CDC25C基因在成年山羊与幼年山羊卵巢颗粒细胞中的表达及功能验证
发布时间：2019-03-27 12:37
【摘要】：卵泡颗粒细胞是卵泡的一种重要组成单位,环绕于卵母细胞周围。随着卵泡的发育,卵泡颗粒细胞会发生相应的变化,其增殖分泌的多种激素及生长因子与卵母细胞的生长和成熟有着极为重要的作用。本研究前期发现,幼龄母羊与成年母羊的卵母细胞在体外成熟过程中,成熟率有着显著性差异,并且幼龄母羊的卵母细胞经体外成熟受精后发育至四细胞的比例也显著性低于成年母羊。试验中发现,幼龄母羊与成年母羊的卵泡颗粒细胞有着很大的不同点,无论是数量,还是环绕卵母细胞的形态。另一个发现就是细胞分裂周期蛋白25同源蛋白C基因在幼龄母羊卵母细胞与成年母羊卵母细胞中的表达有显著性差异,这个基因具有调控细胞分裂周期作用,可调节真核生物细胞有丝分裂,是细胞周期进入M期的关键因子之一。综合上述两种发现,本实验决定从卵泡颗粒细胞的角度验证CDC25C基因功能,尝试解开不同年龄阶段卵母细胞成熟率差异的原因。本实验以江苏南通当地白山羊为研究对象,采集1.5年龄以上的成年母羊与2月龄羔羊的卵巢并使用0.01MPBS带回实验室。体外分离卵泡颗粒细胞经培养转染后,对成年母羊与羔羊的卵泡颗粒细胞进行了 CDC25C、CDK1、WEE1相关基因,雌激素和孕激素,转染后细胞周期等相关检测,结果如下:1.山羊CDC25C基因的克隆根据NCBI提供的CDC25C基因序列,设计引物克隆CDC25C基因的CDS区,随后构建该基因的pCMV-HA-CDC25C载体和小分子干扰载体siCDC25C。测序结果表明pCMV-HA-CDC25C载体构建正确,与此同时小分子干扰载体siCDC25C在预实验中被证实有效,皆可用于后续试验。2.原代分离细胞的鉴定依据酶联免疫原理,山羊卵泡颗粒细胞表面拥有特异性FSH受体,试验采用Rabbit Anti-FSHR多克隆抗体作为一抗,SA1074-兔IgG为二抗,经过酶联免疫检测后认定,本实验分离的细胞绝大部分都是所需要的卵泡颗粒细胞,可继续后续试验。3.CDC25C基因对卵泡颗粒细胞的影响构建pCMV-HA-CDC25C载体和小分子干扰载体siCDC25C,转染成年山羊和幼龄山羊卵泡颗粒细胞后,利用实时荧光定量PCR技术对CDC25C、CDK1、WEE1基因的相对表达量进行检测,利用酶联免疫方式检测了 PROG和E2两种激素分泌情况,利用切片技术比较了成年山羊与幼龄山羊卵巢的区别,利用细胞周期检测技术比较了两种山羊卵泡颗粒细胞转染重组载体和干扰载体后的细胞周期区别。(1)转染pCMV-HA-CDC25C载体后,成年山羊的卵泡颗粒细胞的CDC25C、CDK1、WEE1基因的相对表达量都得到了显著性提升;PROG和E2两种激素的分泌量也有所上升,且与空白组比较差异显著;成年山羊卵巢上的卵泡较少,但发育程度更好;细胞中分裂期占间期的比例得到显著性上升。转染pCMV-HA-CDC25C载体后,幼龄山羊的卵泡颗粒细胞的CDC25C基因的相对表达量得到上升,但是CDK1、WEE1基因的相对表达量却出现了下降;PROG和E2两种激素的分泌量也有所上升;幼龄山羊卵巢上的卵泡更多,但发育程度不好;细胞中分裂期占间期的比例得到显著性上升。(2)转染小分子干扰载体siCDC25C后,成年山羊的卵泡颗粒细胞的CDC25C、CDK1、WEE1基因的相对表达量都有所下降;PROG和E2两种激素的分泌量有一定上升;细胞中分裂期占间期的比例有显著性下降。幼龄山羊的卵泡颗粒细胞的CDC25C、CDK1、WEE1基因的相对表达量均有所下降;PROG和E2两种激素的分泌量有一定上升;细胞中分裂期占间期的比例有显著性下降。
[Abstract]:The granulosa cell of the follicle is an important component of the follicle, surrounding the oocyte. As the development of the follicle, the granulosa cell of the follicle can change accordingly, and the proliferation and secretion of various hormones and growth factors play a very important role in the growth and maturation of the oocytes. In the early stage of this study, the maturation rate of the oocytes of the young ewes and the adult ewes was significantly different in the process of in vitro maturation, and the proportion of the oocytes developed to the four cells after the in vitro maturation of the oocytes of the young ewes was significantly lower than that of the adult ewes. It was found in the experiment that the young ewes were different from the granulosa cells of the adult ewes, both in number and in the form of the surrounding oocytes. the other finding is that the expression of the cell division cycle protein 25 homologous protein C gene in the young female sheep oocyte and the adult female sheep oocyte has a significant difference, and the gene has the function of regulating the cell division period, and can regulate the mitosis of the eukaryote cells, It is one of the key factors of the cell cycle entering the M phase. In combination with the above two findings, this experiment decided to verify the function of the CDC25C gene from the angle of the granulosa cell of the follicle, and try to solve the difference of the maturation rate of the oocytes in different age groups. The experiment was carried out with the local white goat of Nantong, Jiangsu Province, and the ovaries of the adult female and the 2-month-old lamb were collected and brought back to the laboratory with 0.01 MPBS. In vitro, the granulosa cells of the follicular granulosa cells were transfected with CDC25C, CDK1, WEE1-related genes, estrogen and progestogen, and the cell cycle after transfection, and the results were as follows:1. The clone of the goat CDC25C gene is designed to clone the CDS region of the CDC25C gene according to the CDC25C gene sequence provided by the NCBI, and then the pCMV-HA-CDC25C vector of the gene and the small-molecule interference vector siCDC25C are constructed. The results