本文選題：綿羊 + 卵母細胞�。� 參考：《河北農(nóng)業(yè)大學》2015年碩士論文

【摘要】：組織蛋白酶B(CTSB)和組織蛋白酶S(CTSS)為存在于溶酶體內(nèi)的半胱氨酸蛋白酶,在細胞凋亡、卵母細胞成熟和胚胎附植等多種生理活動中發(fā)揮著重要作用。caspase 3處于caspase級聯(lián)反應(yīng)的下游,為細胞凋亡的執(zhí)行者,其活化作為細胞凋亡早期的標志。研究這三種蛋白酶的活性和表達量在卵母細胞體外成熟過程中變化及與卵母細胞質(zhì)量和發(fā)育能力的相關(guān)性,能反應(yīng)卵母細胞在體外成熟過程中所受到凋亡刺激的強度,同時,研究半胱氨酸蛋白酶抑制劑E-64對卵母細胞內(nèi)CTSB、CTSS和caspase 3活性和表達量的影響,則可為提高卵母細胞體外成熟質(zhì)量和發(fā)育能力提供理論依據(jù)。對于不同來源(成年綿羊及羔羊)、不同質(zhì)量的綿羊卵母細胞,利用RT-PCR檢測在體外成熟(IVM)過程中CTSB、CTSS和caspase 3基因表達量,利用雙夾心ELISA對CTSB、CTSS和caspase 3蛋白表達量和活性進行檢測,結(jié)果表明:在IVM 24h時,質(zhì)量正常的卵母細胞內(nèi)CTSB、CTSS和caspase 3基因、蛋白表達量與活性均低于質(zhì)量差的卵母細胞(P0.05;P0.01)。成年綿羊卵母細胞與羔羊卵母細胞相比,在IVM 24h時,成年綿羊卵母細胞內(nèi)CTSB、CTSS和caspase 3基因和蛋白表達量及CTSB活性均低于羔羊卵母細胞(P0.05;P0.01)。成年綿羊及羔羊的卵母細胞在體外成熟過程中,CTSB、CTSS和caspase 3基因表達量的研究結(jié)果表明:在成年綿羊卵母細胞內(nèi),CTSB基因表達量保持穩(wěn)定(P0.05),成熟液中添加1μmol/L E-64和5μmol/L E-64對CTSB基因表達量無顯著影響(P0.05),添加10μmol/L E-64則使CTSB基因表達量極顯著高于對照組(P0.01);CTSS基因表達量在IVM 16h以前一直處于無法被檢測到的水平,并且1、5和10μmol/L E-64對CTSS基因表達量均無顯著影響(P0.05);caspase 3基因表達量呈先降低后升高的趨勢(P0.05),1μmol/L E-64對caspase 3基因表達量無顯著影響(P0.05),5μmol/L E-64對基因表達起到下調(diào)作用(P0.05),而10μmol/L E-64提高了卵母細胞內(nèi)caspase基因表達量(P0.05)。對于羔羊卵母細胞,CTSB基因表達量呈上升趨勢(P0.01),1μmol/L E-64和5μmol/L E-64顯著或極顯著降低了CTSB基因表達量(P0.05;P0.01),10μmol/L E-64則極顯著促進了CTSB的基因表達(P0.01);與成年綿羊卵母細胞不同,對照組CTSS基因在IVM 0h已經(jīng)有所表達,IVM 24h時CTSS基因表達量極顯著高于IVM 0h(P0.01),1μmol/L E-64和5μmol/L E-64對CTSS基因表達量無顯著影響(P0.05),而10μmol/L E-64極顯著促進了CTSS基因表達(P0.01);caspase 3基因表達量變化與CTSS相似,不同之處是1μmol/L E-64和5μmol/L E-64組內(nèi)caspase 3基因表達量均與IVM 0h無顯著差異(P0.05)。成年綿羊及羔羊的卵母細胞內(nèi)CTSB、CTSS和caspase 3蛋白表達量和活性的結(jié)果表明:成年綿羊卵母細胞內(nèi),CTSB蛋白表達量和活性均保持穩(wěn)定(P0.05),5μmol/L E-64對CTSB蛋白表達量無顯著影響(P0.05),但在IVM 8h時CTSB活性顯著降低(P0.05),10μmol/L E-64使CTSB蛋白表達量上調(diào)(P0.05),同時使CTSB活性降低(P0.01);成年綿羊卵母細胞內(nèi)CTSS蛋白表達量和活性在體外成熟過程中無顯著變化(P0.05),5μmol/L和10μmol/L E-64可不同程度地降低CTSS活性(P0.05;P0.01),而對CTSS蛋白表達量無顯著影響(P0.05);caspase 3蛋白表達量保持穩(wěn)定(P0.05),活性于IVM 8h降至最低點后(P0.01),又于IVM16h升高至與IVM 0h和IVM 24h無顯著差異的水平(P0.05),5μmol/L和10μmol/L E-64分別在不同程度上降低了caspase 3活性(P0.05;P0.01),5μmol/L E-64對caspase 3蛋白表達量無顯著影響(P0.05),10μmol/L E-64組的caspase 3蛋白表達量升高(P0.05)。羔羊卵母細胞內(nèi),CTSB蛋白表達量無顯著變化(P0.05),而活性呈上升趨勢(P0.01),5μmol/L E-64組的CTSB蛋白表達量極顯著降低(P0.01),對CTSB活性無顯著影響(P0.05),10μmol/L E-64組的CTSB蛋白表達極顯著升高(P0.01),并且CTSB活性顯著降低(P0.05);CTSS蛋白表達量和活性均無顯著變化(P0.05),5μmol/L E-64和10μmol/L E-64組的CTSS蛋白表達量發(fā)生不同程度降低(P0.05;P0.01),5μmol/L E-64組的CTSS活性極顯著降低(P0.01),而10μmol/L E-64組的CTSS活性無顯著變化(P0.05);caspase 3蛋白表達量和活性保持穩(wěn)定(P0.05),5μmol/L E-64組的caspase 3蛋白表達量和活性均降低(P0.05),10μmol/L E-64組內(nèi)caspase 3蛋白表達量和活性無顯著變化(P0.05)。
[Abstract]:Cathepsin B (CTSB) and cathepsin S (CTSS) is a cysteine protease that exists in the enzyme soluble enzyme. It plays an important role in many physiological activities such as apoptosis, oocyte maturation and embryo implantation..caspase 3 is downstream of caspase cascade reaction. It is the executor of apoptosis, and its activation is the early stage of apoptosis. The correlation between the activity and expression of these three proteases during oocyte maturation in vitro and the correlation with oocyte quality and development ability can reflect the intensity of oocyte apoptosis stimulated in the process of maturation in vitro, and the study of cysteine protease inhibitor E-64 on CTSB, CTSS and CTSS in oocytes. The effect of caspase 3 activity and expression can provide a theoretical basis for improving the quality and development ability of oocytes in vitro. For different sources (adult sheep and lambs), different quality sheep oocytes are used to detect the expression of CTSB, CTSS and caspase 3 in the process of in vitro maturation (IVM), and the double sandwich ELISA is used. The expression and activity of CTSB, CTSS and caspase 3 protein were detected. The results showed that at IVM 24h, the CTSB, CTSS and caspase 3 genes in normal oocytes were lower than