【摘要】：個(gè)體AMH的水平與其卵巢儲(chǔ)備能力直接相關(guān),AMH水平越高的個(gè)體,卵巢儲(chǔ)備能力越強(qiáng)。現(xiàn)已證實(shí),AMH水平可以作為評(píng)價(jià)一些物種卵巢儲(chǔ)備能力的標(biāo)準(zhǔn)。關(guān)于AMH的表達(dá)調(diào)控關(guān)系,國(guó)內(nèi)外研究已證實(shí),BMPs家族可以通過(guò)SMAD通路對(duì)AMH的表達(dá)起調(diào)控作用,且已有文獻(xiàn)報(bào)道,BMP15可以上調(diào)人和綿羊卵巢中AMH的表達(dá)。但關(guān)于BMP15調(diào)控AMH的具體機(jī)制尚不清楚。為了研究BMP15調(diào)控AMH表達(dá)的機(jī)制,本實(shí)驗(yàn)以性成熟期的重慶本地山羊?yàn)椴蓸訉?duì)象,采集其兩側(cè)卵巢,收集顆粒細(xì)胞進(jìn)行培養(yǎng)。先用不同濃度的外源人重組BMP15蛋白對(duì)顆粒細(xì)胞進(jìn)行處理,通過(guò)測(cè)定處理后顆粒細(xì)胞中AMH的表達(dá)量變化和p38 MAPK通路的磷酸化情況,確定了10 ng/mL的BMP15對(duì)AMH的表達(dá)具有上調(diào)作用,且對(duì)p38 MAPK的磷酸化具有激活作用。在此基礎(chǔ)上,采用p38 MAPK通路的抑制劑SB203580和p38 MAPK基因的干擾片段與10 ng/m L的BMP15同時(shí)作用于顆粒細(xì)胞,測(cè)定顆粒細(xì)胞中AMH和AMH轉(zhuǎn)錄相關(guān)因子SOX9的基因和蛋白表達(dá)量,根據(jù)各因子表達(dá)量的變化情況確定p38 MAPK通路在該過(guò)程所起的作用。研究結(jié)果如下:1、BMP15蛋白可以上調(diào)山羊卵巢的顆粒細(xì)胞中AMH的表達(dá),且濃度越大,其對(duì)AMH的上調(diào)作用越強(qiáng);同時(shí),濃度為10 ng/m L的BMP15的刺激可以激活p38 MAPK通路的磷酸化。2、抑制劑濃度在25μM以下時(shí),不會(huì)對(duì)顆粒細(xì)胞的活性產(chǎn)生影響,且當(dāng)抑制劑濃度為20μM時(shí)對(duì)通路的抑制效果最佳。同時(shí)加入BMP15和p38 MAPK通路抑制劑后,p38 MAPK通路被有效抑制,此時(shí)從基因和蛋白水平檢測(cè)AMH和SOX9的表達(dá)量相對(duì)于只加入BMP15的實(shí)驗(yàn)組都顯著降低(P0.05),說(shuō)明p38 MAPK通路的阻斷影響了BMP15對(duì)AMH和SOX9的上調(diào)作用。3、采用干擾片段對(duì)p38 MAPK基因的沉默顯著降低了p38 MAPK基因的表達(dá)量,先對(duì)p38 MAPK基因沉默后再加入BMP15蛋白,相對(duì)只加入BMP15蛋白的實(shí)驗(yàn)組,AMH和SOX9的表達(dá)也在基因和蛋白水平顯著降低了(P0.05),進(jìn)一步證明了p38 MAPK通路在該過(guò)程中起著重要作用。綜合以上結(jié)果得出結(jié)論,濃度為10 ng/m L的BMP15可以上調(diào)AMH的表達(dá),這一作用主要通過(guò)激活p38 MAPK通路實(shí)現(xiàn),SOX9參與了該過(guò)程。本研究為AMH的相關(guān)研究提供了基礎(chǔ)數(shù)據(jù),也為明確AMH的調(diào)控的相關(guān)分子機(jī)理打下了堅(jiān)實(shí)的基礎(chǔ)。
[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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S827

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