發(fā)布時(shí)間：2018-02-27 20:53

  本文關(guān)鍵詞： 雞 卵巢顆粒細(xì)胞 AMH 基因敲除 過(guò)表達(dá)　出處：《西北農(nóng)林科技大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：抗繆勒氏管激素(anti-Müllerian hormone,AMH)作為雌性動(dòng)物體內(nèi)目前已知的唯一抑制始基卵泡生長(zhǎng)的細(xì)胞因子,能夠抑制芳香化酶活性、減少原始卵泡初始募集、降低卵泡FSH敏感性進(jìn)而抑制卵巢功能。但禽類(lèi)AMH的研究較少。本研究主要通過(guò)對(duì)雞原代卵巢顆粒細(xì)胞中AMH基因進(jìn)行靶向敲除和過(guò)表達(dá),改變顆粒細(xì)胞中AMH及相關(guān)基因的表達(dá)水平,進(jìn)而分析雞卵巢顆粒細(xì)胞AMH在卵泡發(fā)育中的功能。結(jié)果如下:1.構(gòu)建靶向雞AMH基因敲除表達(dá)載體pll3.7-U6-SgAMH-spCas9和與之對(duì)應(yīng)的SSA-RPG雙熒光篩選報(bào)告載體pB-CMV-DsRed-CAG-AMH.200bp repeat.Puro-T2A-GFP,共轉(zhuǎn)染雞DF-1細(xì)胞系,報(bào)告載體水平敲除陽(yáng)性細(xì)胞約占轉(zhuǎn)染細(xì)胞的5%。T7E1酶切結(jié)果表明經(jīng)Puromycin篩選可以明顯富集陽(yáng)性細(xì)胞,TA克隆測(cè)序表明篩選富集后DF-1中靶位點(diǎn)突變效率高達(dá)50%。2.雞卵巢顆粒細(xì)胞體外培養(yǎng)容易自發(fā)凋亡,喪失特征性表型,同時(shí)失去FSH處理效應(yīng)。通過(guò)改進(jìn)分離方法、選擇基礎(chǔ)培養(yǎng)基、優(yōu)化血清濃度,添加適宜濃度的激素、生長(zhǎng)因子、維生素以及預(yù)鋪鼠尾膠原,對(duì)培養(yǎng)條件進(jìn)行優(yōu)化。最終建立了在預(yù)鋪鼠尾膠原作為胞外基質(zhì),采用含10%FBS、1×非必需氨基酸、1×復(fù)合維生素溶液和1×Anti-anti的DMEM培養(yǎng)液的優(yōu)化培養(yǎng)體系,培養(yǎng)雞小黃卵泡GCs 4代后仍能夠保持其細(xì)胞特性。3.為了研究AMH基因在雞卵巢顆粒細(xì)胞中的功能,將構(gòu)建的敲除表達(dá)載體和SSA-RPG篩選報(bào)告載體通過(guò)Lipofectamine?3000共轉(zhuǎn)至雞SYF卵巢顆粒細(xì)胞中,48h后報(bào)告載體水平轉(zhuǎn)染效率約有30%,敲除效率約占轉(zhuǎn)染細(xì)胞的5%;經(jīng)0.5μg/ml的Puromycin篩選進(jìn)行富集后提取細(xì)胞基因組,TA克隆測(cè)序結(jié)果顯示突變效率約為2%。構(gòu)建過(guò)表達(dá)載體pcDNA3.1-AMH與敲除載體分別轉(zhuǎn)染雞GCs,轉(zhuǎn)染24 h后添加2.5units/ml FSH處理細(xì)胞48 h,分析相關(guān)基因表達(dá)變化后發(fā)現(xiàn):在雞卵巢顆粒細(xì)胞中,AMH能夠明顯抑制目標(biāo)基因Cyp19a1的表達(dá),而此過(guò)程中Smad5和ALK2轉(zhuǎn)錄水平并沒(méi)有明顯改變。綜上,通過(guò)相應(yīng)載體敲除和過(guò)表達(dá)AMH基因能夠明顯改變雞SYF的卵巢顆粒細(xì)胞中目標(biāo)基因Cyp19a1的表達(dá),而Smad5和ALK2轉(zhuǎn)錄水平卻并沒(méi)有發(fā)生明顯變化。本研究結(jié)果有助于揭示AMH基因在雞卵巢顆粒細(xì)胞中的作用機(jī)制,有望進(jìn)一步了解家禽卵泡發(fā)育和卵泡選擇的分子機(jī)理,其結(jié)果可為蛋禽育種提供實(shí)驗(yàn)依據(jù)與理論支撐。
[Abstract]:As the only known cytokine to inhibit the growth of primordial follicles in female animals, anti-M 眉 llerian hormone AMH can inhibit aromatase activity and reduce initial recruitment of primordial follicles. However, there are few studies on AMH in poultry. In this study, the target knockout and overexpression of AMH gene in chicken primary ovarian granulosa cells were studied. To change the expression level of AMH and related genes in granulosa cells, The function of AMH in follicular development of chicken ovarian granulosa cells was analyzed. The results are as follows: 1. Construct the target chicken AMH knockout expression vector pll3.7-U6-SgAMH-spCas9 and corresponding SSA-RPG double fluorescent screening report vector pB-CMV-DsRed-CAG-AMH.200bp repeat. Puro-T2A-GFP. then co-transfect the chicken DF-1 cell line. The result of enzyme digestion of 5. T7E1 showed that the positive cells could be significantly enriched by Puromycin screening. The result of sequencing showed that the efficiency of target mutation in DF-1 was as high as 50%. 