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Abstract：
OBJECTIVE: To observe the effect of human umbilical cord mesenchymal stem cells (hUCMSCs) secretions on the relevant factors in mouse retinal astrocytes, and to investigate the effect of hUCMSCs on the expression of vascular endothelial growth factor-A (VEGF-A) and to observe the therapeutic effect on the mouse model of retinopathy of prematurity (ROP).
METHODS: Cultured hUCMSCs and extracted exosomes from them and then retinal astrocytes were divided into control group and hypoxia group. MTT assay, flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to detect related indicators. Possible mechanisms by which hUCMSCs exosomes affect VEGF-A expression in hypoxia-induced mouse retinal astrocytes were explored. At last, the efficacy of exosomes of UCMSCs in a mouse ROP model was explored. Graphpad6 was used to comprehensively process data information.
RESULTS: The secretion was successfully extracted from the culture supernatant of hUCMSCs by gradient ultracentrifugation. Reactive oxygen species (ROS) and hypoxia inducible factor-1α (HIF-1α) of mice retinal astrocytes under different hypoxia time and the expression level of VEGF-A protein and VEGF-A mRNA increased, and the ROP cell model was established after 6h of hypoxia. The secretions of medium and high concentrations of hUCMSCs can reduce ROS and HIF-1α, the expression levels of VEGF-A protein and VEGF-A mRNA are statistically significant and concentration dependent. Compared with the ROP cell model group, the expression of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signal pathway related factors in the hUCMSCs exocrine group is significantly decreased. The intravitreal injection of the secretions of medium and high concentrations of hUCMSCs can reduce VEGF-A and HIF-1α in ROP model tissues. HE staining shows that the number of retinal neovascularization in ROP mice decreases with the increase of the dose of hUCMSCs secretion.
CONCLUSIONS: In a hypoxia induced mouse retinal astrocyte model, hUCMSCs exosomes are found to effectively reduce the expression of HIF-1α and VEGF-A, which are positively correlated with the concentration of hUCMSCs exosomes. HUCMSCs exosomes can effectively reduce the number of retinal neovascularization and the expression of HIF-1α and VEGF-A proteins in ROP mice, and are positively correlated with drug dosage. Besides, they can reduce the related factors on the PI3K/AKT/mTOR signaling pathway.
METHODS: Cultured hUCMSCs and extracted exosomes from them and then retinal astrocytes were divided into control group and hypoxia group. MTT assay, flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to detect related indicators. Possible mechanisms by which hUCMSCs exosomes affect VEGF-A expression in hypoxia-induced mouse retinal astrocytes were explored. At last, the efficacy of exosomes of UCMSCs in a mouse ROP model was explored. Graphpad6 was used to comprehensively process data information.
RESULTS: The secretion was successfully extracted from the culture supernatant of hUCMSCs by gradient ultracentrifugation. Reactive oxygen species (ROS) and hypoxia inducible factor-1α (HIF-1α) of mice retinal astrocytes under different hypoxia time and the expression level of VEGF-A protein and VEGF-A mRNA increased, and the ROP cell model was established after 6h of hypoxia. The secretions of medium and high concentrations of hUCMSCs can reduce ROS and HIF-1α, the expression levels of VEGF-A protein and VEGF-A mRNA are statistically significant and concentration dependent. Compared with the ROP cell model group, the expression of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signal pathway related factors in the hUCMSCs exocrine group is significantly decreased. The intravitreal injection of the secretions of medium and high concentrations of hUCMSCs can reduce VEGF-A and HIF-1α in ROP model tissues. HE staining shows that the number of retinal neovascularization in ROP mice decreases with the increase of the dose of hUCMSCs secretion.
CONCLUSIONS: In a hypoxia induced mouse retinal astrocyte model, hUCMSCs exosomes are found to effectively reduce the expression of HIF-1α and VEGF-A, which are positively correlated with the concentration of hUCMSCs exosomes. HUCMSCs exosomes can effectively reduce the number of retinal neovascularization and the expression of HIF-1α and VEGF-A proteins in ROP mice, and are positively correlated with drug dosage. Besides, they can reduce the related factors on the PI3K/AKT/mTOR signaling pathway.