showed that the vector of pCMV-HA-CDC25C was constructed correctly, while the small-molecular-interference vector siCDC25C was proved to be effective in the pre-experiment and can be used for subsequent test. The identification of primary isolated cells according to the enzyme-linked immune principle, the surface of the granulosa cell of the goat has specific FSH receptor, the test adopts the RabbitAnti-FSHR polyclonal antibody as an anti-, SA1074-rabbit IgG as a second anti-virus, and after the enzyme-linked immunoassay, 3. The effect of the CDC25C gene on the granulosa cells of the follicle was constructed with pCMV-HA-CDC25C vector and the small-molecular-interference vector siCDC25C, and then the adult goat and the young goat's follicular granulosa cells were transfected. The relative expression of CDC25C, CDK1 and WEE1 gene was detected by real-time fluorescence quantitative PCR, and the secretion of both PROG and E2 was detected by enzyme-linked immunosorbent assay. Cell cycle detection was used to compare the cell cycle difference between the two goat's granulosa cells transfected with the recombinant vector and the interfering vector. (1) After transfection of pCMV-HA-CDC25C vector, the relative expression of CDC25C, CDK1 and WEE1 gene in the granulosa cells of the adult goat was significantly improved; the secretion of both PROG and E2 was also increased, and the difference between PROG and E2 was significant. But the degree of development is better; the proportion of the split phase in the cell is significantly increased. After the pCMV-HA-CDC25C vector was transfected with pCMV-HA-CDC25C vector, the relative expression of the CDC25C gene of the follicle-granulosa cells of the young goat was increased, but the relative expression of the CDK1 and the WEE1 gene decreased; the secretion of both PROG and E2 was also increased; and the follicles on the ovary of the young goat were more, But the degree of development is not good, and the proportion of the split phase in the cell is significantly increased. (2) The relative expression of CDC25C, CDK1 and WEE1 in the granulosa cells of the adult goat decreased after transfection of the small-molecular-interference vector siCDC25C, and the secretion of both PROG and E2 was increased; the proportion of the split-phase in the cells was significantly decreased. The relative expression of CDC25C, CDK1 and WEE1 in the follicle-granulosa cells of the young goat decreased; the secretion of both PROG and E2 was increased; the proportion of the split phase in the cells decreased significantly.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S827
本文编号：2448186
[Abstract]:The granulosa cell of the follicle is an important component of the follicle, surrounding the oocyte. As the development of the follicle, the granulosa cell of the follicle can change accordingly, and the proliferation and secretion of various hormones and growth factors play a very important role in the growth and maturation of the oocytes. In the early stage of this study, the maturation rate of the oocytes of the young ewes and the adult ewes was significantly different in the process of in vitro maturation, and the proportion of the oocytes developed to the four cells after the in vitro maturation of the oocytes of the young ewes was significantly lower than that of the adult ewes. It was found in the experiment that the young ewes were different from the granulosa cells of the adult ewes, both in number and in the form of the surrounding oocytes. the other finding is that the expression of the cell division cycle protein 25 homologous protein C gene in the young female sheep oocyte and the adult female sheep oocyte has a significant difference, and the gene has the function of regulating the cell division