Connexin43促进缺血性脑卒中溶栓后出血转化的上游分子机制研究

Recombinant tissue plasminogen activator(rtPA) is the most effective drug for acute ischemic stroke in super early stage, but it can increase the risk of hemorrhagic transformation (HT). In previous experiment we have found that connexin43(Cx43) plays a role in HT. Bioinformatics analysis revealed that lncRNA H19 might regulate Cx43 via microRNAs(miR-130a-3p、miR-301a-3p、miR-130b-3p). We have proved that H19 increased in HT model and knock-down of H19 led to decrease of Cx43, which suggests that H19 might affect HT through regulating Cx43 via microRNAs. Cx43+/- and wild-type mice animal model of HT as well as oxygen and glucose deprivation /reoxygenation (OGD/R) and rtPA intervention cell model will be established. With the molecular biological techniques such as siRNA, molecular cloning, luciferase assay and RIP, this project intends to evaluate whether H19 functions in HT via Cx43, whether Cx43 is the downstream target of H19 and whether H19 regulates Cx43 via microRNAs. We hope to gradually reveal the roles of H19 on Cx43 as the upstream mechanism and lay the foundation for future drug development.

重组组织型纤溶酶原激活剂（rtPA）是缺血性脑卒中超早期最有效治疗药物，但会增加出血转化（HT）风险。我们前期研究发现connexin43(Cx43)参与HT。生物信息学分析表明lncRNAH19可通过microRNAs(miR-130a-3p、miR-301a-3p、miR-130b-3p)调控Cx43。经前期验证，动物模型中H19升高，细胞模型中下调H19致Cx43降低。据此我们提出假说，H19可能通过microRNAs调控Cx43参与HT。本研究拟建立Cx43+/-及野生型小鼠HT模型、氧糖剥夺/复氧复糖+rtPA细胞模型，采用RNAi、分子克隆、双荧光素酶和RIP等分子生物学技术，阐明H19是否通过调控Cx43参与HT、Cx43是否为H19下游靶点、H19是否通过microRNAs调控Cx43表达，逐步揭示H19作为上游分子调控Cx43的机制，为后续药物研究奠定基础。

重组组织型纤溶酶原激活剂（rtPA）是缺血性脑卒中超早期最有效治疗药物，但会增加出血转化（HT）风险，我们前期研究发现connexin43(Cx43)参与这一过程并发挥重要作用，但其上游的调控机制尚不完全明确，限制了对其应用的开发，需要深入挖掘潜在机制。生物信息学分析表明lncRNA H19可通过microRNAs (miR-130a-3p、miR-301a-3p、miR-130b-3p)调控Cx43。本研究通过动物模型和细胞模型验证了lncRNA H19 、miRNAs、Cx43在溶栓后出血转化及血脑屏障破坏中起作用，在外周血样本中也检测出部分指标表达差异明显，通过双荧光素酶实验验证了lncRNA H19、miRNAs、Cx43相互之间存在靶向结合的作用方式，并发现Wnt信号通路与之存在一定关系，最终阐明了H19-miRNAs-Cx43这一调控轴的作用机制，为缺血性脑卒中预防及治疗方面增加了新理论资料，可为后续进一步探讨急性缺血性脑卒中溶栓后出血转化的治疗改善相关分子机制提供一定理论基础，也可为出血转化风险预测提供新的可选项。