鸡卵巢颗粒细胞中AMH基因功能的研究

发布时间：2018-02-27 20:53

本文关键词： 鸡卵巢颗粒细胞 AMH 基因敲除过表达　出处：《西北农林科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：抗缪勒氏管激素(anti-Müllerian hormone,AMH)作为雌性动物体内目前已知的唯一抑制始基卵泡生长的细胞因子,能够抑制芳香化酶活性、减少原始卵泡初始募集、降低卵泡FSH敏感性进而抑制卵巢功能。但禽类AMH的研究较少。本研究主要通过对鸡原代卵巢颗粒细胞中AMH基因进行靶向敲除和过表达,改变颗粒细胞中AMH及相关基因的表达水平,进而分析鸡卵巢颗粒细胞AMH在卵泡发育中的功能。结果如下:1.构建靶向鸡AMH基因敲除表达载体pll3.7-U6-SgAMH-spCas9和与之对应的SSA-RPG双荧光筛选报告载体pB-CMV-DsRed-CAG-AMH.200bp repeat.Puro-T2A-GFP,共转染鸡DF-1细胞系,报告载体水平敲除阳性细胞约占转染细胞的5%。T7E1酶切结果表明经Puromycin筛选可以明显富集阳性细胞,TA克隆测序表明筛选富集后DF-1中靶位点突变效率高达50%。2.鸡卵巢颗粒细胞体外培养容易自发凋亡,丧失特征性表型,同时失去FSH处理效应。通过改进分离方法、选择基础培养基、优化血清浓度,添加适宜浓度的激素、生长因子、维生素以及预铺鼠尾胶原,对培养条件进行优化。最终建立了在预铺鼠尾胶原作为胞外基质,采用含10%FBS、1×非必需氨基酸、1×复合维生素溶液和1×Anti-anti的DMEM培养液的优化培养体系,培养鸡小黄卵泡GCs 4代后仍能够保持其细胞特性。3.为了研究AMH基因在鸡卵巢颗粒细胞中的功能,将构建的敲除表达载体和SSA-RPG筛选报告载体通过Lipofectamine?3000共转至鸡SYF卵巢颗粒细胞中,48h后报告载体水平转染效率约有30%,敲除效率约占转染细胞的5%;经0.5μg/ml的Puromycin筛选进行富集后提取细胞基因组,TA克隆测序结果显示突变效率约为2%。构建过表达载体pcDNA3.1-AMH与敲除载体分别转染鸡GCs,转染24 h后添加2.5units/ml FSH处理细胞48 h,分析相关基因表达变化后发现:在鸡卵巢颗粒细胞中,AMH能够明显抑制目标基因Cyp19a1的表达,而此过程中Smad5和ALK2转录水平并没有明显改变。综上,通过相应载体敲除和过表达AMH基因能够明显改变鸡SYF的卵巢颗粒细胞中目标基因Cyp19a1的表达,而Smad5和ALK2转录水平却并没有发生明显变化。本研究结果有助于揭示AMH基因在鸡卵巢颗粒细胞中的作用机制,有望进一步了解家禽卵泡发育和卵泡选择的分子机理,其结果可为蛋禽育种提供实验依据与理论支撑。
[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.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S831

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