发情期牛子宫液外泌体的提取与鉴定及其对体外受精胚胎发育的影响

发布时间：2018-03-04 22:07

本文选题：子宫液　切入点：外泌体　出处：《西北农林科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：外泌体是由细胞通过胞吞胞吐的方式向外环境中释放的一种纳米级囊泡,其内包含重要的生物信息物质,如蛋白质、脂质、mRNA和mi RNA等。这种小囊泡作为“信使”广泛参与着机体生理或病理的生物学活动。越来越多的研究表明,外泌体几乎存在于所有的体液当中,暗示其对生物生命活动的重要性以及必要性。有证据表明从哺乳动物生命的最开始——胚胎,外泌体就扮演了很重要的角色。在本研究中,首先对发情期牛的子宫液中的外泌体进行提取和鉴定,之后将其添加至IVF培养液中,来观察其对IVF胚胎发育的影响,主要研究结果如下:1.利用导管向发情期的牛子宫注入生理盐水进行冲洗,每次2 ml,重复6-8次,收集到子宫冲洗液;2.通过改良后的超速离心法,获得了纯度较高的子宫液外泌体;3.在透射电子显微镜观察到外泌体形态:直径在100 nm左右,大小均一,形态较为规则,呈圆形或椭圆形;4.外泌体特异性膜蛋白免疫荧光染色显示为CD9+,Western Blot曝光出CD9条带;5.将外泌体样品添加至IVF培养液中,结果显示实验(exosomes)组与对照组的卵裂率(81.2%vs79.1%)以及囊胚率(33.3%vs32.5%)均未见明显差异;6.经囊胚的凋亡染色,在囊胚细胞数(110.21±4.52 vs 113.11±3.20)无差异的情况下,实验(exosome)组囊胚凋亡细胞数显著低于对照组(2.68±0.30 vs 5.83±0.44,P0.05)。综上所述,发情期的牛子宫液外泌体能够降低体外培养囊胚细胞凋亡的比例。
[Abstract]:Exosomes are nanometer-sized vesicles released by cells into the outer environment by emesis. They contain important biological information substances, such as proteins. These vesicles, as messengers, are widely involved in the biological activities of the body's physiology or pathology. More and more studies have shown that exocrine bodies are present in almost all body fluids. Suggesting its importance and necessity for biological activities. There is evidence that exosomes play a significant role from the very beginning of mammalian life, the embryo. In order to observe the effect of estrus on the development of IVF embryos, the exocrine in the uterine fluid of estrous cattle was extracted and identified, and then added into the IVF culture medium to observe its effect on the development of IVF embryos. The main results are as follows: 1. Irrigating bovine uterus during estrus with normal saline with catheter, 2 ml each time, repeat 6 to 8 times, collect uterine irrigation fluid 2. Through the improved ultracentrifugation method, The exocrine of uterine fluid with high purity was obtained. The morphology of exocrine was observed by transmission electron microscope (TEM): the diameter was about 100 nm, the size was uniform, and the morphology was regular. The exocrine specific membrane protein immunofluorescence staining showed that the CD9 band was exposed by CD9 Blot. The exocrine sample was added to the culture medium of IVF. The results showed that there was no significant difference in cleavage rate (81.2 vs 79.1) and blastocyst rate (33.3vs32.5) between the experimental group and the control group. There was no significant difference in blastocyst cell number (110.21 卤4.52 vs 113.11 卤3.20). The number of blastocyst apoptotic cells in the estrus group was significantly lower than that in the control group (2.68 卤0.30 vs 5.83 卤0.44p 0.05). In conclusion, the exocrine of bovine uterus during estrus could reduce the percentage of apoptosis of blastocyst cells cultured in vitro.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S823

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