不同濃度的igf-1,分析獲能后精子的活性、評(píng)估不同處理組受精后胚胎的卵裂率,并采用real-timepcr、wb和間接免疫熒光方法從基因和蛋白水平檢測(cè)igf-1對(duì)牦牛精子bax和bcl-2表達(dá)的變化。研究發(fā)現(xiàn)24月齡牦牛睪丸組織中egf和egfr的表達(dá)水平顯著高于6月齡睪丸組織(p?0.05),egfr的水平高于egf,24月齡睪丸組織中表達(dá)更廣,睪丸間質(zhì)細(xì)胞、曲精小管肌上皮細(xì)胞、睪丸支持細(xì)胞、生殖細(xì)胞和精子均可表達(dá)egf和egfr。100ng·ml-1igf-1可以顯著提高牦牛的精子活性(p?0.05),該處理組中受精后胚胎卵裂率也顯著提高(p?0.05),而精子中bax基因的表達(dá)顯著降低,bcl-2表達(dá)增加。綜上表明生長(zhǎng)因子可調(diào)控牦牛精子的生成和活性,從而促進(jìn)后續(xù)胚胎的發(fā)育,且這種調(diào)控作用與細(xì)胞凋亡有關(guān)。(4)牦牛卵母細(xì)胞體外成熟過(guò)程中加入不同濃度的bmp6,分析卵母細(xì)胞成熟率、用成熟的卵母細(xì)胞生產(chǎn)克隆胚胎,并評(píng)估其發(fā)育能力,采用real-timepcr和間接免疫熒光方法分析2細(xì)胞胚胎和囊胚中h3k9ac,h3k18ac,和h3k9me3表達(dá)的變化,同時(shí)分析囊胚bax和bcl-2的表達(dá)。研究發(fā)現(xiàn)100ng·ml-1bmp6可以顯著提高卵母細(xì)胞成熟率、克隆胚胎卵裂率和囊胚形成率(p?0.05),囊胚質(zhì)量也有所提高。在bmp6處理組中,h3k9ac、h3k18ac、bcl-2基因表達(dá)增加、而bax、h3k9me3基因表達(dá)降低,h3k9ac,h3k18ac,和h3k9me3在2細(xì)胞克隆胚胎的表達(dá)差異更顯著(p?0.05)。研究結(jié)果表明bmp6可以提高牦牛卵母細(xì)胞和后續(xù)克隆胚胎的發(fā)育潛能,其作用類似于其他osfs,且這種調(diào)節(jié)作用與其調(diào)控組蛋白修飾和細(xì)胞凋亡有關(guān)。(5)收集牦牛cocs,采用ct和ssv方法對(duì)其進(jìn)行玻璃化冷凍,冷凍-解凍后分析不同處理組卵母細(xì)胞的成熟率和胚胎發(fā)育能力,采用real-timepcr、wb從基因和蛋白水平檢測(cè)成熟卵母細(xì)胞、卵裂胚胎、和囊胚中hsp90的表達(dá)變化;同時(shí)在牦牛卵母細(xì)胞體外成熟過(guò)程中加入不同濃度的igf-1,分析卵母細(xì)胞的成熟率和CIRP的表達(dá)水平,將不同處理組成熟的卵母細(xì)胞采用CT法玻璃化冷凍,冷凍解凍后進(jìn)行孤雌激活,分析胚胎的發(fā)育能力。研究發(fā)現(xiàn)CT和SSV冷凍組卵母細(xì)胞成熟率、卵裂率和囊胚率比較接近、其中成熟率和卵裂率顯著低于對(duì)照組(p?0.05),HSP90的表達(dá)水平也低于對(duì)照組,而三組囊胚率和HSP90的表達(dá)水平比較接近。100 ng·mL-1 IGF-1可以提高成熟卵母細(xì)胞CIRP表達(dá)和卵母細(xì)胞的成熟率,冷凍-解凍后孤雌激活胚胎的卵裂率和囊胚率有所提高,但囊胚質(zhì)量差異不顯著。結(jié)果表明CT和SSV玻璃化冷凍方法對(duì)牦牛未成熟卵母細(xì)胞具有相同的冷凍效果,且其對(duì)發(fā)育能力的影響主要集中在成熟期到早期卵裂期,并與HSP90的表達(dá)水平有一定關(guān)聯(lián)性,而卵母細(xì)胞成熟過(guò)程中IGF-1可以調(diào)控CIRP的表達(dá),提高成熟卵母細(xì)胞對(duì)低溫的適應(yīng)能力,降低低溫對(duì)卵母細(xì)胞損傷,從而提高后續(xù)發(fā)育能力。
[Abstract]:As an important species resource in the Qinghai Tibet Plateau, yak is very important to the development of the animal husbandry of the "Silk Road Economic Belt" in China through assisted reproduction technology. In order to improve the quality of the yak embryo in the production of yak in vitro, the regulation effect of growth factor on the development of yak in vitro embryo and its effect on the cell apoptosis are clarified. In order to improve yak oocyte and embryo cryopreservation through growth factors, the following experiments have been carried out and some results have been achieved. (1) Real-time PCR and indirect immunofluorescence were used to detect the expression of EGF and EGFR during the development of COCs and in vitro fertilization embryos. The maturation rate of yak oocytes and embryos were analyzed by EGF. The effects of developmental ability, blastocyst quality and the expression of apoptosis related genes were detected. The study found that EGF and EGFR could be expressed at all stages of oocyte maturation and embryo development, and the expression level of mature oocytes was higher than that of immature oocytes. The expression of blastocyst was higher than that of other developmental embryos, and EGFR expressed water. The oocyte maturation rate, embryo development ability and blastocyst quality were significantly higher than that of EGF., and the best action concentration was 100 ng. ML-1.EGF, the expression of Bax was significantly reduced (P? 0.05) in mature oocytes (P? 0.05), while the expression level of anti apoptotic gene Baxi increased significantly (P? 0.05), HSP70 and Survivi in blastocysts. The