间充质干细胞来源外泌体对视网膜光感受器细胞PI3K/AKT通路及Ang-1蛋白水平作用的研究

Effects of mesenchymal stem cells sourced exosomes on PI3K/AKT pathway and Ang-1 protein level of retinal photoreceptor cells

目的 探讨间充质干细胞(mesenchymal stem cell,MSC)下外泌体对视网膜光感受器细胞PI3K/AKT通路及Ang-1蛋白水平的影响。方法 应用免疫印迹检测人脐带间充质干细胞(human umbilical cord derived mesenchymal stem cells,h UCMSCs)外泌体表面标志蛋白CD63及CD90,应用流式细胞仪检测光损伤661W细胞凋亡,应用免疫荧光检测Ang-1,应用免疫印迹检测PI3K、p-PI3K、AKT、p-AKT蛋白表达。结果 形态学及免疫印迹检测CD63、CD90表达结果证实h UCMSCs外泌体。线粒体膜电位检测结果以及细胞形态学鉴定661W细胞建立光损伤模型成功。流式细胞仪、免疫荧光检测结果显示,与正常组比较模型组细胞凋亡率及Ang-1阳性表达增加,在经过h UCMSCs干预后有所降低,且呈现出明显的浓度依赖性(P<0.05)。免疫印迹结果显示,与正常组比较,模型组PI3K、AKT、p-PI3K、p-AKT表达降低,经过h UCMSCs干预后有所升高(P<0.05),且高浓度组PI3K、AKT、pPI3K、p-AKT表达水平与激动剂组比较无差异(P>0.05),模型组PI3K、AKT、p-PI3K、p-AKT表达水平高于抑制剂组(P<0.05)。结论 间充质干细胞下外泌体可能是通过激活PI3K/AKT通路,抑制了视网膜光感受器细胞的凋亡以及Ang-1的水平,从而对光损伤视网膜起保护作用。

Objective To investigate the effects of mesenchymal stem cell(MSC) sourced exosomes on PI3K/AKT pathway and ANG-1 protein level of retinal photoreceptor cells. Methods Western blotting was used to detect CD63 and CD90. Flow cytometry was used to detect apoptosis of 661W cells after light injury. Immunofluorescence was used to detect ANG-1, and western blotting was used to detect the expression of PI3K, p-PI3K, AKT, and pAKT. Results The expression of CD63 and CD90 in human umbilical cord derived mesenchymal stem cells was confirmed by morphology and western blot. Mitochondrial membrane potential detection results and cell morphology identification 661W cells successfully established the light damage model. The results of flow cytometry and immunofluorescence showed that compared to the normal group, the apoptosis rate and ang-1 positive expression of the model group increased, but decreased after the intervention of HUCMscs, and presented a significant concentration dependence(P<0.05). Western blotting results showed that compared with the normal group, the expressions of PI3K, AKT,P-PI3K and P-Akt in the model group decreased, but increased after the intervention of HUCMscs(P<0.05), and the expression levels of PI3K, AKT, p-PI3K and p-AKT in the high concentration group had no difference compared with the agonist group(P>0.05). The expression levels of PI3K, AKT, p-PI3K and p-AKT in the model group were higher than those in the inhibitor group(P<0.05). Conclusion Exosomes from mesenchymal stem cells may inhibit the apoptosis of retinal photoreceptor cells and the level of ANG-1 by activating the PI3K/AKT pathway, thus protecting the photodamaged retina.