period, and can regulate the mitosis of the eukaryote cells, It is one of the key factors of the cell cycle entering the M phase. In combination with the above two findings, this experiment decided to verify the function of the CDC25C gene from the angle of the granulosa cell of the follicle, and try to solve the difference of the maturation rate of the oocytes in different age groups. The experiment was carried out with the local white goat of Nantong, Jiangsu Province, and the ovaries of the adult female and the 2-month-old lamb were collected and brought back to the laboratory with 0.01 MPBS. In vitro, the granulosa cells of the follicular granulosa cells were transfected with CDC25C, CDK1, WEE1-related genes, estrogen and progestogen, and the cell cycle after transfection, and the results were as follows:1. The clone of the goat CDC25C gene is designed to clone the CDS region of the CDC25C gene according to the CDC25C gene sequence provided by the NCBI, and then the pCMV-HA-CDC25C vector of the gene and the small-molecule interference vector siCDC25C are constructed. The results showed that the vector of pCMV-HA-CDC25C was constructed correctly, while the small-molecular-interference vector siCDC25C was proved to be effective in the pre-experiment and can be used for subsequent test. The identification of primary isolated cells according to the enzyme-linked immune principle, the surface of the granulosa cell of the goat has specific FSH receptor, the test adopts the RabbitAnti-FSHR polyclonal antibody as an anti-, SA1074-rabbit IgG as a second anti-virus, and after the enzyme-linked immunoassay, 3. The effect of the CDC25C gene on the granulosa cells of the follicle was constructed with pCMV-HA-CDC25C vector and the small-molecular-interference vector siCDC25C, and then the adult goat and the young goat's follicular granulosa cells were transfected. The relative expression of CDC25C, CDK1 and WEE1 gene was detected by real-time fluorescence quantitative PCR, and the secretion of both PROG and E2 was detected by enzyme-linked immunosorbent assay. Cell cycle detection was used to compare the cell cycle difference between the two goat's granulosa cells transfected with the recombinant vector and the interfering vector. (1) After transfection of pCMV-HA-CDC25C vector, the relative expression of CDC25C, CDK1 and WEE1 gene in the granulosa cells of the adult goat was significantly improved; the secretion of both PROG and E2 was also increased, and the difference between PROG and E2 was significant. But the degree of development is better; the proportion of the split phase in the cell is significantly increased. After the pCMV-HA-CDC25C vector was transfected with pCMV-HA-CDC25C vector, the relative expression of the CDC25C gene of the follicle-granulosa cells of the young goat was increased, but the relative expression of the CDK1 and the WEE1 gene decreased; the secretion of both PROG and E2 was also increased; and the follicles on the ovary of the young goat were more, But the degree of development is not good, and the proportion of the split phase in the cell is significantly increased. (2) The relative expression of CDC25C, CDK1 and WEE1 in the granulosa cells of the adult goat decreased after transfection of the small-molecular-interference vector siCDC25C, and the secretion of both PROG and E2 was increased; the proportion of the split-phase in the cells was significantly decreased. The relative expression of CDC25C, CDK1 and WEE1 in the follicle-granulosa cells of the young goat decreased; the secretion of both PROG and E2 was increased; the proportion of the split phase in the cells decreased significantly.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S827
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