山羊卵巢中BMP15通过p38 MAPK通路调控AMH的表达
发布时间：2018-12-25 08:24
【摘要】：个体AMH的水平与其卵巢储备能力直接相关,AMH水平越高的个体,卵巢储备能力越强。现已证实,AMH水平可以作为评价一些物种卵巢储备能力的标准。关于AMH的表达调控关系,国内外研究已证实,BMPs家族可以通过SMAD通路对AMH的表达起调控作用,且已有文献报道,BMP15可以上调人和绵羊卵巢中AMH的表达。但关于BMP15调控AMH的具体机制尚不清楚。为了研究BMP15调控AMH表达的机制,本实验以性成熟期的重庆本地山羊为采样对象,采集其两侧卵巢,收集颗粒细胞进行培养。先用不同浓度的外源人重组BMP15蛋白对颗粒细胞进行处理,通过测定处理后颗粒细胞中AMH的表达量变化和p38 MAPK通路的磷酸化情况,确定了10 ng/mL的BMP15对AMH的表达具有上调作用,且对p38 MAPK的磷酸化具有激活作用。在此基础上,采用p38 MAPK通路的抑制剂SB203580和p38 MAPK基因的干扰片段与10 ng/m L的BMP15同时作用于颗粒细胞,测定颗粒细胞中AMH和AMH转录相关因子SOX9的基因和蛋白表达量,根据各因子表达量的变化情况确定p38 MAPK通路在该过程所起的作用。研究结果如下:1、BMP15蛋白可以上调山羊卵巢的颗粒细胞中AMH的表达,且浓度越大,其对AMH的上调作用越强;同时,浓度为10 ng/m L的BMP15的刺激可以激活p38 MAPK通路的磷酸化。2、抑制剂浓度在25μM以下时,不会对颗粒细胞的活性产生影响,且当抑制剂浓度为20μM时对通路的抑制效果最佳。同时加入BMP15和p38 MAPK通路抑制剂后,p38 MAPK通路被有效抑制,此时从基因和蛋白水平检测AMH和SOX9的表达量相对于只加入BMP15的实验组都显著降低(P0.05),说明p38 MAPK通路的阻断影响了BMP15对AMH和SOX9的上调作用。3、采用干扰片段对p38 MAPK基因的沉默显著降低了p38 MAPK基因的表达量,先对p38 MAPK基因沉默后再加入BMP15蛋白,相对只加入BMP15蛋白的实验组,AMH和SOX9的表达也在基因和蛋白水平显著降低了(P0.05),进一步证明了p38 MAPK通路在该过程中起着重要作用。综合以上结果得出结论,浓度为10 ng/m L的BMP15可以上调AMH的表达,这一作用主要通过激活p38 MAPK通路实现,SOX9参与了该过程。本研究为AMH的相关研究提供了基础数据,也为明确AMH的调控的相关分子机理打下了坚实的基础。
[Abstract]:The level of AMH is directly related to the ability of ovarian reserve. The higher the level of AMH, the stronger the ability of ovarian reserve. It has been confirmed that AMH level can be used as a criterion for evaluating the ovarian reserve capacity of some species. Concerning the expression and regulation of AMH, domestic and foreign studies have confirmed that BMPs family can regulate the expression of AMH through the SMAD pathway, and it has been reported that BMP15 can up-regulate the expression of AMH in human and sheep ovaries. However, the specific mechanism of BMP15 regulating AMH is not clear. In order to study the mechanism of BMP15 regulating AMH expression, Chongqing local goats in sexual maturity were sampled, their bilateral ovaries were collected and granulosa cells were collected for culture. Granulosa cells were treated with different concentrations of recombinant human BMP15 protein. The expression of AMH and phosphorylation of p38 MAPK pathway in granulosa cells were measured after treatment. It was determined that 10 ng/mL BMP15 could up-regulate the expression of AMH. The phosphorylation of p38 MAPK was activated. On this basis, the interference fragment of SB203580 and p38 MAPK gene, which is the inhibitor of p38 MAPK pathway, was used to act on granulosa cells simultaneously with 10 ng/m L BMP15. The expression of AMH and AMH transcription related factor SOX9 in granulosa cells was measured. The role of p38 MAPK pathway in this process was determined according to the changes in the expression of various factors. The results are as follows: 1BMP15 protein can up-regulate the expression of AMH in granulosa cells of goat ovary, and the higher the concentration, the stronger the up-regulation of AMH; At the same time, the stimulation of 10 ng/m L BMP15 could activate the phosphorylation of p38 MAPK pathway. When the inhibitor concentration was below 25 渭 M, the activity of granulosa cells was not affected. When the concentration of the inhibitor was 20 渭 M, the inhibitory effect on the pathway was the best. After the addition of BMP15 and p38 MAPK pathway inhibitors, the p38 MAPK pathway was effectively inhibited. At this time, the expression of AMH and SOX9 in the gene and protein levels was significantly lower than that in the experimental group which only added BMP15 (P0.05). It was suggested that the blocking of p38 MAPK pathway affected the upregulation of AMH and SOX9 by BMP15. (3) silencing p38 MAPK gene by interference fragment significantly reduced the expression of p38 MAPK gene, and then added BMP15 protein after silencing p38 MAPK gene. Compared with the experimental group which only added BMP15 protein, the expression of AMH and SOX9 also decreased significantly (P0.05), which further proved that p38 MAPK pathway plays an important role in this process. It is concluded that 10 ng/m L BMP15 can up-regulate the expression of AMH by activating p38 MAPK pathway, and SOX9 is involved in this process. This study provided the basic data for the study of AMH and laid a solid foundation for clarifying the molecular mechanism of the regulation of AMH.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S827