2. Chicken ovary granules were fine. Cell culture in vitro is prone to spontaneous apoptosis. By improving the separation method, selecting the base medium, optimizing the serum concentration, adding the appropriate concentration of hormones, growth factors, vitamins and precoated rat tail collagen, we lost the characteristic phenotype and the effect of FSH treatment. The culture conditions were optimized. Finally, the optimal culture system was established in which rat tail collagen was precoated as extracellular matrix, and DMEM medium containing 10s 1 脳 non-essential amino acid and 1 脳 Anti-anti was used to optimize the culture system. In order to study the function of AMH gene in chicken ovarian granulosa cells, the constructed knockout expression vector and SSA-RPG screening report vector were passed through Lipofectamine. The transfection efficiency of the report vector was about 30% and the knockout efficiency was about 5% after transfection into chicken SYF ovary granulosa cells for 48 h, and the genomic TA clone sequencing showed mutation effect after enrichment of 0.5 渭 g / ml Puromycin. The rate of expression was about 2.The overexpression vector pcDNA3.1-AMH and knockout vector were constructed and transfected into chicken GCsrespectively. After 24 h transfection, the cells were treated with 2.5units-1 FSH for 48 h. The expression of related genes was analyzed. The results showed that the expression of target gene Cyp19a1 was significantly inhibited in chicken ovarian granulosa cells. However, the transcriptional level of Smad5 and ALK2 did not change significantly. In conclusion, the expression of the target gene Cyp19a1 in the ovarian granulosa cells of chicken SYF could be significantly changed by knockout and overexpression of AMH gene in the corresponding vector. However, the transcriptional levels of Smad5 and ALK2 have not changed significantly. This study is helpful to reveal the mechanism of AMH gene acting in chicken ovarian granulosa cells and to further understand the molecular mechanism of follicular development and follicular selection in poultry. The results can provide experimental basis and theoretical support for egg poultry breeding.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S831

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 陳悅?cè)?黃荷鳳;;顆粒細(xì)胞增殖分化中卵泡刺激素的cAMP-PKA通路[J];國(guó)際生殖健康/計(jì)劃生育雜志;2011年01期

2 于洪川,張書(shū)起,曹麗蓉,張振漢;禽類(lèi)卵泡生長(zhǎng)發(fā)育和閉鎖的調(diào)節(jié)機(jī)制[J];寧夏農(nóng)學(xué)院學(xué)報(bào);1999年01期

相關(guān)博士學(xué)位論文 前1條

1 金艷梅;雞等級(jí)前卵泡顆粒細(xì)胞發(fā)育的激素和營(yíng)養(yǎng)調(diào)控及機(jī)理研究[D];浙江大學(xué);2007年

，

本文編號(hào)：1544263