猪子宫腔液外泌体来源的TIMP2蛋白对胚胎附植的影响

doi:10.11843/j.issn.0366-6964.2022.04.012

摘要/Abstract

摘要： 旨在研究猪子宫腔液外泌体来源的TIMP2蛋白对妊娠早期胚胎附植的影响。本研究采用超速离心法分离妊娠第9天(n=3)和第12天(n=3)大白猪子宫腔液外泌体，利用Western blot (WB)检测外泌体中TIMP2蛋白的变化，并通过免疫组化试验研究TIMP2蛋白在子宫中的表达定位。进一步用外泌体与猪胚胎滋养层细胞(porcine trophoblast,PTr2)共培养试验研究外泌体对TIMP2蛋白的靶向运输作用，最后在PTr2细胞中干扰和超表达TIMP2基因，利用CCK-8、EdU及划痕试验研究TIMP2蛋白对PTr2细胞增殖和迁移的影响。通过超速离心法成功分离到子宫腔液外泌体，其粒径主要分布在30～150 nm之间，WB试验结果表明，妊娠第12天猪子宫腔液外泌体中TIMP2蛋白含量显著增高(P<0.05)，外泌体与滋养层细胞共培养试验表明，TIMP2蛋白能通过外泌体靶向运输到滋养层细胞。免疫组化结果发现，TIMP2蛋白在子宫内膜和胚胎中均表达，且在妊娠第12天子宫内膜中的表达量极显著高于妊娠第9天(P<0.01)。进一步通过体外细胞试验发现，干扰PTr2中的TIMP2基因，细胞增殖能力极显著增强(P<0.01)，迁移能力显著增强(P<0.05)；超表达该基因，细胞增殖能力极显著减弱(P<0.01)，迁移能力也显著减弱(P<0.05)。以上结果表明，子宫腔液外泌体来源TIMP2蛋白可能由母体子宫内膜所分泌，并能通过外泌体靶向进入胚胎滋养层细胞调控其增殖和迁移，从而影响胚胎附植。

关键词: 外泌体, TIMP2, PTr2, 胚胎附植

Abstract: This study aimed to investigate the effect of TIMP2 protein derived from porcine uterine fluid exosomes on embryo implantation during early pregnancy. Exosomes were isolated from the uterine fluid of Yorkshire pigs at day 9 (n=3) and 12 (n=3) of pregnancy by ultracentrifugation. The relative abundance of TIMP2 protein in exosomes was determined using Western blot (WB)， and the expression and location of TIMP2 in uterus was detected by immunohistochemistry. Subsequently, co-culture experiments of exosomes and porcine trophoblast cells (PTr2) were used to investigate the targeted transport of exosomes for TIMP2 protein. Finally, the TIMP2 gene was interfered or overexpressed in PTr2, and the effects of TIMP2 on proliferation and migration of PTr2 cells were determined using CCK-8, EdU and wound healing assay. The exosomes were successfully isolated from uterine fluid by ultracentrifugation, and their particle sizes were mainly distributed from 30 to 150 nm. WB results showed that the TIMP2 protein abundance in exosomes of pig uterine fluid at day 12 of pregnancy was significantly increased (P<0.05). While the co-culture experiments of exosomes and trophoblast cells showed that TIMP2 protein could be targeted transport to trophoblast cells through exosomes. Immunohistochemical results showed that TIMP2 protein was found in both the embryo and endometrium, and the abundance level in endometrium at day 12 of pregnancy was significantly higher than that at day 9 of pregnancy (P<0.01). In addition, in vitro cell culture experiments found that interfering with TIMP2 gene in PTr2 significantly enhanced cell proliferation (P<0.01) and migration ability (P<0.05)， while the overexpression of TIMP2 impaired the proliferation (P<0.01) and migration ability (P<0.05). These results suggest that TIMP2 protein in uterine fluid exosomes may be secreted by the maternal endometrium, and could be targeted into the trophoblast cells through exosomes to regulate their proliferation and migration, thereby affecting embryo implantation.

Key words: exosomes, TIMP2, PTr2, embryo implantation

中图分类号:

S828.3

匡婧靖, 贺艳娟, 胡群, 顾婷, 吴珍芳, 蔡更元, 洪林君. 猪子宫腔液外泌体来源的TIMP2蛋白对胚胎附植的影响[J]. 畜牧兽医学报, 2022, 53(4): 1122-1132.

KUANG Jingjing, HE Yanjuan, HU Qun, GU Ting, WU Zhenfang, CAI Gengyuan, HONG Linjun. Effect of TIMP2 Protein Derived from Porcine Uterine Fluid Exosomes on Embryo Implantation During Early Pregnancy[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(4): 1122-1132.

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