骨桥蛋白调控骨髓间充质干细胞抑制宫腔粘连内膜纤维化的作用研究

Effect of osteopontin regulating bone marrow mesenchymal stem cells on inhibiting endometrial fibrosis of intrauterine adhesions

摘要:

目的： 探究骨桥蛋白（osteopontin，OPN）在抑制小鼠宫腔粘连（intrauterine adhesions，IUA）后纤维化中的作用。方法： 分离培养C57 BL/6小鼠和OPN^-/-C57 BL/6小鼠的原代骨髓间充质干细胞（mesenchymal stem cells，MSCs）；利用流式细胞仪检测分离的细胞表面抗原。将30只雌性小鼠随机分为假手术组、模型组、OPN^-/-MSCs组、MSCs组、溶剂组，每组6只。造模1周后，将MSCs、OPN^-/-MSCs分别注射至小鼠左右宫角；治疗结束2周后，二维超声诊断小鼠子宫形态，HE染色和Masson染色检测子宫组织内膜形态变化及纤维化情况，免疫组化染色检测α平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）表达，Western blot检测转化生长因子-β1（transforming growth factor-β1，TGF-β1）、血管内皮生长因子（vascular endothelial growth factor，VEGF）、Ⅰ型胶原α1（typeⅠcollagen α1，COL1A1）、Ⅲ型胶原α1（typeⅢcollagen α1，COL3A1）蛋白表达。结果： 成功分离并培养得到MSCs与OPN^-/-MSCs细胞，且在2组细胞中均检测到CD29、CD44和CD90呈阳性表达，CD34呈阴性表达。与模型组比较，OPN^-/-MSCs组和MSCs组子宫形态病变得到改善，腺体数目不同程度增多，纤维化面积有所减少，α-SMA表达明显下降（P=0.000），子宫组织中TGF-β1、VEGF、COL1A1及COL3A1蛋白表达水平均明显下降（均P=0.000）。与OPN^-/-MSCs组比较，MSCs组子宫形态改善更为明显，腺体数目增多，而纤维化面积减少，α-SMA表达明显下降（P=0.000），子宫组织中TGF-β1、VEGF、COL1A1及COL3A1蛋白表达水平也明显下降（均P=0.000）。结论： MSCs比OPN^-/-MSCs注射宫腔粘连小鼠后抑制宫腔粘连内膜纤维化的作用更明显，表明OPN具有调控MSCs抑制小鼠宫腔粘连内膜纤维化的作用。

Abstract:

Objective: To explore the role of osteopontin (OPN) in inhibiting fibrosis after intrauterine adhesions (IUA) in mice. Methods: Primary bone marrow mesenchymal stem cells (MSCs) of C57 BL/6 mice and OPN^-/-C57 BL/6 mice were isolated and cultured, and the surface antigens of separated cells were detected by flow cytometry. Thirty female mice were randomized into sham operation group, model group, OPN^-/-MSCs group, MSCs group, and solvent group, with 6 mice in each group. One week after modeling, MSCs and OPN^-/-MSCs were injected into the left and right uterine horns of the mice respectively. Two weeks after the treatment, two-dimensional ultrasound was conducted for the diagnosis of mouse uterus morphology, HE staining and Masson staining for the detection of the morphological changes and fibrosis of the endometrium of the uterus, immunohistochemical staining for the detection of the expression of α-smooth muscle actin (α-SMA), and Western blot for the detection of the protein expression of transforming growth factor-β1 (TGF-β1), vascular endothelial growth factor (VEGF), typeⅠcollagen α1 (COL1A1), and typeⅢcollagen α1 (COL3A1). Results: MSCs and OPN^-/-MSCs cells were successfully isolated and cultured, and expression of CD29, CD44 and CD90 were positive and expression of CD34 was negative in both groups. Compared with the model group, the morphological changes of the uterus in the OPN^-/-MSCs group and the MSCs group were improved, the number of glands increased in varying degrees, the area of fibrosis decreased, and the positive expression of α-SMA decreased significantly (P=0.000). TGF-β1, VEGF, COL1A1 and COL3A1 protein expression levels were significantly decreased (all P=0.000). Compared with the OPN^-/-MSCs group, the uterine morphology of the MSCs group improved more markedly, the number of glands increased, and the area of fibrosis decreased, the positive expression of α-SMA decreased significantly (P=0.000), and the expression levels of TGF-β1, VEGF, COL1A1 and COL3A1 proteins also decreased significantly (all P=0.000). Conclusion: MSCs are more effective than OPN^-/-MSCs in inhibiting intrauterine adhesions and endometrial fibrosis in mice, indicating that OPN has the effect of regulating bone marrow mesenchymal stem cells to inhibit endometrial fibrosis of intrauterine adhesions in mice.