[Treatment of intrauterine adhesions in rats with hypoxia-cultured BMSC-derived exosomes].

Objective: To perform intrauterine adhesion modeling, and to investigate the repair effect of hypoxic treated bone marrow mesenchymal stem cells (BMSC) and their derived exosomes (BMSC-exo) on endometrial injury. Methods: BMSC and their exosomes BMSC-exo extracted from rats' femur were cultured under conventional oxygen condition (21%O2) or hypoxia condition (1%O2). Intrauterine adhesion modeling was performed on 40 healthy female SD rats by intrauterine injection of bacterial lipopolysaccharide after curettage. On the 28th day of modeling, 40 rat models were randomly divided into five groups, and interventions were performed: (1) NC group: 0.2 ml phosphate buffered solution was injected into each uterine cavity; (2) BMSC group: 0.2 ml BMSC (1×106/ml) with conventional oxygen culture was injected intrauterine; (3) L-BMSC group: 0.2 ml of hypoxic cultured BMSC (1×106/ml) was injected intrauterine; (4) BMSC-exo group: 0.2 ml of BMSC-exo cultured with conventional oxygen at a concentration of 500 μg/ml was injected into the uterine cavity; (5) L-BMSC-exo group: 0.2 ml hypoxic cultured BMSC-exo (500 μg/ml) was injected intrauterine. On the 14th and 28th day of treatment, four rats in each group were sacrificed by cervical dislocation after anesthesia, and endometrial tissues were collected. Then HE and Masson staining were used to observe and calculate the number of glands and fibrosis area in the endometrium. The expressions of angiogenesis related cytokines [vascular endothelial growth factor A (VEGFA) and CD31], and fibrosis-related proteins [collagen-Ⅰ, collagen-Ⅲ, smooth muscle actin α (α-SMA), and transforming growth factor β1 (TGF-β1)] in endometrial tissues were detected by western blot. Results: (1) HE and Masson staining showed that the number of endometrial glands in L-BMSC group, BMSC-exo group and L-BMSC-exo group increased and the fibrosis area decreased compared with NC group on the 14th and 28th day of treatment (all P<0.05). Noteworthily, the changes of L-BMSC-exo group were more significant than those of BMSC-exo group (all P<0.05), and the changes of BMSC-exo group were greater than those of BMSC group (all P<0.05). (2) Western blot analysis showed that, compared with NC group, the expressions of collagen-Ⅲ and TGF-β1 in BMSC group, L-BMSC group, BMSC-exo group and L-BMSC-exo group decreased on the 14th and 28th day of treatment (all P<0.05). As the treatment time went on, the expressions of fibrosis-related proteins were different. Compared with BMSC group, the expressions of collagen-Ⅲ, α-SMA and TGF-β1 in the BMSC-exo group and L-BMSC group decreased on the 