JMJD1C对牛卵丘细胞增殖与凋亡的影响

发布时间：2018-11-14 20:55

【摘要】：卵丘细胞作为颗粒细胞的一种，其在卵母细胞成熟过程中的扩张状态以及凋亡比率对卵母细胞的成熟具有重要作用。已有研究表明，BMP15、IGF-I与FSH均可影响其增殖、生长与分化，，从而影响卵母细胞正常生长和成熟。JMJD1C是KDM3家族中的一员，其中KDM3A、KDM3B即JMJD1A、JMJD1B能够特异去除H3K9me1与H3K9me2的甲基化修饰，但JMJD1C是否也具有对H3K9的催化作用或活性，且在BMP15、IGF-I、FSH介导下对卵丘细胞增殖与凋亡的影响则罕有报道。为明确JMJD1C对H3K9的去甲基化作用，本研究在对牛卵丘细胞进行体外培养后，利用RNAi方法抑制JMJD1C的表达，并通过免疫荧光方法检测H3K9me1、H3K9me2的表达变化。结果表明：对牛卵丘细胞中JMJD1C mRNA表达成功地进行抑制后，H3K9me1、H3K9me2表达量无明显变化，即JMJD1C对去除H3K9me1与H3K9me2的甲基化修饰均并无明显作用。为检测BMP15、IGF-I和FSH对JMJD1C表达的影响，分别在培养液中添加不同浓度BMP15、IGF-I和FSH并检测JMJD1C的表达变化，实时荧光定量PCR结果表明，添加FSH和IGF-1对JMJD1C的mRNA表达水平无显著影响；而50ng/ml与100ng/ml的BMP15添加会显著降低JMJD1C的mRNA表达水平（p㩳0.01）。MTS细胞增殖检测结果也表明，BMP15的添加量为50ng/ml时，可明显促进卵丘细胞增殖，即BMP15的添加浓度为50ng/ml时显著抑制了JMJD1C的表达并促进了卵丘细胞增殖。因此进一步选取添加量为50ng/mlBMP15探究JMJD1C在BMP15介导下对卵丘细胞凋亡与增殖的影响。基于以上结果，利用siRNA敲减JMJD1C基因的表达，同时从mRNA水平和蛋白水平检测凋亡标记基因Bcl2和Bax表达量变化。实时荧光定量PCR与Western Blot结果均表明，BMP15添加后沉默JMJD1C基因，凋亡标记基因Bcl2与Bax的比值都明显升高，即卵丘细胞的凋亡率降低。表明了BMP15与JMJD1C之间的间接联系可以影响卵丘细胞的凋亡，JMJD1C可抑制卵丘细胞中BMP15介导的抗凋亡作用。综上所述，本研究明确了牛卵丘细胞中JMJD1C在mRNA水平上不具有直接调控组蛋白H3K9me1、H3K9me2的去甲基化作用，而JMJD1C可影响BMP15在牛卵丘细胞中介导的抗凋亡作用。研究结果有助于识别KDM3密切相关的蛋白质之间的差别，对JMJD1C的功能进行了新的探究并为揭示卵丘细胞凋亡的分子机制提供一定基础。
[Abstract]:As one of granulosa cells, cumulus cells play an important role in oocyte maturation. Studies have shown that both BMP15,IGF-I and FSH can affect the proliferation, growth and differentiation of oocytes, thus affecting the normal growth and maturation of oocytes. JMJD1C is a member of the KDM3 family, in which KDM3A,KDM3B is JMJD1A,. JMJD1B can specifically remove the methylation modification of H3K9me1 and H3K9me2, but whether JMJD1C also has the catalytic effect or activity on H3K9, and the effect of BMP15,IGF-I,FSH on the proliferation and apoptosis of cumulus cells is rarely reported. In order to clarify the demethylation effect of JMJD1C on H3K9, the expression of JMJD1C was inhibited by RNAi method after cultured bovine cumulus cells in vitro, and the expression of H3K9me1, H3K9me2 was detected by immunofluorescence. The results showed that after successfully inhibiting the expression of JMJD1C mRNA in bovine cumulus cells, the expression of H3K9me1 and H3K9me2 did not change significantly, that is, JMJD1C had no significant effect on the removal of methylation modification of H3K9me1 and H3K9me2. In order to detect the effect of BMP15,IGF-I and FSH on the expression of JMJD1C, different concentrations of BMP15,IGF-I and FSH were added to the culture medium and the expression of JMJD1C was detected. The addition of FSH and IGF-1 had no significant effect on the mRNA expression of JMJD1C. BMP15 addition of 50ng/ml and 100ng/ml significantly decreased the mRNA expression level of JMJD1C (pp0.01). MTS cell proliferation test also showed that the addition of BMP15 to 50ng/ml could significantly promote the proliferation of cumulus cells. The addition of BMP15 to 50ng/ml significantly inhibited the expression of JMJD1C and promoted the proliferation of cumulus cells. Therefore, 50ng/mlBMP15 was added to investigate the effect of JMJD1C on apoptosis and proliferation of cumulus cells mediated by BMP15. Based on the above results, the expression of JMJD1C gene was reduced by siRNA knockout, and the changes of Bcl2 and Bax expression levels of apoptotic marker genes Bcl2 and Bax were detected at the level of mRNA and protein at the same time. The results of real-time fluorescence quantitative PCR and Western Blot showed that the ratio of Bcl2 to Bax was significantly increased after BMP15 was added to silencing the JMJD1C gene, that is, the apoptotic rate of cumulus cells was decreased. The results indicated that the indirect connection between BMP15 and JMJD1C could affect the apoptosis of cumulus cells, and JMJD1C could inhibit the anti-apoptotic effect of BMP15 in cumulus cells. In conclusion, JMJD1C in bovine cumulus cells did not directly regulate the demethylation of histone H3K9me1H3K9me2 at the mRNA level, while JMJD1C could affect the anti-apoptosis effect of BMP15 in bovine cumulus cells. The results are helpful to recognize the differences between proteins closely related to KDM3, and to explore the function of JMJD1C and provide a basis for revealing the molecular mechanism of cumulus cell apoptosis.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823

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