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基质胶和激活素A可促进培养的人视网膜色素上皮细胞的细胞间接触和抗凋亡活性

Guo X, Zhu D, Lian R, et al.

期刊名称:Experimental Eye Research

卷期:2016年第146卷

关键词:
细胞 基质 上皮

摘要

年龄相关性黄斑变性(AMD)是老龄化人群失明的首要原因。目前,采用移植健康RPE细胞替代患病视网膜色素上皮(RPE)细胞可能是治疗AMD的一种可行的方法。然而,保持体外培养的视网膜色素上皮细胞的细胞 - 细胞间接触和良好存活率很困难的,从根本上决定RPE细胞移植的成功。本研究分析了基质胶和激活素AMA)在调节人RPE细胞 - 细胞间接触和抗细胞凋亡活性中的作用,通过原子力显微镜(AFM),扫描电子显微镜(SEM),免疫荧光染色,定量聚合酶链反应(qPCR)分析,膜联蛋白V /碘化丙啶(PI)分析,线粒体膜电位(△Ψ米)测定法,细胞内活性氧(ROS)测定法和Western印迹来评估。体外培养的hRPE细胞可在MA处理后保持其上皮形态至少4代。 MA可促进hRPE 细胞N-钙粘蛋白向外侧细胞边缘的接触。MA处理后可增强培养的hRPE细胞的紧密连接相关基因和蛋白质的表达,如紧密连接蛋白-1,紧密连接蛋白3occludinZO-1的表达,以及偏振ZO-1蛋白的分布和屏障功能。此外,血清撤离诱导的细胞凋亡情况下,MA处理可减少细胞凋亡,降低ROSBax,增加hRPE细胞的△Ψ m and Bcl2。此外,MA处理可升高β连环蛋白和其靶蛋白的蛋白表达水平,包括细胞周期素D1c-MycSurvivin,以及ZO-1,β连环蛋白,SurvivinTCF-4的基因表达水平,所有这些都可以通过Wnt /β-catenin途径抑制剂XAV-939下调。总之,MA处理能有效的促进视网膜色素上皮细胞的细胞 - 细胞间接触和抗细胞凋亡活性,部分涉及Wnt /β-catenin通路。这项研究将对理解hRPE细胞核将来的细胞治疗具有益处。

Age-related macular degeneration (AMD) is a leading cause of blindness among the aging population. Currently, replacement of diseased retinal pigment epithelium (RPE) cells with transplanted healthy RPE cells could be a feasible approach for AMD therapy. However, maintaining cell-cell contact and good viability of RPE cells cultured invitro is difficult and fundamentally determines the success of RPE cell transplantation. This study was conducted to examine the role of Matrigel and Activin A (MA) in regulating cell-cell contact and anti-apoptotic activity in human RPE (hRPE) cells, as assessed by atomic force microscopy (AFM), scanning electron microscope (SEM), immunofluorescence staining, quantitative polymerase chain reaction (qPCR) analysis, Annexin V/propidium iodide (PI) analysis, mitochondrial membrane potential (△Ψ m) assays, intracellular reactive oxygen species (ROS) assays and Western blotting. hRPE cells cultured invitro could maintain their epithelioid morphology after MA treatment over at least 4 passages. The contact of N-cadherin to the lateral cell border was promoted in hRPE cells at P2 by MA. MA treatment also enhanced the expression of tight junction-associated genes and proteins, such as Claudin-1, Claudin-3, Occludin and ZO-1, as well as polarized ZO-1 protein distribution and barrier function, in cultured hRPE cells. Moreover, MA treatment decreased apoptotic cells, ROS and Bax and increased △Ψ m and Bcl2 in hRPE cells under serum withdrawal-induced apoptosis. In addition, MA treatment elevated the protein expression levels of β-catenin and its target proteins, including Cyclin D1, c-Myc and Survivin, as well as the gene expression levels of ZO-1, β-catenin, Survivin and TCF-4, all of which could be down-regulated by the Wnt/β-catenin pathway inhibitor XAV-939. Taken together, MA treatment could effectively promote cell-cell contact and anti-apoptotic activity in hRPE cells, partly involving the Wnt/β-catenin pathway. This study will benefit the understanding of hRPE cells and future cell therapy.


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