28th day (all P<0.05). Moreover, the expressions of collagen-Ⅲ and TGF-β1 in L-BMSC-exo group were lower than those in BMSC-exo group on the 28th day (all P<0.05). And the expressions of collagen-Ⅰ, α-SMA and TGF-β1 in L-BMSC-exo group were lower than those in L-BMSC group on the 28th day (all P<0.05). (3) The results of western blot analysis of VEGFA and CD31 showed that, the expressions of VEGFA and CD31 in BMSC group, L-BMSC group, BMSC-exo group and L-BMSC-exo group increased on the 14th and 28th day of treatment compared with NC group (all P<0.05). Treatment for 28 days, the expressions of VEGFA and CD31 in BMSC-exo group and CD31 in L-BMSC group were higher than those in BMSC group (all P<0.05). Moreover, the expressions of VEGFA and CD31 in L-BMSC-exo group were higher than those in BMSC-exo group and L-BMSC group on the 28th day (all P<0.05). Conclusions: Treatment of BMSC and their exosomes BMSC-exo with hypoxia could promote endometrial gland hyperplasia, inhibit tissue fibrosis, and further repair the damaged endometrium in rats with intrauterine adhesion. Importantly, hypoxic treatment of BMSC-exo is the most effective in intrauterine adhesion rats.目的： 构建宫腔粘连动物模型，并探讨低氧处理骨髓间充质干细胞（BMSC）及其来源的外泌体（BMSC-exo）对子宫内膜损伤的修复作用。 方法： 在常规氧（21%O2）或低氧（1%O2）条件下培养从大鼠股骨中提取的BMSC及其BMSC-exo。采用刮宫+脂多糖宫腔内注射双重损伤的方法对40只健康雌性SD大鼠进行宫腔粘连建模，并于建模第28天，将40只模型大鼠采用随机数字表法随机分为5组（每组各8只），并进行干预：（1）NC组：每侧宫腔注射0.2 ml磷酸盐缓冲液；（2）BMSC组：每侧宫腔注射0.2 ml浓度1×106/ml常规氧培养的BMSC；（3）L-BMSC组：每侧宫腔注射0.2 ml浓度1×106/ml低氧培养的BMSC；（4）BMSC-exo组：每侧宫腔注射0.2 ml浓度500 μg/ml常规氧培养的BMSC-exo；（5）L-BMSC-exo组：每侧宫腔注射0.2 ml浓度500 μg/ml低氧培养的BMSC-exo。分别于治疗第14、28天，采用麻醉后颈椎脱臼法各处死每组大鼠4只，取子宫内膜组织，进行HE和Masson染色观察计算子宫内膜的腺体数量和纤维化情况，蛋白印迹法（western blot）检测大鼠子宫内膜组织中血管生成相关细胞因子［血管内皮生长因子A（VEGFA）、CD31］和纤维化相关蛋白［胶原蛋白Ⅰ（collagen-Ⅰ）、胶原蛋白Ⅲ（collagen-Ⅲ）、平滑肌肌动蛋白α（α-SMA）、转化生长因子β1（TGF-β1）］的表达情况。 结果： （1）HE和Masson染色结果显示，治疗第14、28天，与NC组相比，L-BMSC组、BMSC-exo组和L-BMSC-exo组宫腔粘连模型大鼠子宫内膜腺体数量均增加、子宫内膜纤维化面积均减少（P均<0.05），且L-BMSC-exo组较BMSC-exo组变化显著（P均<0.05），BMSC-exo组较BMSC组变化显著（P均<0.05）。（2）western blot检测纤维化相关蛋白（collagen-Ⅰ、collagen-Ⅲ、α-SMA、TGF-β1）表达结果显示，治疗第14、28天时，与NC组相比，BMSC组、L-BMSC组、BMSC-exo组和L-BMSC-exo组collagen-Ⅲ、TGF-β1的表达均降低（P均<0.05）。随着治疗时间的延长，纤维化相关蛋白表达情况不同，治疗第28天，与BMSC组相比，BMSC-exo组和L-BMSC组collagen-Ⅲ、α-SMA和TGF-β1的表达均降低（P均<0.05）；且L-BMSC-exo组collagen-Ⅲ和TGF-β1的表达均较BMSC-exo组更低（P均<0.05）；L-BMSC-exo组collagen-Ⅰ、α-SMA和TGF-β1的表达均较L-BMSC组更低（P均<0.05）。（3）western blot检测血管生成相关细胞因子（VEGFA、CD31）表达的结果显示，治疗第14、28天时，与NC组相比，BMSC组、L-BMSC组、BMSC-exo组和L-BMSC-exo组VEGFA、CD31的表达均升高（P均<0.05）。治疗第28天，与BMSC组相比，BMSC-exo组VEGFA和CD31的表达以及L-BMSC组CD31的表达均升高（P均<0.05）；且L-BMSC-exo组VEGFA和CD31的表达均较BMSC-exo组和L-BMSC组更高（P均<0.05）。 结论： 低氧处理的BMSC及其BMSC-exo均能促进宫腔粘连模型大鼠子宫内膜腺体增生，抑制纤维化，促进子宫内膜血管生成，进一步修复受损的子宫内膜。低氧处理的BMSC-exo对宫腔粘连模型大鼠具有更好的治疗效果。.