本文编号：2390927
[Abstract]:The level of AMH is directly related to the ability of ovarian reserve. The higher the level of AMH, the stronger the ability of ovarian reserve. It has been confirmed that AMH level can be used as a criterion for evaluating the ovarian reserve capacity of some species. Concerning the expression and regulation of AMH, domestic and foreign studies have confirmed that BMPs family can regulate the expression of AMH through the SMAD pathway, and it has been reported that BMP15 can up-regulate the expression of AMH in human and sheep ovaries. However, the specific mechanism of BMP15 regulating AMH is not clear. In order to study the mechanism of BMP15 regulating AMH expression, Chongqing local goats in sexual maturity were sampled, their bilateral ovaries were collected and granulosa cells were collected for culture. Granulosa cells were treated with different concentrations of recombinant human BMP15 protein. The expression of AMH and phosphorylation of p38 MAPK pathway in granulosa cells were measured after treatment. It was determined that 10 ng/mL BMP15 could up-regulate the expression of AMH. The phosphorylation of p38 MAPK was activated. On this basis, the interference fragment of SB203580 and p38 MAPK gene, which is the inhibitor of p38 MAPK pathway, was used to act on granulosa cells simultaneously with 10 ng/m L BMP15. The expression of AMH and AMH transcription related factor SOX9 in granulosa cells was measured. The role of p38 MAPK pathway in this process was determined according to the changes in the expression of various factors. The results are as follows: 1BMP15 protein can up-regulate the expression of AMH in granulosa cells of goat ovary, and the higher the concentration, the stronger the up-regulation of AMH; At the same time, the stimulation of 10 ng/m L BMP15 could activate the phosphorylation of p38 MAPK pathway. When the inhibitor concentration was below 25 渭 M, the activity of granulosa cells was not affected. When the concentration of the inhibitor was 20 渭 M, the inhibitory effect on the pathway was the best. After the addition of BMP15 and p38 MAPK pathway inhibitors, the p38 MAPK pathway was effectively inhibited. At this time, the expression of AMH and SOX9 in the gene and protein levels was significantly lower than that in the experimental group which only added BMP15 (P0.05). It was suggested that the blocking of p38 MAPK pathway affected the upregulation of AMH and SOX9 by BMP15. (3) silencing p38 MAPK gene by interference fragment significantly reduced the expression of p38 MAPK gene, and then added BMP15 protein after silencing p38 MAPK gene. Compared with the experimental group which only added BMP15 protein, the expression of AMH and SOX9 also decreased significantly (P0.05), which further proved that p38 MAPK pathway plays an important role in this process. It is concluded that 10 ng/m L BMP15 can up-regulate the expression of AMH by activating p38 MAPK pathway, and SOX9 is involved in this process. This study provided the basic data for the study of AMH and laid a solid foundation for clarifying the molecular mechanism of the regulation of AMH.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S827
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