脊髓损伤是极其严重的中枢创伤，伴有缺氧及血脊髓屏障(Blood-Spinal Cord Barrier ，BSCB)的破坏。研究发现羊膜干细胞（Human Amniotic Stem Cells，HASCs）能耐受缺氧，显著改善脊髓微环境。课题组在体外OGD模型上证实：HASCs和HSMECs中miR-429均显著下调，HIF-1A和KLF6的3’UTR与miR-429存在互补结合位点，且miR-429抑制可上调HIFA和KLF6的表达；我们推测miR-429可靶定HIFA和KLF6调控HASCs耐受缺氧、改进BSCB结构及功能。本项目首先研究miRNA-429在HASCs对抗缺氧、调节BSCB通透性中的作用, 明确miRNA-429在HASCs调节BSCB中的机制；进一步探索移植转染antagomiR429的HASCs治疗对脊髓损伤后对缺氧耐受及BSCB通透性影响。本项目首次揭示HASCs在改善脊髓微环境中的作用和调控机制，丰富lHASCs治疗脊髓损伤的理论依据，为脊髓损伤的治疗开创新靶点。

Spinal cord injury is very serious with CNS injury, accompanied with hypoxia and the disruption of blood-spinal cord barrier (BSCB). The study demonstrated that hypoxia and the disruption of BSCB is involved in the pathophysiologyical process of SCI. The study demonstrated that amniotic stem cells (HASCs) can significantly increase the tolerance to hypoxia and improve the micro-environment in spinal cord. Recently, microRNAs (miRNA) have emerged as a critical class of negative regulators of gene expression. miRNA are endogenous small 21-23 nucleotide nonocoding RNAs that possess remarkable evolutionary conservation. These small molecules elicit their regulatory effects by base-pairing to partially complementary mRNAs and function by at least two mechanisms: degradation of target mRNA transcripts and inhibition of mRNA translation. Emerging evidence indicates that miRNAs play critical roles in the regulation of various biological and pathologic processes. To search for miRNAs which may be involved in the regulation of BSCB permeability induced by SCI, we used miRCURYLNSTM miRNA-array analysis to screen differentially expressed miRNAs in HASCs and HSMECs in OGD condition. We found that the level of miR-429 were significantly down regulated in HASCs and HSMECs in vitro BSCB model. To explore fuction of miR-429 , we applied common used bioinformatics algorithms (TargetScan, PicTar and miRanda) to identify its potential target genes. We demonstrated that miRNA-429 target to 3’UTR of HIF-1A and KLF6. In addition, inhibitor of miRNA-429 could significantly increase the expression of HIF-1A and KLF6; we speculate that miR-429 could regulate the hypoxia tolerance of HASCs, improved structure and function of BSCB by targeting HIFA and KLF6. The first aim of this study is to investigate the effects of miRNA-429 on HASCs-mediated hypoxia and regulation of BSCB permeability, elucidate the mechanism miRNA-429 on HASCs-mediated hypoxia and regulation of BSCB permeability; further study will focus on the effects of HASCs transplantation treatment with antagomiR429 on hypoxia tolerance and the permeability of BSCB after spinal cord injury. The project was the first to reveal the role and mechanism of HASCs in the improvement in the micro-environment after SCI, providing the theoretical and experimental evidences to fully understand the related mechanisms, and create a new target for drug development in SCI.

项目背景： 脊髓损伤是极其严重的中枢创伤，脊髓损伤后因局部缺血、缺氧而容易导致神经细胞发生坏死，轴突再生被抑制。 因而，如何有效改善脊髓损伤后的微环境、促进脊髓功能恢复成为研究的热点。研究发现人羊膜干细胞（HASCs）能耐受缺氧，显著改善脊髓微环境。但是，关于 HASCs 改进脊髓微环境的机制，国内外尚未见报。课题组在体外OGD模型上证实：HASCs和HSMECs中miR-429均显著下调，HIF-1α的3’UTR与miR-429存在互补结合位点，且miR-429抑制可上调HIF-1α的表达；我们推测miR-429可靶定HIF-1α调控HASCs耐受缺氧。主要研究内容：研究缺氧条件下miR-429调控HIF-1ɑ对HASCs存活、增殖和凋亡的影响，进一步明确miR-429调控HASCs耐受缺氧的作用与机制。重要结果：1、成功分离与培养HASCs，并诱导细胞缺氧、建立缺氧模型。2、成功构建真核表达载体 pcDNA3.1-HIF-1α/shRNA-HIF-1α。3、HASCs成功转染pcDNA3.1-HIF-1α/shRNA-HIF-1α、antagomiR-429。4、HASCs通过增加HIF-1α活性，调节VEGF的表达，增加细胞耐受缺氧。5、在缺氧条件下antagomiR-429通过调节HIF-1α表达，调控下游VEGF表达，使HASCs在缺氧条件下耐受缺氧。6、antagomiR-429参与HIF-1α表达上调，同时也参与相关凋亡蛋白Bcl-2、Bax 及caspase-3调节，并具有增加细胞存活率、增值率和抗凋亡作用。7、miR-429 可能通过调控 HASCs 细胞中 HIF-1α 转录，上调 HIF-1α 表达，增加 HASCs 对缺氧的耐受力。关键数据：1、200umol/l Cocl2、24h作为诱导HASCs缺氧的理想条件。2、miR-429 共转染48h时，上调 HIF-1α 和VEGF表达量最高。3、miR-429 共转染48h时，Bcl-2表达量最高、Bax表达量最低， Bcl-2/Bax表达量数值最大；caspase-3表达量最低。科学意义：本项目首次揭示HASCs在改善脊髓微环境中的作用和调控机制，丰富HASCs治疗脊髓损伤的理论依据，为脊髓损伤的治疗开创新靶点。