PWMI后少突-血管单元重建对移植人少突胶质前体细胞成髓鞘的影响

Oligodendrocyte progenitor cell (OPC) loses and differentiation failure lead to dysmyelination which is the typical pathological features of preterm white matter injury (PWMI). Until now, no treatment has shown efficacy. Cell replacement therapy based on OPC is expected to become an effective strategy for PWMI. We have obtained the human OPC with the world’s leading technique and performed OPC transplant in PWMI anmimal model. However, we found that myelination rate is very low after OPC transplant. Previous studies indicated that “oligovascular unit” not only is an important microenviroment for OPC development and angiogenesis, but also is the “soil” for transplanted OPC survival. OPC differentiation microenviroment might be from promoting to inhibiting after PWMI. However, the typical pathological changes in “oligovascular unit”, the mechanisms for OPC differentiation inhibition, the strategies for OPC differentiation disinhibition and “oligovascular unit” restoration remain to be clarified. We intend to take transgenic mice and peterm autopsy sample as the object of the research, and use two-photon imaging, proteomics and Crispr/Cas9 gene editing technique, etc. to clarify the typical pathological changes in “oligovascular unit” after PWMI. Then the key function targets to inhibit OPC differentiation and myelination will be revealed. Moreover, it will be clarified whether marrow stromal cell (MSC) and MSC derived exosome and endothelial cell(EC) promote OPC differentiation and myelination by function targets regulation, “oligovascular unit” restoration and microenviroment improvement. The project will contibute to human OPC translational research: from basic research to clinical application.

少突胶质前体细胞（OPC）丢失及其分化失败导致髓鞘形成障碍是早产儿脑白质损伤(PWMI)的典型病理特征，目前尚无有效疗法，以OPC为基础的细胞替代治疗有望成为有效策略。我们以国际领先的技术优势获得人OPC并进行了PWMI后移植研究，但发现移植OPC成髓鞘效率很低。研究表明“少突-血管单元”不仅是OPC发育和血管新生的微环境，也是移植OPC赖以生存的“土壤”，损伤后OPC分化微环境可能由促进转为抑制；但PWMI后单元特征性改变、OPC分化抑制机制、“去抑制”及单元重建策略尚有待明确。本项目拟以转基因鼠和早产儿尸检样本为研究对象，利用双光子活体成像、蛋白组学和Crispr/Cas9等技术明确PWMI后单元的特征性改变；获得抑制OPC分化成髓鞘的关键靶点；阐明基质干细胞（MSC）及其外泌体和内皮祖细胞（EPC）是否通过靶点调控、单元重建和微环境改善促OPC成髓鞘；本研究有助于人OPC的转化研究。

少突胶质前体细胞（oligodendrocyte precursor cell，OPC）丢失及其分化失败导致髓鞘形成障碍是早产儿脑白质损伤(preterm white matter injury，PWMI)的典型病理特征，目前尚无有效疗法。本研究用缺血缺氧方法建立C57BL/6（P3）小鼠PWMI模型，发现模型小鼠学习记忆能力下降，损伤侧OPC数目和血管数量显著减少，血管内皮细胞形态改变以及髓鞘形成障碍，且PI3K/AKT/mTOR信号通路参与了缺血缺氧后的血管及髓鞘形成（数据发表在Brain Res Bull. 2020, 155, 81-91）。磁珠分选PWMI小鼠脑微血管内皮细胞，蛋白组学显示305个蛋白上调，295个下调，聚类分析表明损伤侧内皮细胞来源的轴突导向因子（Netrin4，Ntn4）表达显著增加，WB进一步证实PWMI后Ntn4表达显著增加，给予小鼠重组蛋白Ntn4可诱导OPC数目增加。在此基础上，特异性脑微血管内皮细胞Ntn4，敲除小鼠表现为学习记忆能力下降， OPC数目和髓鞘碱性蛋白（MBP）的表达均明显减少。细胞实验发现，内皮细胞Ntn4敲减后的条件培养基促进了OPC向成熟少突胶质细胞分化。此外，脑微血管内皮细胞缺氧处理后的EPCs移植到PWMI小鼠脑内，可以更有效的促进OPC分化和髓鞘形成，其机制可能与EPCs分泌的CXCL12激活了 OPCs中的CXCL12-CXCR4 信号轴有关（数据发表在Stem Cell Research &Therapy. 2021 ;12(1):118）。 用小分子化合物RIP1/RIP3抑制剂ZJU-37联合人OPC脑内移植，ZJU-37通过抑制NLRP3炎性小体活化促进了PWMI新生大鼠人OPC移植后的髓鞘形成（数据发表在Stem Cell Research &Therapy. 2021 ;12(1):462）。