靶向性调节性ODNR01减轻心肌缺血再灌注损伤及其机制研究

Recent studies have suggested that CD3+ T lymphocytes played an important role in myocardial ischemia/reperfusion injury (MIRI). IFN- - γ secreted by Th1 and IL-17 secreted by γδ T/Th17, which are regulated by STAT1/4 and STAT3 pathway respectively, exacerbated MIRI. Regulatory ODNR01 designed by our group supressed differentiation of Th1 and Th17 through modulating phosphorylation of STAT1/3/4. However, suppressing STAT3 phosphorylation in myocardium could aggravated MIRI, which means this is not a targeting effect. The present research plans to 1) prepare targeted regulatory ODNR01 which is a kind of immunoliposome (IML) with CD3 antibody on its surface and regulatory ODNR01 inside, examine the CD3+ T cell targeting effect and influence on differentiation of Th1 and Th17, and secretion of IL-17 by γδT cell of this IML; 2) explore the effect of targeted regulatory ODNR01 on MIRI by evaluating MIRI index, T cell differentiation and inflammatory process and further clarify the detailed mechanism by in vitro experiments. Our study will construct a CD3+ T cell-targeting compound which could be used to regulate MIRI and provide the theoretical and experimental basis for the development of targeted therapy in MIRI.

最近的研究证实CD3+T淋巴细胞参与心肌缺血再灌注损伤（MIRI），Th1细胞分泌的IFN-γ和-γδ T/Th17细胞分泌的IL-17促进MIRI进程，其分化分别受到STAT1/4和STAT3通路调控。我们设计的调节性ODNR01通过干预STAT1/3/4磷酸化而抑制Th1和Th17细胞分化，但其对心肌组织STAT3磷酸化的抑制可能加重MIRI，即作用缺乏靶向性。本课题拟制备表面耦联CD3单抗并载带调节性ODNR01的免疫脂质体，即靶向性调节性ODN R01，观察其对CD3+T细胞的靶向性、Th1和Th17细胞分化和 γδT分泌IL-17的影响；应用靶向性调节性ODN R01干预MIRI小鼠，观察其对MIRI指标、T细胞亚群分化及炎症的影响，并通过体外实验阐明其机制。本课题的研究可望构建一种靶向CD3+ T细胞的调控MIRI炎症的化合物，为MIRI的免疫调节和靶向治疗奠定新的理论基础。

最近的研究证实CD3+T淋巴细胞参与心肌缺血再灌注损伤（MIRI），Th1细胞分泌的IFN-γ和γδT/Th17细胞分泌的IL-17促进MIRI进程，其分化分别受到STAT1/4和STAT3通路调控。我们设计的调节性ODNR01通过干预STAT1/3/4磷酸化而抑制Th1和Th17细胞分化（已申请获得发明专利）），但其对心肌组织STAT3磷酸化的抑制可能加重MIRI，即作用缺乏靶向性。本课题制备了表面耦联CD3单抗并载带调节性ODNR01的免疫脂质体，即靶向性调节性ODNR01，并探讨了靶向性调节性ODN R01在MIRI中的作用和机制。我们首先成功制备而了anti-CD3-ODN脂质体，其在体外与CD3阳性T细胞有特异性的结合能力，即具有特异性的T细胞靶向能力。我们用对照ODN1612、调节性ODNR01、靶向性调节性ODNR01分别干预MIRI模型小鼠，发现与对照ODN1612相比，调节性ODNR01能减轻小鼠MIRI，抑制心肌细胞凋亡，减少中性粒细胞趋化因子KC、MIP-2和LIX的表达，抑制中性粒细胞的浸润，并减少促炎因子IFN-γ、TNF-α、IL-17的表达，而与调节性ODNR01相比，靶向性调节性ODNR01显示出更好的心脏保护作用，对心肌细胞凋亡，中性粒细胞浸润和炎症因子表达的抑制作用也更强。在探索靶向性调节性ODNR01调控MIRI的机制时我们发现靶向性调节性ODNR01对Rag1-/-（缺乏T/B淋巴细胞）小鼠的MIRI无保护作用，而给Rag1-/-回输T细胞后靶向性调节性ODNR01对小鼠MIRI的保护作用也恢复，这说明靶向性调节性ODNR01对MIRI的保护作用依赖于T细胞。我们还阐明了靶向性调节性ODNR01调控T细胞分化的机制。我们发现靶向性调节性ODNR01抑制Th1，Th17以及γδT17的分化，其机制为抑制STAT1/3/4磷酸化，并抑制STAT1/3/4与DNA的结合活性。本课题的研究成功构建了一种靶向CD3+ T细胞的调控MIRI炎症的化合物，为MIRI的免疫调节和靶向治疗提供了新的理论基础和实验依据。