Objective: To investigate the repair effect of human umbilical cord blood mesenchymal stem cells (hUCB-MSC) on endometrium of intrauterine adhesion (IUA) by establishing animal model. Methods: Eighteen healthy female New Zealand white rabbits were divided into a control group, an IUA group and an hUCB-MSC transplantation group according to random number table method. The control group underwent laparotomy only. The IUA group underwent IUA modeling surgery using curettage and lipopolysaccharide (LPS) cotton placed in uterine cavity for double injury. The hUCB-MSC transplantation group received the same IUA modeling surgery followed by multipoint injection of hUCB-MSC suspension (2×106 cells/ml, 500 μl) into the bilateral uterine myometrium one week after surgery. Four weeks after the IUA modeling surgery, the rabbits were euthanized and samples were collected. Hematoxylin and eosin (HE) staining and Masson staining were used to assess the number of endometrial glands and the fibrosis rate. RNA sequencing was performed on the endometrium of the IUA group and hUCB-MSC transplantation group.The mRNA and protein expression of the fibrosis markers [transforming growth factor β1 (TGF-β1) and tissue inhibitors of metalloproteinase 1 (TIMP1)] and RNA sequencing-related parameters were detected by quantitative real-time PCR (qRT-PCR) and Western blot. Results: Compared with the control group, the number of glands in IUA group decreased (26.33±1.53 vs 4.33±1.53, P<0.001), and the fibrosis rate increased (18.01%±2.21% vs 69.55%±2.42%, P<0.001), indicating successful modeling. HE and Masson staining revealed that the number of glands in the hUCB-MSC transplantation group was increased compared with that in IUA group (17.33±2.52 vs 4.33±1.53, P<0.001), and the fibrosis rate decreased (69.55%±2.42% vs 41.55%±1.99%,P=0.001). Western blot analyses of fibrosis-related proteins (TGF-β1 and TIMP1) showed elevated levels of TGF-β1 (0.91±0.05) and TIMP1 (0.99±0.01) proteins in the IUA group compared to control group (0.61±0.04, 0.68±0.07) (P=0.001, 0.015). The hUCB-MSC transplantation group exhibited reduced expression of TGF-β1 (0.69±0.04) and TIMP1 (0.62±0.08) proteins compared to the IUA group (P=0.005, 0.014). Western Blot results related to the PI3K/AKT signaling pathway [phosphorylated phosphatidylinositol 3-kinase (p-PI3K), phosphorylated protein kinase B (p-AKT)] showed that the expression of p-PI3K(1.05±0.05) and p-AKT(1.17±0.06) in the IUA group increased compared to the control group (0.78±0.03, 0.85±0.05) (P=0.002, 0.002). The expression of p-PI3K (0.74±0.02) and p-AKT (0.93±0.04) proteins in the hUCB-MSC transplantation group decreased compared to the IUA group (P=0.003, 0.005). Conclusion: hUCB-MSC can repair the damaged endometrium in rabbit intrauterine adhesion model by increasing the number of endometrial glands and improving its level of fibrosis.
目的： 构建宫腔粘连动物模型，探究人脐血间充质干细胞（hUCB-MSC）对宫腔粘连（IUA）模型子宫内膜的修复效果。 方法： 将18只健康雌性新西兰大白兔按照随机数字表法平均分为对照组、IUA组和hUCB-MSC移植组。对照组仅开腹处理，IUA组采用刮宫及放置脂多糖（LPS）棉线双重损伤的方法行IUA造模手术，hUCB-MSC移植组行IUA造模手术，术后1周于双侧子宫肌壁间多点注射hUCB-MSC悬液（2×106/ml，500 μl）。IUA造模术后4周处死大白兔进行取材，通过苏木精-伊红（HE）染色和Masson染色检测子宫内膜腺体数量和纤维化率。对IUA组及hUCB-MSC 移植组子宫内膜行RNA转录组测序。通过实时定量逆转录聚合酶链反应（qRT-PCR）及Western印迹法等方法检测纤维化指标［转化生长因子（TGF-β1）及基质金属蛋白酶抑制因子1（TIMP1）］和磷脂酰肌醇3-激酶/蛋白激酶B（PI3K/AKT）信号相关mRNA及蛋白表达。 结果： 与对照组相比，IUA组腺体数量减少（26.33±1.53比4.33±1.53，P<0.001），纤维化率增高（18.01%±2.21%比69.55%±2.42%，P<0.001），证明造模成功；与IUA组相比，hUCB-MSC移植组腺体数目增加（4.33±1.53比17.33±2.52，P<0.001），纤维化率降低（69.55%±2.42%比41.55%±1.99%，P=0.001）。与对照组相比，IUA组TGF-β1及TIMP1蛋白表达水平升高（分别为：0.91±0.05比0.61±0.04、0.99±0.01比0.68±0.07）（P=0.001、0.015）；hUCB-MSC移植组TGF-β1（0.69±0.04）和TIMP1（0.62±0.08）蛋白表达水平与IUA组相比下降（P=0.005、0.014）。IUA组磷酸化PI3K（p-PI3K）（1.05±0.05）及磷酸化AKT（p-AKT）（1.17±0.06）表达与对照组相比（0.78±0.03、0.85±0.05）升高（P=0.002、0.002）；hUCB-MSC移植组p-PI3K（0.74±0.02）及p-AKT（0.93±0.04）蛋白与IUA组相比降低（P=0.003、0.005）。 结论： hUCB-MSC能促进兔宫腔粘连模型子宫内膜腺体增生，改善其纤维化水平，从而修复受损的子宫内膜。.