骨髓间充质细胞源性exosomes调控视网膜变性过程中小胶质细胞活性及其对视功能的修复作用研究

Transplantation of retinal stem cells offers hope for treating retinal degeneration. But maintaining visual recovery after transplantation has been problematic. Modulation microglial cells phenotype and optimizing the microenvironment for stem cells survival will contribute to overcoming these problems. Here, for the first time, we injected retinal progenitor cells (RPC) with bone marrow mesenchymal stem cells (BM-MSC) into the degenerated retina. We also showed that BM-MSC significantly impair microglia activition, accordingly, visual function was restored with a longer time. So we hypothesized that BM-MSC derived exsomes shaped the microglial activyty and promoted neural survival, which emerged as a potential therapy for retinal degeneration. In this study, further investigation will include: 1) Effect of BM-MSC derived exsomes on the phenotype of retinal microglia. 2) MicroRNAs (miR-124 and miR-155) played a key role during this activity. 3) Efficacy of RPC combining with B-MSC derived exosomes or miR-124 when transplanted into the subretinal space. Together our study strongly implicates microRNAs in mediating BM-MSC stimulated functional recovery in animal models of retinal degeneration, and miR-124 may therefore have a wide application in neurodegenerative diseases.

干细胞移植为视网膜变性疾病的治疗带来希望，但突出问题在于移植后的宿主视功能不能长期维持。移植微环境中小胶质细胞功能是决定影响移植疗效的关键因素。本课题组前期研究显示，视网膜干细胞移植中掺入骨髓间充质干细胞（BM-MSC）,能明显提高和延长移植疗效，提出"BM-MSC源性exosomes通过miR-124/miR-155表达变化，调控小胶质细胞活性，改善视网膜移植微环境"的假设。基于此，本项目拟：1)观察BM-MSC源性exosomes对视网膜小胶质细胞表型的影响，证实其神经保护作用；2）探讨BM-MSC源性miR-124、miR-155及信号通路在调控小胶质细胞活性中的作用；3）观察视网膜前体细胞联合exosomes或miR-124治疗RCS大鼠的疗效，明确BM-MSC源性miR-124的生物学作用。本项目不仅为BM-MSC应用提供明确实验依据,也是microRNA临床应用的有价值探索。

视网膜祖细胞(RPC)移植为视网膜变性疾病治疗带来希望，但突出问题是移植后宿主视功能不能长期维持，主要原因是移植微环境中小胶质细胞活化导致慢性炎症环境，不利于RPC存活。鉴于骨髓间充质干细胞(BMSC)独特的免疫调控功能，本课题探讨BMSC联合RPC移植治疗视网膜变性的效应，并探讨其机制。主要研究结果如下：.1.发现BMSC联合RPC移植治疗视网膜变性动物RCS大鼠，疗效优于任何一种单独细胞移植。在移植术后的闪光视网膜电图检查结果显示联合移植组视功能明显高于RPC和BMSC单独移植组，形态学显示联合移植组移植细胞的存活数量和视网膜外核层厚度明显高于单独移植组，且移植RPC能分化为光感受器。进一步研究发现，联合移植组视网膜中小胶质细胞的活化明显减轻。表明，BMSC通过调控视网膜小胶质细胞活性而提高RPC细胞的移植效果。.2.发现BMSC的外泌体(exosomes)可以被小胶质细胞吞噬而发挥直接调控小胶质细胞的作用。对exosomes进行miRNA测序，发现炎性环境中exosomes的miRNA种类发生了很大的变化，综合表达量、表达差异、炎性相关通路及RCS大鼠视网膜缺乏的miRNA，我们认为miR-143-3p、miR-221-3p、miR-155-5p、miR-146a-5p可能联合移植治疗视网膜变性疾病的关键。.3. 经伦理委员会批准，共开展16例BMSC联合RPC移植治疗视网膜色素变性(RP)的临床试验，联合移植组10例，RPC单独移植组6例。移植后随访一年的结果提示：16例患者均未出现局部肿瘤生长及排斥反应，RPC单独移植组在术后3-6月患者的视力有提高，随后逐渐下降，而联合移植组移植疗效与RPC单独移植并无明显差异。表明，RPC移植治疗RP是安全的，短期有一定的疗效，但本次临床试验中BMSC联合RPC移植没有表现出优越性。