新型双功能超声微泡增效CD47高表达的骨髓间充质干细胞干预心肌纤维化的研究

Pathological myocardial fibrosis is the necessary stage in the progression of ischemic heart disease to heart failure. Stem cells have showed unique advantages in the treatment of myocardial fibrosis in recent years. However, inefficient stem cell transplantation is the bottleneck in cellular cardiomyoplasty. This study intends to use lipid microbubbles as a carrier, assemble the anti-myosin light chain antibody (AMLCA) and CD29 antibody onto the lipid microbubbles by covalent binding method, and thus prepare a new bifunctional echogenic immunoliposomes preparation which could guide mesenchymal stem cells to the injured target.With the aid of new ultrasonic wave irradiated microbubbles, intravenous infusion of mesenchymal stem cells with CD47 high expression to observe the improvement of transplantation efficiency and intervention effect on myocardial fibrosis after optimization of the "seed" and "soil".CD47 is the cell marker to evade phagocytosis by the immune system, mesenchymal stem cells with CD47 high expression are with the "amnesty" sign;Bifunctional echogenic immunoliposomes are expected to have the capability to find target initiatively and specific adhesion to the injured myocardium, the combination of the two functions are expected to prolong the survival time of stem cell after transplantation and to promote targeted homing. Objectives are:1. To develop a new bifunctional immune lipid microbubbles;2. To further explore the effect and mechanism of the new microbubbles to enhance the homing of mesenchymal stem cell with CD47 high expression and intervention on myocardial fibrosis. The study aims to improve defects in low efficiency of stem cell transplantation, and study results will provide new thought to improve efficiency in cellular cardiomyoplasty.

病理性心肌纤维化是缺血性心脏病向心衰发展的必经阶段。干细胞移植给心肌纤维化的治疗带来了新的希望，但是干细胞移植效率低下是心肌成形术中面临的瓶颈问题。本研究拟将脂质微泡作为载体，通过共价结合法将抗肌凝蛋白轻链抗体（AMCLA）及CD29抗体装配在脂质微泡上，制备能导引骨髓间充质干细胞至损伤靶位的新型双功能免疫脂质微泡制剂。在超声辐照新型微泡辅助下，静脉输入CD47高表达的间充质干细胞，观察优化干细胞和促进靶向归巢后对移植效率的改善及心肌纤维化的干预效果。CD47是能躲避免疫细胞吞噬的"细胞免死"标志，高表达CD47的间充质干细胞获有"特赦"标志；双功能免疫脂质微泡可望具有主动寻靶、特异性黏附于损伤心肌的能力，二者联合应用试图延长干细胞移植后的存活时间并促进靶向归巢。本研究着力于改善干细胞移植效率低、归巢量少的缺陷，将为提高干细胞移植效率提供新的思路。

背景及研究内容： . 病理性心肌纤维化是缺血性心脏病向心衰发展的必经阶段。干细胞移植给心肌纤维化的治疗带来了新的希望，但是干细胞移植效率低下是心肌成形术中面临的瓶颈问题。本研究拟在超声辐照新型微泡辅助下，静脉输入CD47高表达的间充质干细胞，观察优化干细胞和促进靶向归巢后对移植效率的改善及心肌纤维化的干预效果。CD47是能躲避免疫细胞吞噬的“细胞免死”标志，高表达CD47的间充质干细胞获有“特赦”标志；双功能免疫脂质微泡可望具有主动寻靶、特异性黏附于损伤心肌的能力，二者联合应用试图延长干细胞移植后的存活时间并促进靶向归巢。本研究着力于改善干细胞移植效率低、归巢量少的缺陷，将为提高干细胞移植效率提供新的思路。 .. 重要结果：.(1) 经质粒介导可成功将大鼠 CD47 目的基因片段转染至大鼠骨髓间充质干细胞，并能够在体外有效的过表达。.(2) CD47过表达能提高骨髓间充质干细胞的归巢效率及修复效果，联合双功能微泡，干细胞移植效率增强。骨髓间充质干细胞归巢后可干预心肌纤维化、改善MMP-TIMP表达，其机制与STAT介导的信号通路有关。.. 关键数据及其科学意义：.(1)成功构建了 pYr-ads-4-ratCD47 质粒，经酶切、 测序结果表明 pYr-ads-4-ratCD47 重组质粒构建成功；RT-PCR、 Western blot 显示实验组转染至大鼠骨髓间充质干细胞后 CD47 的 mＲNA 和蛋白水平表达明显增高(P <0.05)；功能检测提示过表达 CD47 分子的骨髓间充质干细胞成功逃避巨噬细胞吞噬。.(2) 干细胞归巢数最多的是过表达CD47联合双功能微泡组，其次为过表达CD47组，单纯干细胞组归巢数最少。与未治疗组相比，各治疗组的心肌胶原纤维含.量、MMP-9、TIMP-1、STAT1下降(P<0.05)，STAT3表达上调(P<0.05)。