expression of N can also be significantly improved in the 100 ng. ML-1 EGF treatment group. The results show that EGF and EGFR are important autocrine or paracrine factors during the maturation and embryonic development of yak oocytes, which can improve the development of yak oocytes and in vitro fertilization embryos by regulating the expression of apoptosis related genes such as Bax, Baxi, HSP70 and Survivin. (2) Real-time PCR, WB and indirect immunofluorescence were used to detect the expression of IGF-1 and IGF-1R during the development of COCs and IVF embryos from gene and protein levels. At the same time, the culture of yak cumulus cells in vitro, and in vitro embryo development were added to different concentrations of IGF-1, and analyzed the apoptosis rate and the apoptosis related groups of the cumulus cells. The expression of IGF-1 and IGF-1R can be expressed at all stages of oocyte maturation and embryo development. The expression level of mature oocytes is higher than that of immature oocytes. The expression level of blastocyst is higher than that of other developmental embryos. The expression level of IGF-1R is higher than that of IGF-1, and the exogenous I is higher than that of the other developmental stages. GF-1 can significantly increase the maturation rate of oocytes, the embryonic development ability and the blastocyst quality, when the concentration is 100 ng. ML-1, the effect is the best. IGF-1 can significantly reduce the apoptosis rate (P? 0.05) in the process of somatic cell culture. In the IGF-1 concentration 100 ng. ML-1 treatment group, the apoptotic rate of the cumulus cells is the lowest, and the expression of Bax is the lowest. The expression level of HSP70 and Bcl-2 is the highest. The results show that IGF-1 can regulate the expression of HSP70, Bax and Bcl-2 in cumulus cells and reduce the rate of apoptosis. And IGF-1 can be used as an important growth factor in the development of yak embryo development, which can improve the formation and proliferation of blastocysts. (3) collect 6 month old and 24 month old yak testis tissue, real-t Imepcr, WB and IHC methods were used to analyze the expression of EGF and EGFR during the development of yak testis from gene and protein levels. In the process of obtaining energy in Yak frozen sperm, the activity of different concentrations of IGF-1 was added to analyze the sperm activity after the acquired energy, and the cleavage rate of different treatment groups after fertilization was evaluated, and the real-timepcr, WB and indirect immunofluorescence methods were used. The changes in the expression of Bax and Bcl-2 of yak sperm by IGF-1 were detected from gene and protein level. The expression level of EGF and EGFR in 24 month old yak testis was significantly higher than that of 6 month old testicular tissue (P? 0.05). The level of EGFR was higher than that of EGF, 24 month old in the testicular tissue, and in the Leydig cells, in the seminiferous tubules, and in the testicular branches. The expression of EGF and egfr.100ng / ml-1igf-1 could significantly increase the sperm activity of Yak (P? 0.05). The cleavage rate of the embryos after fertilization was significantly increased (P? 0.05) in the treatment group (P? 0.05), and the expression of Bax gene in the sperm was significantly reduced and the expression of Bcl-2 increased. The results