背景:宫腔粘连(IUA)是育龄妇女不孕症的最严重原因之一,子宫内膜继发损伤。干细胞疗法可有效治疗受损的子宫内膜。目前的报道主要集中在干细胞通过旁分泌或转分化的治疗效果。分别。这项研究调查了在治疗IUA时是否优先发生旁分泌或转分化。
方法:体外共培养人羊膜间充质干细胞(hAMSCs)和转化生长因子β(TGF-β1)诱导的人子宫内膜基质细胞(THESCs)。纤维连接蛋白(FN)的mRNA和蛋白表达水平,胶原蛋白I,细胞角蛋白19(CK19),定量实时聚合酶链反应(qPCR)检测E-cadherin(E-cad)和波形蛋白,蛋白质印迹(WB)和免疫组织化学染色(IHC)。采用SD大鼠建立IUA模型。hAMSCs,hAMSCs-条件培养基(hAMSCs-CM),将GFP标记的hAMSCs注射到子宫内,分别。通过Masson染色评估子宫内膜的纤维化区域。通过苏木精和伊红(H&E)检测子宫内膜腺体的数量。通过免疫荧光(IF)追踪GFP标记的hAMSCs。hAMSCs,联合PPCNg(hAMSCs/PPCNg),被注射进阴道,与宫腔内注射相比。
结果:qPCR和WB显示,与hAMSCs共培养后,IUA-THESCs中FN和I型胶原水平显著降低。此外,CK19,E-cad,共培养2天后,hAMSCs中波形蛋白的表达无明显差异。共培养后6天,CK19、E-cad和Vimentin在hAMSCs中的表达均有明显变更。组织学测定显示hAMSCs和hAMSCs-CM组的子宫内膜腺体增加和纤维化面积显著减少。然而,这些变化在两组之间没有统计学差异.在体内,荧光成像显示GFP-hAMSCs定位于子宫内膜间质并逐渐发生凋亡。阴道注射hAMSCs的效果与H&E染色评估的宫内注射效果相当,MASSON染色和IHC。
结论:我们的数据表明hAMSCs通过旁分泌促进子宫内膜修复,优先于转分化。
IUA是育龄妇女不孕的关键原因,诊所没有找到令人满意的治疗措施。hAMSCs可以通过旁分泌和转分化机制有效治疗宫腔粘连。本研究在体外和体内证实羊膜间充质干细胞优先通过旁分泌抑制子宫内膜纤维化和促进上皮修复,从而有效治疗宫腔粘连。在体外与hAMSCs共培养2天后,IUA-THESCs中的纤维化标志物蛋白水平显着降低。然而,hAMSCs上皮标记蛋白水平显著升高,需要至少6天的共培养。hAMSCs-CM在抑制IUA大鼠纤维化和促进子宫内膜修复方面具有与hAMSCs相同的疗效,支持hAMSCs通过体内旁分泌促进子宫内膜重塑的观点。此外,GFP标记的hAMSCs持续定植于子宫内膜基质而不是上皮,并逐渐发生凋亡。这些发现证明hAMSCs通过旁分泌改善IUA的子宫内膜纤维化,优先于转分化,为hAMSCs精准治疗IUA提供最新见解,为促进MSCs的“无细胞治疗”提供理论依据。
BACKGROUND: Intrauterine adhesion (IUA) is one of the most severe causes of infertility in women of childbearing age with injured endometrium secondary to uterine performance. Stem cell therapy is effective in treating damaged endometrium. The current reports mainly focus on the therapeutic effects of stem cells through paracrine or transdifferentiation, respectively. This study investigates whether paracrine or transdifferentiation occurs preferentially in treating IUA.
METHODS: Human amniotic mesenchymal stem cells (hAMSCs) and transformed human endometrial stromal cells (THESCs) induced by transforming growth factor beta (TGF-β1) were co-cultured in vitro. The mRNA and protein expression levels of Fibronectin (FN), Collagen I, Cytokeratin19 (CK19), E-cadherin (E-cad) and Vimentin were detected by Quantitative real-time polymerase chain reaction (qPCR), Western blotting (WB) and Immunohistochemical staining (IHC). The Sprague-Dawley (SD) rats were used to establish the IUA model. hAMSCs, hAMSCs-conditional medium (hAMSCs-CM), and GFP-labeled hAMSCs were injected into intrauterine, respectively. The fibrotic area of the endometrium was evaluated by Masson staining. The number of endometrium glands was detected by hematoxylin and eosin (H&E). GFP-labeled hAMSCs were traced by immunofluorescence (IF). hAMSCs, combined with PPCNg (hAMSCs/PPCNg), were injected into the vagina, which was compared with intrauterine injection.
RESULTS: qPCR and WB revealed that FN and Collagen I levels in IUA-THESCs decreased significantly after co-culturing with hAMSCs. Moreover, CK19, E-cad, and Vimentin expressions in hAMSCs showed no significant difference after co-culture for 2 days. 6 days after co-culture, CK19, E-cad and Vimentin expressions in hAMSCs were significantly changed. Histological assays showed increased endometrial glands and a remarkable decrease in the fibrotic area in the hAMSCs and hAMSCs-CM groups. However, these changes were not statistically different between the two groups. In vivo, fluorescence imaging revealed that GFP-hAMSCs were localized in the endometrial stroma and gradually underwent apoptosis. The effect of hAMSCs by vaginal injection was comparable to that by intrauterine injection assessed by H&E staining, MASSON staining and IHC.
CONCLUSIONS: Our data demonstrated that hAMSCs promoted endometrial repair via paracrine, preferentially than transdifferentiation.
IUA is the crucial cause of infertility in women of childbearing age, and no satisfactory treatment measures have been found in the clinic. hAMSCs can effectively treat intrauterine adhesions through paracrine and transdifferentiation mechanisms. This study confirmed in vitro and in vivo that amniotic mesenchymal stem cells preferentially inhibited endometrial fibrosis and promoted epithelial repair through paracrine, thus effectively treating intrauterine adhesions. The level of fibrosis marker proteins in IUA-THESCs decreased significantly after co-culturing with hAMSCs for 2 days in vitro. However, the level of epithelial marker proteins in hAMSCs increased significantly, requiring at least 6 days of co-culture. hAMSCs-CM had the same efficacy as hAMSCs in inhibiting fibrosis and promoting endometrial repair in IUA rats, supporting the idea that hAMSCs promoted endometrial remodeling through paracrine in vivo. In addition, GFP-labeled hAMSCs continuously colonized the endometrial stroma instead of the epithelium and gradually underwent apoptosis. These findings prove that hAMSCs ameliorate endometrial fibrosis of IUA via paracrine, preferentially than transdifferentiation, providing the latest insights into the precision treatment of IUA with hAMSCs and a theoretical basis for promoting the “cell-free therapy” of MSCs.