CDC25C基因在成年山羊與幼年山羊卵巢顆粒細(xì)胞中的表達(dá)及功能驗(yàn)證
發(fā)布時(shí)間：2019-03-27 12:37
【摘要】：卵泡顆粒細(xì)胞是卵泡的一種重要組成單位,環(huán)繞于卵母細(xì)胞周?chē)ｋS著卵泡的發(fā)育,卵泡顆粒細(xì)胞會(huì)發(fā)生相應(yīng)的變化,其增殖分泌的多種激素及生長(zhǎng)因子與卵母細(xì)胞的生長(zhǎng)和成熟有著極為重要的作用。本研究前期發(fā)現(xiàn),幼齡母羊與成年母羊的卵母細(xì)胞在體外成熟過(guò)程中,成熟率有著顯著性差異,并且幼齡母羊的卵母細(xì)胞經(jīng)體外成熟受精后發(fā)育至四細(xì)胞的比例也顯著性低于成年母羊。試驗(yàn)中發(fā)現(xiàn),幼齡母羊與成年母羊的卵泡顆粒細(xì)胞有著很大的不同點(diǎn),無(wú)論是數(shù)量,還是環(huán)繞卵母細(xì)胞的形態(tài)。另一個(gè)發(fā)現(xiàn)就是細(xì)胞分裂周期蛋白25同源蛋白C基因在幼齡母羊卵母細(xì)胞與成年母羊卵母細(xì)胞中的表達(dá)有顯著性差異,這個(gè)基因具有調(diào)控細(xì)胞分裂周期作用,可調(diào)節(jié)真核生物細(xì)胞有絲分裂,是細(xì)胞周期進(jìn)入M期的關(guān)鍵因子之一。綜合上述兩種發(fā)現(xiàn),本實(shí)驗(yàn)決定從卵泡顆粒細(xì)胞的角度驗(yàn)證CDC25C基因功能,嘗試解開(kāi)不同年齡階段卵母細(xì)胞成熟率差異的原因。本實(shí)驗(yàn)以江蘇南通當(dāng)?shù)匕咨窖驗(yàn)檠芯繉?duì)象,采集1.5年齡以上的成年母羊與2月齡羔羊的卵巢并使用0.01MPBS帶回實(shí)驗(yàn)室。體外分離卵泡顆粒細(xì)胞經(jīng)培養(yǎng)轉(zhuǎn)染后,對(duì)成年母羊與羔羊的卵泡顆粒細(xì)胞進(jìn)行了 CDC25C、CDK1、WEE1相關(guān)基因,雌激素和孕激素,轉(zhuǎn)染后細(xì)胞周期等相關(guān)檢測(cè),結(jié)果如下:1.山羊CDC25C基因的克隆根據(jù)NCBI提供的CDC25C基因序列,設(shè)計(jì)引物克隆CDC25C基因的CDS區(qū),隨后構(gòu)建該基因的pCMV-HA-CDC25C載體和小分子干擾載體siCDC25C。測(cè)序結(jié)果表明pCMV-HA-CDC25C載體構(gòu)建正確,與此同時(shí)小分子干擾載體siCDC25C在預(yù)實(shí)驗(yàn)中被證實(shí)有效,皆可用于后續(xù)試驗(yàn)。2.原代分離細(xì)胞的鑒定依據(jù)酶聯(lián)免疫原理,山羊卵泡顆粒細(xì)胞表面擁有特異性FSH受體,試驗(yàn)采用Rabbit Anti-FSHR多克隆抗體作為一抗,SA1074-兔IgG為二抗,經(jīng)過(guò)酶聯(lián)免疫檢測(cè)后認(rèn)定,本實(shí)驗(yàn)分離的細(xì)胞絕大部分都是所需要的卵泡顆粒細(xì)胞,可繼續(xù)后續(xù)試驗(yàn)。3.CDC25C基因?qū)β雅蓊w粒細(xì)胞的影響構(gòu)建pCMV-HA-CDC25C載體和小分子干擾載體siCDC25C,轉(zhuǎn)染成年山羊和幼齡山羊卵泡顆粒細(xì)胞后,利用實(shí)時(shí)熒光定量PCR技術(shù)對(duì)CDC25C、CDK1、WEE1基因的相對(duì)表達(dá)量進(jìn)行檢測(cè),利用酶聯(lián)免疫方式檢測(cè)了 PROG和E2兩種激素分泌情況,利用切片技術(shù)比較了成年山羊與幼齡山羊卵巢的區(qū)別,利用細(xì)胞周期檢測(cè)技術(shù)比較了兩種山羊卵泡顆粒細(xì)胞轉(zhuǎn)染重組載體和干擾載體后的細(xì)胞周期區(qū)別。(1)轉(zhuǎn)染pCMV-HA-CDC25C載體后,成年山羊的卵泡顆粒細(xì)胞的CDC25C、CDK1、WEE1基因的相對(duì)表達(dá)量都得到了顯著性提升;PROG和E2兩種激素的分泌量也有所上升,且與空白組比較差異顯著;成年山羊卵巢上的卵泡較少,但發(fā)育程度更好;細(xì)胞中分裂期占間期的比例得到顯著性上升。轉(zhuǎn)染pCMV-HA-CDC25C載體后,幼齡山羊的卵泡顆粒細(xì)胞的CDC25C基因的相對(duì)表達(dá)量得到上升,但是CDK1、WEE1基因的相對(duì)表達(dá)量卻出現(xiàn)了下降;PROG和E2兩種激素的分泌量也有所上升;幼齡山羊卵巢上的卵泡更多,但發(fā)育程度不好;細(xì)胞中分裂期占間期的比例得到顯著性上升。(2)轉(zhuǎn)染小分子干擾載體siCDC25C后,成年山羊的卵泡顆粒細(xì)胞的CDC25C、CDK1、WEE1基因的相對(duì)表達(dá)量都有所下降;PROG和E2兩種激素的分泌量有一定上升;細(xì)胞中分裂期占間期的比例有顯著性下降。幼齡山羊的卵泡顆粒細(xì)胞的CDC25C、CDK1、WEE1基因的相對(duì)表達(dá)量均有所下降;PROG和E2兩種激素的分泌量有一定上升;細(xì)胞中分裂期占間期的比例有顯著性下降。
[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.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S827
本文編號(hào)：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.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S827
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