间充质干细胞来源的外泌体通过 TGF-β1/Smad 途径逆转 EMT 并促进受损子宫内膜修复。

BACKGROUND: Intrauterine adhesion (IUA) is one of the most serious complications in patients with endometrial repair disorder after injury. Currently, there is no effective treatment for IUA. Stem cell is the main candidate of new therapy, which functions mainly through paracrine mechanism. Stem-derived exosomes (Exo) play an important role in tissue injury. Here, we mainly aim to study the effect of bone marrow mesenchymal stem cell (BMSC)-derived Exo on repairing endometrium of IUA animal models and its effect on TGF-β1 induced EMT in endometrial epithelial cells (EECs). METHODS: Totally, 64 female rabbits were randomly divided into Sham operation group, model group, BMSC treatment group, and Exo treatment group. EMT in EECs was induced by TGF-β1. Then, EECs were treated with Exo (25 μg/ml, 50 μg/ml, 100 μg/ml) for 24 h. HE staining and Masson staining were used to evaluate the changes in glandular number and fibrosis area. The expression levels of CK19 and VIM were detected by immunohistochemistry. Western blotting was used to detect the expression of CK19, VIM, FSP-1, E-cadherin, TGF-β1, TGF-β1R, Smad 2, and P-Smad 2. RT-PCR was used to detect mRNA expression levels of CK19, VIM, FSP-1, E-cadherin, TGF-β1, TGF-β1R, and Smad 2. RESULTS: Compared with the model group, the number of endometrial glands was significantly increased and endometrial fibrosis area was significantly decreased in BMSC and Exo groups (P < 0.05). CK19 level significantly increased whereas VIM level significantly decreased after treatment of BMSCs and Exo (P < 0.05). Additionally, the expressions of TGF-β1, TGF-β1R, and Smad2 mRNA were all significantly decreased after BMSC and Exo treatment (P < 0.05). Besides, phosphorylation levels of TGF-β1, TGF-β1R, and Smad2 were also significantly decreased in BMSC and Exo treatment groups (P < 0.05). Furthermore, there was no significant difference between BMSC and Exo treatment groups (P > 0.05). EMT was induced in EECs by 60 ng/ml TGF-β1 for 24 h. After Exo treatment for 24 h, mRNA expressions of CK-19 and E-cadherin increased, while those of VIM, FSP-1, TGF-β1, and Smad2 decreased. Additionally, protein expressions of CK-19 and E-cadherin increased, while those of VIM, FSP-1, TGF-β1, Smad2, and P-Smad2 decreased. CONCLUSIONS: BMSC-derived Exo is involved in the repair of injured endometrium, with similar effect to that of BMSC, and can reverse EMT in rabbit EECs induced by TGF-β1. BMSC-derived Exo may promote endometrial repair by the TGF-β1/Smad signaling pathway.

背景：宫腔粘连（IUA）是子宫内膜修复障碍患者在损伤后最严重的并发症之一。目前，IUA 尚无有效的治疗方法。干细胞是新疗法的主要候选者，主要通过旁分泌机制发挥作用。干细胞衍生的外泌体（Exo）在组织损伤中发挥重要作用。在这里，我们主要旨在研究骨髓间充质干细胞（BMSC）衍生的 Exo 对 IUA 动物模型子宫内膜修复的影响及其对 TGF-β1 诱导的子宫内膜上皮细胞（EEC）上皮间质转化（EMT）的影响。

方法：共 64 只雌性兔子随机分为假手术组、模型组、BMSC 治疗组和 Exo 治疗组。用 TGF-β1 诱导 EEC 发生 EMT。然后，用 Exo（25μg/ml、50μg/ml、100μg/ml）处理 EEC 24 小时。用 HE 染色和 Masson 染色评估腺体数量和纤维化面积的变化。免疫组织化学检测 CK19 和 VIM 的表达。Western blot 检测 CK19、VIM、FSP-1、E-cadherin、TGF-β1、TGF-β1R、Smad 2 和 P-Smad 2 的表达。RT-PCR 检测 CK19、VIM、FSP-1、E-cadherin、TGF-β1、TGF-β1R 和 Smad 2 的 mRNA 表达水平。

结果：与模型组相比，BMSC 和 Exo 组子宫内膜腺体数量明显增加，子宫内膜纤维化面积明显减少（P<0.05）。BMSC 和 Exo 处理后 CK19 水平明显升高，而 VIM 水平明显降低（P<0.05）。此外，BMSC 和 Exo 处理后 TGF-β1、TGF-β1R 和 Smad2mRNA 的表达均明显降低（P<0.05）。此外，BMSC 和 Exo 处理组 TGF-β1、TGF-β1R 和 Smad2 的磷酸化水平也明显降低（P<0.05）。此外，BMSC 和 Exo 治疗组之间无显著差异（P>0.05）。用 60ng/ml TGF-β1 诱导 EEC 发生 EMT 24 小时。用 Exo 处理 24 小时后，CK-19 和 E-cadherin 的 mRNA 表达增加，而 VIM、FSP-1、TGF-β1 和 Smad2 的表达减少。此外，CK-19 和 E-cadherin 的蛋白表达增加，而 VIM、FSP-1、TGF-β1、Smad2 和 P-Smad2 的蛋白表达减少。

结论：BMSC 衍生的 Exo 参与受损子宫内膜的修复，作用与 BMSC 相似，并能逆转 TGF-β1 诱导的兔 EEC 的 EMT。BMSC 衍生的 Exo 可能通过 TGF-β1/Smad 信号通路促进子宫内膜修复。