本文關鍵詞： 雞 卵巢顆粒細胞 AMH 基因敲除 過表達　出處：《西北農(nóng)林科技大學》2017年碩士論文　論文類型：學位論文

【摘要】：抗繆勒氏管激素(anti-Müllerian hormone,AMH)作為雌性動物體內目前已知的唯一抑制始基卵泡生長的細胞因子,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敏感性進而抑制卵巢功能。但禽類AMH的研究較少。本研究主要通過對雞原代卵巢顆粒細胞中AMH基因進行靶向敲除和過表達,改變顆粒細胞中AMH及相關基因的表達水平,進而分析雞卵巢顆粒細胞AMH在卵泡發(fā)育中的功能。結果如下:1.構建靶向雞AMH基因敲除表達載體pll3.7-U6-SgAMH-spCas9和與之對應的SSA-RPG雙熒光篩選報告載體pB-CMV-DsRed-CAG-AMH.200bp repeat.Puro-T2A-GFP,共轉染雞DF-1細胞系,報告載體水平敲除陽性細胞約占轉染細胞的5%。T7E1酶切結果表明經(jīng)Puromycin篩選可以明顯富集陽性細胞,TA克隆測序表明篩選富集后DF-1中靶位點突變效率高達50%。2.雞卵巢顆粒細胞體外培養(yǎng)容易自發(fā)凋亡,喪失特征性表型,同時失去FSH處理效應。通過改進分離方法、選擇基礎培養(yǎng)基、優(yōu)化血清濃度,添加適宜濃度的激素、生長因子、維生素以及預鋪鼠尾膠原,對培養(yǎng)條件進行優(yōu)化。最終建立了在預鋪鼠尾膠原作為胞外基質,采用含10%FBS、1×非必需氨基酸、1×復合維生素溶液和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胞特性。3.為了研究AMH基因在雞卵巢顆粒細胞中的功能,將構建的敲除表達載體和SSA-RPG篩選報告載體通過Lipofectamine?3000共轉至雞SYF卵巢顆粒細胞中,48h后報告載體水平轉染效率約有30%,敲除效率約占轉染細胞的5%;經(jīng)0.5μg/ml的Puromycin篩選進行富集后提取細胞基因組,TA克隆測序結果顯示突變效率約為2%。構建過表達載體pcDNA3.1-AMH與敲除載體分別轉染雞GCs,轉染24 h后添加2.5units/ml FSH處理細胞48 h,分析相關基因表達變化后發(fā)現(xiàn):在雞卵巢顆粒細胞中,AMH能夠明顯抑制目標基因Cyp19a1的表達,而此過程中Smad5和ALK2轉錄水平并沒有明顯改變。綜上,通過相應載體敲除和過表達AMH基因能夠明顯改變雞SYF的卵巢顆粒細胞中目標基因Cyp19a1的表達,而Smad5和ALK2轉錄水平卻并沒有發(fā)生明顯變化。本研究結果有助于揭示AMH基因在雞卵巢顆粒細胞中的作用機制,有望進一步了解家禽卵泡發(fā)育和卵泡選擇的分子機理,其結果可為蛋禽育種提供實驗依據(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.
【學位授予單位】：西北農(nóng)林科技大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S831

【參考文獻】

相關期刊論文 前2條

1 陳悅群;黃荷鳳;;顆粒細胞增殖分化中卵泡刺激素的cAMP-PKA通路[J];國際生殖健康/計劃生育雜志;2011年01期

2 于洪川,張書起,曹麗蓉,張振漢;禽類卵泡生長發(fā)育和閉鎖的調節(jié)機制[J];寧夏農(nóng)學院學報;1999年01期

相關博士學位論文 前1條

1 金艷梅;雞等級前卵泡顆粒細胞發(fā)育的激素和營養(yǎng)調控及機理研究[D];浙江大學;2007年

，

本文編號：1544263