摘要：
目的：观察人脐带间充质干细胞(hUCMSCs)分泌对小鼠视网膜星形胶质细胞相关因子的影响。探讨hUCMSCs对血管内皮生长因子-A(VEGF-A)表达的影响,并观察其对早产儿视网膜病变(ROP)小鼠模型的治疗作用。
方法：培养hUCMSCs并从中提取外泌体，然后将视网膜星形胶质细胞分为对照组和缺氧组。MTT测定，流式细胞术,采用逆转录-聚合酶链反应(RT-PCR)和Westernblot检测相关指标。探讨了hUCMSCs外泌体影响缺氧诱导的小鼠视网膜星形胶质细胞VEGF-A表达的可能机制。最后,研究了UCMSCs外泌体在小鼠ROP模型中的功效。Graphpad6用于综合处理数据信息。
结果：通过梯度超速离心从hUCMSCs的培养上清液中成功提取分泌物。不同缺氧时间下小鼠视网膜星形胶质细胞的活性氧(ROS)和缺氧诱导因子-1α(HIF-1α)表达水平及VEGF-A蛋白和VEGF-AmRNA表达水平升高,缺氧6h后建立ROP细胞模型。中、高浓度hUCMSCs的分泌可降低ROS和HIF-1α,VEGF-A蛋白和VEGF-AmRNA的表达水平具有统计学意义和浓度依赖性。与ROP细胞模型组相比，磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路相关因子在hUCMSCs外分泌组中的表达显著降低。玻璃体内注射中、高浓度的hUCMSCs可以减少ROP模型组织中的VEGF-A和HIF-1α。HE染色显示ROP小鼠视网膜新生血管的数量随着hUCMSCs分泌剂量的增加而减少。
结论：在缺氧诱导的小鼠视网膜星形胶质细胞模型中，发现hUCMSCs外泌体能有效降低HIF-1α和VEGF-A的表达,与hUCMSCs外泌体浓度呈正相关。HUCMSCs外泌体能有效降低ROP小鼠视网膜新生血管的数量及HIF-1α和VEGF-A蛋白的表达,与药物剂量呈正相关。此外,能降低PI3K/AKT/mTOR信号通路的相关因子。
方法：培养hUCMSCs并从中提取外泌体，然后将视网膜星形胶质细胞分为对照组和缺氧组。MTT测定，流式细胞术,采用逆转录-聚合酶链反应(RT-PCR)和Westernblot检测相关指标。探讨了hUCMSCs外泌体影响缺氧诱导的小鼠视网膜星形胶质细胞VEGF-A表达的可能机制。最后,研究了UCMSCs外泌体在小鼠ROP模型中的功效。Graphpad6用于综合处理数据信息。
结果：通过梯度超速离心从hUCMSCs的培养上清液中成功提取分泌物。不同缺氧时间下小鼠视网膜星形胶质细胞的活性氧(ROS)和缺氧诱导因子-1α(HIF-1α)表达水平及VEGF-A蛋白和VEGF-AmRNA表达水平升高,缺氧6h后建立ROP细胞模型。中、高浓度hUCMSCs的分泌可降低ROS和HIF-1α,VEGF-A蛋白和VEGF-AmRNA的表达水平具有统计学意义和浓度依赖性。与ROP细胞模型组相比，磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路相关因子在hUCMSCs外分泌组中的表达显著降低。玻璃体内注射中、高浓度的hUCMSCs可以减少ROP模型组织中的VEGF-A和HIF-1α。HE染色显示ROP小鼠视网膜新生血管的数量随着hUCMSCs分泌剂量的增加而减少。
结论：在缺氧诱导的小鼠视网膜星形胶质细胞模型中，发现hUCMSCs外泌体能有效降低HIF-1α和VEGF-A的表达,与hUCMSCs外泌体浓度呈正相关。HUCMSCs外泌体能有效降低ROP小鼠视网膜新生血管的数量及HIF-1α和VEGF-A蛋白的表达,与药物剂量呈正相关。此外,能降低PI3K/AKT/mTOR信号通路的相关因子。
