MiR-21促进骨髓间充质干细胞向肾小管靶向迁移在急性肾损伤中的作用及机制研究

Therapeutic potential of bone marrow-derived mesenchymal stem cells (BMSCs) in acute kidney injury (AKI) was seriously limited by low rates of cell homing and engraftment after transplantation. In previous work, we significantly increased renal retention of grafted BMSCs through renal arterial administration; however, most of BMSCs homing to the kidney were localized primarily to the renal vessels and failed to migrate and integrate into the host renal tubules. It has been suggested that high level of miR-21 is associated with the enhanced migration, invasion of tumor cells. In pre-experiment, we found that increasing the expression of miR-21 could improve the number of BMSCs localized in the injured renal tubules. Thus, we hypothesized that up-regulation of miR-21 in BMSCs might promote their intra-renal migration into injured renal tubules, which may further enhance their therapeutic effects in AKI. To prove this hypothesis, we will: 1）use lentiviral transfection to overexpress miR-21 in BMSCs and CRISPR/cas9 to knockout miR-21 in BMSCs; 2）further validate the in vitro and in vivo effect of miR-21 on the intra-renal migration and the recovery on the AKI; 3) investigate whether the miR-21 regulates the migration of BMSCs through the PTEN/PI3K/Akt pathway. Therefore, this project may provide further optimized strategy for cell therapy of AKI from the view of improving intra-renal cell migration.

骨髓间充质干细胞（BMSCs）在肾脏的归巢率和肾小管定植率低严重限制其对急性肾损伤（AKI）的疗效。我们前期采用肾动脉显微注射法显著提高了BMSCs归巢率，但其定植率仍低。已有研究提示肿瘤细胞miR-21高表达量与其迁移能力呈正相关。我们预实验发现，提高BMSCs的miR-21表达可促使干细胞迁移至肾小管。因此提出假说：上调miR-21表达可能提高干细胞肾内迁移力，促进归巢细胞迁移并定植至肾小管，从而更有效地修复AKI。为此，本项目拟：1）利用慢病毒转染和CRISPR/cas9体系分别建立miR-21过表达和缺失的BMSCs细胞系；2）通过体内外实验验证miR-21对BMSCs肾内迁移能力的影响及对AKI修复作用；3）研究miR-21是否通过PTEN/PI3K/Akt信号通路提高BMSCs的迁移能力。本研究将以“提高干细胞肾内迁移”为切入点，为进一步优化AKI干细胞治疗策略提供新思路。

急性肾损伤（Acute Kidney Injury, AKI）是临床常见的危重症，因其起病急、发病率高和死亡率高等特点，已成为全球公共健康问题。因此，探索建立在AKI发病机制上的有效干预措施是AKI防治的重要问题。包括本课题组在内的多项国内外研究证实输注骨髓间充质干细胞（BMSCs）可有效缓解AKI，但BMSCs在肾脏的归巢率和肾小管定植率低严重限制其对AKI的疗效。我们前期采用肾动脉显微注射法显著提高了BMSCs归巢率，但其定植率仍低。有研究提示肿瘤细胞miR-21高表达量与其迁移能力呈正相关。我们预实验发现，提高BMSCs的miR-21表达可促使干细胞迁移至肾小管。因此提出假说：上调miR-21表达可能提高干细胞肾内迁移力，促进归巢细胞迁移并定植至肾小管，从而更有效地修复AKI。首先，我们通过miR-21 mimics转染构建miR-21过表达BMSCs（miR-21/BMSCs）；RNA锁核苷酸抑制技术，敲减BMSCs中miR-21（anti-miR-21/BMSCs），并通过Real time PCR检测细胞miR-21表达丰度、流式检测细胞表面标记、诱导细胞体外成脂成骨分化，明确细胞修饰成功且维持干性。随后，我们进行细胞划痕实验、transwell小室等体外实验，评估BMSCs的迁移能力。结果表明miR-21/BMSCs的体外迁移能力较对照组明显增强。为探索miR-21丰度变化对BMSCs迁移能力影响的分子机制，我们聚焦PTEN/PI3K/Akt信号通路。研究发现，上述通路特异性抑制剂能取消miR-21高丰度对BMSCs迁移能力的上调作用，即miR-21的丰度与细胞PTEN的表达量呈负相关，并调控其下游PI3K/Akt信号转导途径。接着，我们将细胞输入大鼠肾脏AKI模型中，检验miR-21/BMSCs的生物学作用。共聚焦显微镜观察到miR-21/BMSCs肾内迁移能力有一定程度增强，少量细胞可迁移至肾间质，而对照组基本定植在肾小球。然而比较肾功能时发现，输注BMSCs能促进AKI损伤修复，对照BMSCs和miR-21/BMSCs两组之间没有明显差异。为何更精准定植的细胞不能获得匹配的疗效的提高？在肾内大量定植的细胞又是如何发挥治疗作用的？进一步深入研究阐述上述问题将为提高干细胞靶向、精准定植奠定基础，为临床探索高效的AKI干细胞治疗策略提供新思路。