Department of Pediatric Surgery and Research Laboratory, Hospital Universitario La Paz, 28046 Madrid, Spain.
Cell Transplant. 2013;22(9):1683-94. doi: 10.3727/096368912X657756. Epub 2012 Oct 8.
Lung hypoplasia can be prevented in vitro by retinoic acid (RA). Recent evidence suggests that amniotic fluid stem (AFS) cells may integrate injured lungs and influence their recovery. We tested the hypothesis that AFS cells might improve lung growth and motility by paracrine mechanisms. Pregnant rats received either nitrofen or vehicle on E9.5. In vitro E13 embryonic lungs were cultured in the presence of culture medium alone or with RA, basophils, or AFS cells. In vivo green fluorescent protein-expressing (GFP(+)) rat AFS cells were transplanted in nitrofen-exposed rats on E10.5. E13 lung explants were cultured before analysis. The surface, the number of terminal buds, and the frequency of bronchial contractions were assessed. Protein gene product 9.5 (PGP 9.5) and α-actin protein levels were measured. The lung explants transplanted with AFS cells were stained for α-actin, PGP 9.5, and TTF-1. The levels of FGF-10, VEGFα, and TGF-β1 secreted by the AFS cells in the culture medium were measured. Comparison between groups was made by ANOVA. In vitro, the surface, the number of terminal buds, and the bronchial peristalsis were increased in nitrofen+AFS cell explants in comparison with nitrofen-exposed lungs. While nitrofen+RA lungs were similar to nitrofen+AFS ones, basophils did not normalize these measurements. PGP 9.5 protein was decreased in nitrofen lungs, but after adding AFS cells, the value was similar to controls. No differences were found in the expression of α-actin. In vivo, the surface, number of terminal buds, and peristalsis were similar to control after injection of AFS cells in nitrofen-exposed rats. Colocalization with TTF-1-positive cells was found. The levels of FGF-10 and VEGFα were increased in nitrofen+AFS cell explants, while the levels of TGF-β1 were similar to controls. Lung growth, bronchial motility, and innervation were decreased in nitrofen explants and rescued by AFS cells both in vitro and in vivo, similarly to that observed before with RA. The AFS cell beneficial effect was probably related to paracrine action of growth factor secretion.
肺发育不全可以通过维甲酸（RA）在体外预防。最近的证据表明，羊水干细胞（AFS）可能整合受损的肺部并影响其恢复。我们测试了这样一种假设，即 AFS 细胞可能通过旁分泌机制改善肺生长和运动。妊娠大鼠在 E9.5 时接受尼氟酸或载体。体外 E13 胚胎肺在单独培养基或 RA、嗜碱性粒细胞或 AFS 细胞存在下培养。在 E10.5 时，体内绿色荧光蛋白表达（GFP(+）大鼠 AFS 细胞被移植到尼氟酸暴露的大鼠中。E13 肺外植体在分析前进行培养。评估表面、终末芽的数量和支气管收缩的频率。测量蛋白基因产物 9.5（PGP 9.5）和α-肌动蛋白蛋白水平。用α-肌动蛋白、PGP 9.5 和 TTF-1 对移植 AFS 细胞的肺外植体进行染色。测量培养基中 AFS 细胞分泌的 FGF-10、VEGFα 和 TGF-β1 的水平。通过 ANOVA 比较组间差异。在体外，与尼氟酸暴露的肺相比，尼氟酸+AFS 细胞外植体的表面、终末芽的数量和支气管蠕动增加。虽然尼氟酸+RA 肺与尼氟酸+AFS 肺相似，但嗜碱性粒细胞不能使这些测量值正常化。PGP 9.5 蛋白在尼氟酸肺中减少，但加入 AFS 细胞后，其值与对照相似。α-肌动蛋白的表达无差异。在体内，在尼氟酸暴露的大鼠中注射 AFS 细胞后，表面、终末芽的数量和蠕动与对照相似。与 TTF-1 阳性细胞的共定位被发现。在尼氟酸+AFS 细胞外植体中，FGF-10 和 VEGFα 的水平增加，而 TGF-β1 的水平与对照相似。在体外和体内，尼氟酸外植体的肺生长、支气管运动和神经支配减少，并且被 AFS 细胞挽救，这与以前观察到的 RA 相似。AFS 细胞的有益作用可能与生长因子分泌的旁分泌作用有关。