showed that growth factor could regulate the production of yak sperm and the growth factor. Activity, thus promoting the development of subsequent embryos, and this regulatory role is related to cell apoptosis. (4) yak oocyte in vitro maturation in the process of adding different concentrations of BMP6, analysis of oocyte maturation rate, the use of mature oocytes to produce cloned embryos, and evaluate their development ability, using real-timepcr and indirect immunofluorescence method analysis. The changes in the expression of H3K9Ac, H3K18ac, and h3k9me3 in the 2 cell embryos and blastocysts, and the analysis of the expression of Bax and Bcl-2 in the blastocyst. The study found that 100ng ml-1bmp6 can significantly increase the oocyte maturation rate, clone embryo cleavage rate and blastocyst formation rate (P? 0.05), and the blastocyst quality also increased. In the BMP6 treatment group, H3K9Ac, H3K18ac, bcl-2 gene table The expression of Bax, h3k9me3, H3K9Ac, H3K18ac, and h3k9me3 in the 2 cell cloned embryos was more significant (P? 0.05). The results showed that BMP6 could improve the developmental potential of yak oocytes and subsequent cloned embryos. The effect was similar to that of other OSFs, and this regulation was related to the regulation of histone modification and cells. Apoptosis related. (5) collection of yak COCs, using CT and SSV methods to vitrification, freezing and thawing to analyze the maturation rate and embryonic development ability of different treatment group oocytes, real-timepcr, WB from gene and protein level detection of mature oocyte, cleavage embryo, and blastocyst of Hsp90 expression changes; at the same time in Yak eggs The mature rate of oocyte and the expression level of CIRP were analyzed by adding different concentrations of IGF-1 in the process of maternal cell maturation in vitro. The mature oocytes of different treatment groups were vitrified by CT method, parthenogenetic activation was carried out after freezing thawing, and the developmental ability of embryos was analyzed. The maturation rate and cleavage rate of the oocyte in CT and SSV cryopreservation group were studied. Compared with the blastocyst rate, the maturation rate and cleavage rate were significantly lower than that of the control group (P? 0.05), and the expression level of HSP90 was also lower than that of the control group. The three blastocyst rate and the expression level of HSP90 were close to.100 ng. ML-1 IGF-1, which could improve the CIRP expression and oocyte maturation rate of the mature oocyte, and the parthenogenetic activation of the embryos after freezing thawing. The cleavage rate and blastocyst rate increased, but the difference of blastocyst quality was not significant. The results showed that the CT and SSV vitrification methods had the same freezing effect on Yak immature oocytes, and the effects on the developmental ability mainly concentrated in the mature stage to the early cleavage stage, and had some correlation with the expression level of HSP90, and the oocyte was oocyte. In the process of maturation, IGF-1 can regulate the expression of CIRP, improve the adaptability of mature oocyte to low temperature, reduce the damage to oocyte at low temperature, and thus improve the following developmental ability.

【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S823.85
，

本文編號(hào)：1793764