Secreted protein of Ly6 domain 1 enhanced bovine trophoblastic cell migration activity
Secreted protein of Ly6 domain 1 enhanced bovine trophoblastic cell migration activity
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DOI：
10.1007/s11626-020-00521-x
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发表时间：
2020-11-02
影响因子：
2.1
通讯作者：
Hashizume，Kazuyoshi
中科院分区：
文献类型：
--
作者：
Awad，Mahmoud;Kizaki，Keiichiro;Hashizume，Kazuyoshi
在反刍动物胎盘中，滋养层双核细胞(BNC)通过滋养层单核细胞(TMC)之间的细胞连接并面向子宫内膜表面的迁移，与单个子宫内膜上皮细胞建立了一个复杂的调节单位，而不穿过基底膜(Wooding和Flint 1994)。这一现象可以通过限制滋养层细胞的侵入而分别在牛和羊中形成杂交的母胎三核细胞和合体细胞斑块来解释(Wooding等人。1980)。细胞迁移通常由各种因素指导，例如包括基质金属蛋白酶、激素、趋化因子、受体和其他在内的酶(Dimitriadis等人。2005年；Majali-Martinez等人。2016年；Pollheimer等人。2018年)。在先前的研究中，淋巴细胞抗原-6(Ly6)结构域1的分泌蛋白(SOLD1)在牛滋养层细胞系中表达，并在体外调节细胞的侵袭性(Awad等人)。2014年)。因此，SOLD1可能刺激滋养层细胞向子宫内膜运动，包括侵袭、迁移和增殖。SOLD1是Ly6/尿激酶型纤溶酶原激活剂受体(UPAR)超家族中的一个新的分泌型成员，由于其在绒毛间质中的分布模式以及对牛绒毛膜成纤维细胞增殖和凋亡的调控作用，在胎盘结构和胎膜发育中起着至关重要的作用(Ushizawa等人)。2009、2010)。SOLD1在整个妊娠期间在母体组织中被发现，并且它的表达随着奶牛妊娠的进展而增加(Awad等人。然而，SOLD1的生理功能仍不清楚。在本研究中，我们研究了SOLD1对滋养层细胞在体外迁移的影响。2011)，在本研究中使用。简而言之，按照前面描述的方法，将细胞培养在Dulbecco的改良Eagle‘s培养液/F-12培养液(DMEM/F12，Sigma，Saint Louis，MO)中，其中含有100IU/mL青霉素和100μg/mL链霉素(Sigma)，并添加10%胎牛血清(FBS；Biowest，Nuaillé，法国)，在37℃，5%二氧化碳的气氛中培养(Suzuki et al.2011年)。每隔2~3d更换一次培养液。将培养瓶与酸溶猪I型胶原(3 mg/mLI型胶原，日本大阪Nitta Gelatin)孵育，用蒸馏水稀释10倍，倒入瓶中1h以上，制成胶原包被烧瓶，然后用普通培养基洗净。在磷酸盐缓冲液分离洗涤后，BT-K和-C细胞聚集体被放置在胶原蛋白涂层的烧瓶中。由于BT-K细胞高表达SOLD1，具有较强的侵袭能力，因此选择该细胞株进行迁移实验。Bt-C在之前的一项研究中报告了SOLD1的表达水平较低，仅被用作基因表达的对照(Awad等人。2014年)。使用Transwell系统(细胞培养插入物，编号353182，BD Bioscience，富兰克林湖，新泽西州)在放置在12孔板(BD Bioscience)中的多孔透明聚对苯二甲酸乙二醇膜(孔径8.0cm2，1×105孔/μ)上评估细胞迁移。如前所述，实验是根据制造商的说明进行的(Grasso等人。2014年)。迁移的细胞用0.5%的HE染色。
In ruminant placenta, the migration of trophoblastic binucleate cells (BNCs) through the cellular junction between trophoblastic mononucleate cells (TMCs) and facing the endometrial surface establish a complex adjustment unit with individual endometrial epithelial cells without passing through the basement membrane (Wooding and Flint 1994). This phenomenon may be explained by restricted trophoblast invasion to form hybrid feto-maternal trinucleate cells and syncytial plaques in cows and sheep, respectively (Wooding et al. 1980). Cell migration is usually directed by various factors, such as enzymes including matrix metalloproteinases, hormones, chemokines, receptors, and others (Dimitriadis et al. 2005; Majali-Martinez et al. 2016; Pollheimer et al. 2018). In a previous study, the secreted protein of lymphocyte antigen-6 (Ly6) domain 1 (SOLD1) was expressed in bovine trophoblastic cell lines, and its protein regulated cell invasiveness in vitro (Awad et al. 2014). Therefore, SOLD1 may stimulate trophoblast movement to endometrium, including invasion, migration, and proliferation. SOLD1 is a novel secreted member of the Ly6/urokinase-type plasminogen activator receptor (uPAR) superfamily, and plays a crucial role in placental structure and fetal membrane development due to its distribution pattern in the mesenchyme of the chorionic villi as well as its modulatory effect on the proliferation and apoptosis of bovine chorionic fibroblast cells (Ushizawa et al. 2009, 2010). SOLD1 was found in the maternal tissue throughout gestation and its expression increased as gestation progressed in cows (Awad et al. 2013); however, the physiological function of SOLD1 remains unclear. In the present study, we examined the effect of SOLD1 on the migration of trophoblast cells in vitro.The bovine trophoblast cell lines, BT-K and-C, which were previously established from in vitro fertilized bovine blastocysts (Suzuki et al. 2011), were used in this study. Briefly, the cells were cultured in Dulbecco’s modified Eagle’s medium/F-12 medium (DMEM/F12, Sigma, Saint Louis, MO) containing 100 IU/mL penicillin and 100 μg/mL streptomycin (Sigma), supplemented with 10% fetal bovine serum (FBS; Biowest, Nuaillé, France), at 37 C in an atmosphere of 5% CO2 according to a previously described method (Suzuki et al. 2011). The medium was changed every 2–3 d. Collagen-coated flasks were prepared by incubating the flasks with acid-soluble porcine type I collagen (3 mg/mL type IC collagen; Nitta Gelatin Osaka, Japan), diluted 10-fold with distilled water, and poured into flasks for more than 1 h. The flasks were then washed with general culture medium. After a phosphate-buffered saline dissociated wash, the BT-K and-C cell aggregates were plated in collagen-coated flasks. BT-K cells were selected for the migration assay because this cell line highly expressed SOLD1 and had a better invasion power. BT-C, which expressed lower levels of SOLD1 as reported in a previous study, was used only as a control for gene expression (Awad et al. 2014). Cell migration was assessed using Transwell systems (cell culture inserts, no. 353182, BD Biosciences, Franklin Lakes, NJ) on a porous transparent polyethylene terephthalate membrane (8.0 μm pore size, 1× 105 pores/cm2) placed into 12-well plates (BD Biosciences). Experiments were performed according to the manufacturer’s instructions, as previously described (Grasso et al. 2014). The migrated cells were stained using 0.5% he-