绵羊卵母细胞内CTSB、CTSS和caspase 3基因和蛋白表达及活性变化规律

发布时间：2018-04-29 13:22

本文选题：绵羊 + 卵母细胞　；参考：《河北农业大学》2015年硕士论文

【摘要】：组织蛋白酶B(CTSB)和组织蛋白酶S(CTSS)为存在于溶酶体内的半胱氨酸蛋白酶,在细胞凋亡、卵母细胞成熟和胚胎附植等多种生理活动中发挥着重要作用。caspase 3处于caspase级联反应的下游,为细胞凋亡的执行者,其活化作为细胞凋亡早期的标志。研究这三种蛋白酶的活性和表达量在卵母细胞体外成熟过程中变化及与卵母细胞质量和发育能力的相关性,能反应卵母细胞在体外成熟过程中所受到凋亡刺激的强度,同时,研究半胱氨酸蛋白酶抑制剂E-64对卵母细胞内CTSB、CTSS和caspase 3活性和表达量的影响,则可为提高卵母细胞体外成熟质量和发育能力提供理论依据。对于不同来源(成年绵羊及羔羊)、不同质量的绵羊卵母细胞,利用RT-PCR检测在体外成熟(IVM)过程中CTSB、CTSS和caspase 3基因表达量,利用双夹心ELISA对CTSB、CTSS和caspase 3蛋白表达量和活性进行检测,结果表明:在IVM 24h时,质量正常的卵母细胞内CTSB、CTSS和caspase 3基因、蛋白表达量与活性均低于质量差的卵母细胞(P0.05;P0.01)。成年绵羊卵母细胞与羔羊卵母细胞相比,在IVM 24h时,成年绵羊卵母细胞内CTSB、CTSS和caspase 3基因和蛋白表达量及CTSB活性均低于羔羊卵母细胞(P0.05;P0.01)。成年绵羊及羔羊的卵母细胞在体外成熟过程中,CTSB、CTSS和caspase 3基因表达量的研究结果表明:在成年绵羊卵母细胞内,CTSB基因表达量保持稳定(P0.05),成熟液中添加1μmol/L E-64和5μmol/L E-64对CTSB基因表达量无显著影响(P0.05),添加10μmol/L E-64则使CTSB基因表达量极显著高于对照组(P0.01);CTSS基因表达量在IVM 16h以前一直处于无法被检测到的水平,并且1、5和10μmol/L E-64对CTSS基因表达量均无显著影响(P0.05);caspase 3基因表达量呈先降低后升高的趋势(P0.05),1μmol/L E-64对caspase 3基因表达量无显著影响(P0.05),5μmol/L E-64对基因表达起到下调作用(P0.05),而10μmol/L E-64提高了卵母细胞内caspase基因表达量(P0.05)。对于羔羊卵母细胞,CTSB基因表达量呈上升趋势(P0.01),1μmol/L E-64和5μmol/L E-64显著或极显著降低了CTSB基因表达量(P0.05;P0.01),10μmol/L E-64则极显著促进了CTSB的基因表达(P0.01);与成年绵羊卵母细胞不同,对照组CTSS基因在IVM 0h已经有所表达,IVM 24h时CTSS基因表达量极显著高于IVM 0h(P0.01),1μmol/L E-64和5μmol/L E-64对CTSS基因表达量无显著影响(P0.05),而10μmol/L E-64极显著促进了CTSS基因表达(P0.01);caspase 3基因表达量变化与CTSS相似,不同之处是1μmol/L E-64和5μmol/L E-64组内caspase 3基因表达量均与IVM 0h无显著差异(P0.05)。成年绵羊及羔羊的卵母细胞内CTSB、CTSS和caspase 3蛋白表达量和活性的结果表明:成年绵羊卵母细胞内,CTSB蛋白表达量和活性均保持稳定(P0.05),5μmol/L E-64对CTSB蛋白表达量无显著影响(P0.05),但在IVM 8h时CTSB活性显著降低(P0.05),10μmol/L E-64使CTSB蛋白表达量上调(P0.05),同时使CTSB活性降低(P0.01);成年绵羊卵母细胞内CTSS蛋白表达量和活性在体外成熟过程中无显著变化(P0.05),5μmol/L和10μmol/L E-64可不同程度地降低CTSS活性(P0.05;P0.01),而对CTSS蛋白表达量无显著影响(P0.05);caspase 3蛋白表达量保持稳定(P0.05),活性于IVM 8h降至最低点后(P0.01),又于IVM16h升高至与IVM 0h和IVM 24h无显著差异的水平(P0.05),5μmol/L和10μmol/L E-64分别在不同程度上降低了caspase 3活性(P0.05;P0.01),5μmol/L E-64对caspase 3蛋白表达量无显著影响(P0.05),10μmol/L E-64组的caspase 3蛋白表达量升高(P0.05)。羔羊卵母细胞内,CTSB蛋白表达量无显著变化(P0.05),而活性呈上升趋势(P0.01),5μmol/L E-64组的CTSB蛋白表达量极显著降低(P0.01),对CTSB活性无显著影响(P0.05),10μmol/L E-64组的CTSB蛋白表达极显著升高(P0.01),并且CTSB活性显著降低(P0.05);CTSS蛋白表达量和活性均无显著变化(P0.05),5μmol/L E-64和10μmol/L E-64组的CTSS蛋白表达量发生不同程度降低(P0.05;P0.01),5μmol/L E-64组的CTSS活性极显著降低(P0.01),而10μmol/L E-64组的CTSS活性无显著变化(P0.05);caspase 3蛋白表达量和活性保持稳定(P0.05),5μmol/L E-64组的caspase 3蛋白表达量和活性均降低(P0.05),10μmol/L E-64组内caspase 3蛋白表达量和活性无显著变化(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).

【学位授予单位】：河北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S826

【共引文献】