腺相关病毒介导SDF-1和VEGF165双基因修饰血管内皮祖细胞治疗脓毒症的实验研究

Sepsis/septic shock is the main cause of death in critical ill patients with multiple organ failure. The damage and activation of endothelial cells play an important role in the development of sepsis and severe sepsis. Thus,understanding the mechanisms that maintain and restore endothelial function may have important clinical implications. Endothelial progenitor cells(EPCs) are critically involved in maintaining vascular homeostasis and in mediating macro- and microvascular repair under both physiological and pathological conditions. Our previous studies have indicated that bone marrow-derived endothelial progenitor cells,as vascular endothelium precursor cells,augmented neovascularization in ischemia hindlimb and myocardium,increased myocardial local contractility and improved cardiac function. Nevertheless,the number and the quality of bone marrow-derived endothelial progenitor cells is limited. Vascular endothelial growth factor (VEGF) has been identified as a key component in the development of blood vessels, but VEGF alone may be insufficient to achieve functional and mature development of the vasculature. Stromal-derived factor-1 (SDF-1) is the only known chemokine capable of migration of hematopoietic stemcells (HSCs), as the fluctuations in SDF-1 expression controlled the fluctuated steady-state of HSCs and their progenitors in peripheral blood . Among these, the SDF-1a and its receptor 4 (CXCR4) play a key role in mobilization and migration of EPCs. This study intends to use adeno-associated virus as vector and to transfect VEGF gene and SDF-1 gene into EPCs by use of the technique of gene modification.Subsequently,to transplant modified EPCs into the body of sepsis mice. The aim of this study is to observe the endothelium repair and angiogenesis and to investigate the protective effects and its molecular mechanisms of EPCs transplantation on sepsis mice.This study will provide a potential way for sepsis cell replacement therapy and a novel therapeutic idea for the application of mature stem cells transplanation in human diseases.

脓毒症/感染性休克是危重病患者多器官功能衰竭和死亡的主要原因。内皮细胞的损伤和激活在脓毒症的发生发展中具有重要的作用。我们前期研究发现：作为血管内皮细胞的前体，来源于骨髓的血管内皮祖细胞(EPCs)能促进缺血后的肢体和心肌新生血管形成，增加心肌局部收缩力，改善心功能。血管内皮生长因子（VEGF）能有效动员EPCs，增加其数目和功能。基质细胞衍生因子-1及其受体（SDF-1/CXCR4）能有效提高EPCs 数量，促进EPCs 黏附、增殖、诱导迁移和抑制凋亡来改善EPCs 的功能。本研究拟以腺相关病毒为载体，通过基因修饰技术将VEGF基因和SDF-1基因共转染入EPCs，移植入脓毒症小鼠体内，观察内皮细胞损伤修复情况和对脓毒症小鼠的保护作用及其机制。为脓毒症的治疗开辟新的途径，也为更好地应用成体干细胞移植治疗人类疾病提供新的思路。

脓毒症及其相关病变感染性休克是一组严重的临床综合征，是当前ICU患者多器官功能衰竭和死亡的主要原因，也是危害人类生命健康的一个全球性难题，而内皮细胞的损伤和激活在脓毒症的发生中具有重要的作用。本研究采用不整合入基因组DNA的腺相关病毒作为载体，使用SDF-1和VEGF165两种基因，采用基因转染技术修饰小鼠血管内皮祖细胞，观察修饰后基因表达水平，评估其安全性。进而将两种基因修饰后的血管内皮祖细胞移植入脓毒症小鼠体内，观察对脓毒症小鼠的保护作用、内皮细胞损伤修复情况及其对PI3K/Akt/eNOS信号转导通路的影响。结果发现，（1）与正常EPCs细胞的增殖情况相比，转染SDF-1后的EPCs细胞，其增殖情况影响较小。（2）采用RT-PCR法检测转染后SDF-1 mRNA水平较空载组和正常组明显升高，说明基因转染技术的实施具有可行性。（3）各组细胞培养上清液行Western blotting测定比较SDF-1蛋白表达水平，结果显示转染组蛋白表达水平明显升高。（4）免疫细胞化学方法检测EPCs肿瘤相关基因的表达