内皮间质转化在2型糖尿病支架内再狭窄中的作用机制研究

In-stent restenosis (ISR) has been demonstrated to be a key factor influencing the prognosis of diabetic cardiovascular complications. Intima hyperplasia was confirmed to play a vital role in promoting ISR. However, the translational medical research targeted on the proliferation of vascular smooth muscle cells has not completely achieved expected results. Recently, related researches showed that endothelial-mesenchymal transition (EndMT) might play an important role in the formation of atherosclerosis. But whether or not EndMT contributes to ISR and the underlying mechanisms remain unclear. According to current reports combined with our previous study, we hypothesize that adipocyte-derived exosomes (ADEs) might be the key point of ISR, which might induce EndMT and accelerate the process of intima hyperplasia by activating the Shh/Snail signaling pathway. In this study, we will verify this hypothesis in clinical study, cell experiments as well as animal experiments, taking advantage of the various techniques such as genetic engineering, vascular ultrasound examination, flow cytometry and molecular biological techniques, etc. Our study will provide new ideas and therapeutic targets on preventing ISR, which would be an effective pathway to decrease the mortality of diabetic cardiovascular disease.

PCI术后支架内再狭窄是严重影响糖尿病心血管并发症患者预后的重要因素。血管内膜增生是支架内再狭窄的主要病理过程。但是以平滑肌细胞增殖为靶点的转化医学应用并未获得预期效果。近来有关研究提示内皮间质转化在促动脉粥样硬化形成中起着重要作用，但其在糖尿病支架内再狭窄形成中的作用及其机制尚不清楚。本课题组综合国内外研究现状并结合前期工作基础，提出脂肪细胞来源的exosomes（ADEs）通过其表面携带的Shh，激活内皮细胞Snail信号通路，促进内皮细胞间质转化，加速血管内膜增生，可能是支架内再狭窄关键环节的假说。本课题拟在临床、细胞、动物水平，应用基因工程、血管超声、流式细胞术、分子生物学等多种技术方法验证这一假说。该研究结果可为抑制糖尿病PCI术后支架内再狭窄、降低糖尿病心血管并发症死亡率提供新的思路和治疗靶点。

经皮冠状动脉介入治疗（PCI）是目前用于治疗冠心病的有效手段，而支架内再狭窄（ISR）严重影响PCI患者预后。研究发现糖尿病是支架内再狭窄的重要危险因素。但糖尿病加速支架内再狭窄发生、发展的确切机制尚未完全阐明。本项目应用分子生物学、细胞生物学、病理组织学等技术，以2型糖尿病血管再狭窄ApoE-/-小鼠为对象，以内皮细胞为切入点，对脂肪细胞来源exosomes（AdExos）介导的内皮间质转化（EndMT）在糖尿病血管再狭窄中的作用进行了研究，结果显示：⑴ 3T3-L1前脂肪细胞通过1-甲基-3-异丁基-黄嘌呤、地塞米松及胰岛素诱导分化为成熟脂肪细胞，高糖高胰岛素共同刺激3T3-L1脂肪细胞24h后能够建立胰岛素抵抗模型，胰岛素抵抗的脂肪细胞胞内沉积更多脂质；⑵超速差速离心法能够从细胞上清和脂肪组织中分离提取纯度较高的AdExos； ⑶AdExos直径分布、形态、表面特征性标志物均符合外泌体一般特征；⑷小鼠主动脉内皮细胞及小鼠动脉均能摄取由PKH26标记的AdExos；⑸2型糖尿病小鼠脂肪组织来源外泌体表明富集Shh；⑹2型糖尿病ApoE-/-小鼠血管再狭窄程度高于非糖尿病组，尾静脉注射IR-AdExos进一步促进血管再狭窄，Shh蛋白敲减后IR-AdExos的促血管再狭窄能力有所下降；⑺2型糖尿病ApoE-/-小鼠股动脉再狭窄部位发生EndMT，尾静脉注射IR-AdExos能够进一步促进EndMT的发生，而敲减Shh的IR-AdExos其促EndMT作用减弱；⑻IR-AdExos能够明显促进内皮细胞发生EndMT，Shh敲减的IR-AdExos促EndMT能力降低，Shh过表达的IR-AdExos促EndMT能力进一步增加；⑼TGF-β和SHH重组蛋白能够促进内皮细胞发生EndMT；⑽IR-AdExos的通过下游Snail和Slug分子促进EndMT。结论：糖尿病状态下脂肪细胞来源外泌体通过其表面携带的Shh，激活内皮细胞Snail信号通路，促进内皮细胞间质转化，加速血管内膜增生，促进支架内再狭窄。