those of poor quality oocytes (P0.05; P0.01). Adult ovine oocytes were compared with lamb oocytes, adult ovine oocytes in IVM 24h. The expression of CTSB, CTSS and caspase 3 genes and protein expression and CTSB activity were lower than that of lamb oocytes (P0.05; P0.01). The expression of CTSB, CTSS and caspase 3 genes in adult sheep and lamb oocytes showed that the expression of CTSB gene remained stable (P0.05) in adult ovine oocytes. The addition of 1 mol/L E-64 and 5 mol/L E-64 in the mature liquid had no significant effect on the expression of CTSB gene (P0.05), and the expression of CTSB gene was significantly higher than that of the control group (P0.01) with the addition of 10 mu mol/L E-64, and the CTSS gene expression was not detected before IVM. The expression of caspase 3 gene showed a tendency to decrease first and then increase (P0.05), and 1 mol/L E-64 had no significant effect on the expression of caspase 3 gene (P0.05), and 5 u mol/L E-64 downregulated gene expression (P0.05), while 10 mu mol/L E-64 increased the expression of gene expression in oocyte. The gene expression increased (P0.01). 1 mol/L E-64 and 5 mol/L E-64 significantly reduced the CTSB gene expression (P0.05; P0.01), and 10 mu mol/L E-64 significantly promoted the CTSB gene expression (P0.01). It was significantly higher than IVM 0h (P0.01). 1 mol/L E-64 and 5 mol/L E-64 had no significant effect on the gene expression of CTSS, while 10 mu mol/L E-64 significantly promoted the expression of CTSS genes. The expression of 3 gene expression was similar to that of 1 mu and 5 mu. P0.05. The expression and activity of CTSB, CTSS and caspase 3 protein in the oocytes of adult sheep and lambs showed that the expression and activity of CTSB protein remained stable in adult ovine oocytes (P0.05), and 5 mu mol/L E-64 had no significant influence on the expression of CTSB protein (P0.05), but the activity decreased significantly in IVM 8h. 10 mol/L E-64 increased the expression of CTSB protein (P0.05), and reduced the activity of CTSB (P0.01). There was no significant change in the expression and activity of CTSS protein in adult ovine oocytes (P0.05) in the process of maturation in vitro (P0.05). The activity of CTSS was reduced by 5 and 10 micron mol/L E-64, but there was no significant effect on the expression of the protein. P0.05), the expression of caspase 3 protein remained stable (P0.05), the activity was reduced to the lowest point of IVM 8h (P0.01), and increased to the level of IVM 0h and IVM 24h (P0.05), 5 mu and 10 micron decreased the 3 activity in varying degrees, 5 micron. The expression of caspase 3 protein in 10 mu E-64 group increased (P0.05). There was no significant change in the expression of CTSB protein in the oocyte of lamb (P0.05), but the activity showed a rising trend (P0.01). The expression of CTSB protein in the 5 mu mol/L E-64 group decreased significantly (P0.01), and there was no significant effect on the activity of 10 micron. The expression of CTSB was significantly increased (P0.01), and the activity of CTSB was significantly decreased (P0.05); there was no significant change in the expression and activity of CTSS protein (P0.05). The expression of CTSS protein in 5 mu E-64 and 10 u mol/L E-64 groups decreased in varying degrees (P0.05; P0.01), and the activity of 5 mu was significantly reduced. There was no significant change (P0.05); the expression and activity of caspase 3 protein remained stable (P0.05), and the expression and activity of caspase 3 protein in the 5 mol/L E-64 group decreased (P0.05), and there was no significant change in the expression and activity of caspase 3 protein in the 10 u mol/L E-64 group (P0.05).

【學位授予單位】：河北農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S826

【共引文獻】

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本文編號：1820108