脓毒症是由致病微生物DNA和毒素诱发的全身性炎症反应综合征，发病率占人口的0.3%，目前仍无有效的防治手段，病死率高达30-70%。而"菌、毒、炎和组织修复"并治的综合策略被认为是脓毒症治疗的最优方案。干细胞除具有再生修复功能外，还具有强大的免疫调节和抗炎功能，因此干细胞移植能综合治疗脓毒症，被认为是脓毒症救治的新希望。然而若脓毒症的诱发因素"菌和毒"未被清除，则干细胞移植的疗效将受限。为此，本项目拟首先将可高效杀菌并能中和内外毒素的BPI蛋白与MSCs可自行分泌的抗菌肽LL-37相融合，同时引入四环素调控启动子，条件性控制融合蛋白的表达，构建慢病毒颗粒，并修饰MSCs，最终通过细胞和动物模型验证，获得可控式表达BPI-LL37，即具有抗菌和排毒能力，又具有组织修复和抗炎功能的增强型MSCs，并以此建立脓毒症治疗的新措施。本项目的完成，将为干细胞移植在脓毒症治疗中的应用探索出新的出路。

Sepsis is a systemic inflammatory response syndrome caused by pathogenic microorganisms DNA and toxins,with 0.3% morbidity in the total population and 30-70% mortality in sepsis patients.Now,there is still no effective treatment and prevention of sepsis.The comprehensive treatment strategy for "bacteria,toxins,inflammation and tissue repair" is considered to be the optimal measure for treatment and prevention of sepsis.Besides the tissue regeneration and repair function,stem cells also have powerful immunomodulatory and anti-inflammatory functions.Therefore stem cell transplantation is considered as a promising strategy for comprehensive treatment os sepsis.However,if the inducements of sepsis,bacteria and toxins, are not removed, the efficacy of stem cell transplantation on sepsis will be reduced.Therefore,we firstly plan to design and construct BPI-LL37 fusion protein with highly effective antibacteral and antitoxic abilities.In addition,we will prepare lentiviral particles,which conditionally express BPI-LL37 fusion protein controlled by tetracycline-regulated promoter to infect and modify MSCs.Finally,we will identify and obtain MSCs with enhanced-antibacteral,antitoxic capacity,tissue repair and anti-inflammatory functions by cell and animal experiments.This project will explore a promising strategy for sepsis treatment.

脓毒症是由感染引起机体免疫反应失调并继发多器官功能损伤、高死亡率的综合佂。鉴于MSCs具有强大免疫调节和组织修复能力，被认为是脓毒症治疗的理想工具，但缺乏抗菌和抗毒能力。我们团队基于慢病毒系统，设计构建了广谱抗菌活性融合蛋白rBPI21-LL37修饰的抗菌型MSCs，制备可以分泌该两种融合蛋白的功能增强型人脐带间充质干细胞（BPI-LL37-hUC-MSCs、LL37-BPI-hUC-MSCs，以下简写为BL-hUC-MSCs、LB-hUC-MSCs）。我们采用多种方法鉴定两种干细胞（BL-hUC-MSCs、LB-hUC）的功能，如：干性、抗炎、抗菌以及内毒素中和作用；另外，我们用BL- hUC –MSCs联合抗生素治疗盲肠结扎穿孔法和LPS诱导建立的小鼠脓毒症模型，生化检验评价肝、肾及胰腺功能， ELISA法检测TNF-α、IL-1β、IL-6、IL-10水平，HE染色评估小鼠肝、肾、肺组织病理变化，免疫组化染色检查小鼠肝、肾、脾及肺组织cleaved-caspase-3蛋白的表达情况。结果显示：抗菌融合蛋白rBPI21-LL37修饰的抗菌型MSCs（BL-hUC-MSCs与LB-hUC-MSCs）均可以向成骨、成软骨和脂肪细胞方向诱导分化；BL-hUC-MSCs和LB-hUC-MSCs的条件培养基可以降低巨噬细胞炎症因子的表达；BL-hUC-MSCs和LB-hUC-MSCs的条件培养基对多种细菌的生长产生抑制作用；BL-hUC-MSCs和LB-hUC-MSCs的条件培养基对内毒素可以起到中和作用;BL-hUC-MSCs对生存率的改善要优于hUC-MSCs；BL-hUC-MSCs移植可以降低血清中TNF-α、IL-1β、IL-6、IL-10的水平;BL-hUC-MSCs移植可以降低血清中丙氨酸氨基转移酶、天门冬氨酸氨基转移酶、淀粉酶、肌酐和尿素等指标的水平；BL-hUC-MSCs移植可以改善肝脏、肾脏和肺受损的组织结构;BL-hUC-MSCs移植可以减少肝脏、肾脏、脾脏及肺cleaved-caspase-3蛋白的表达。本项目通过体内外实验评价了抗菌融合蛋白rBPI21-LL37修饰的抗菌型MSCs的抗菌、抗毒、抗炎和组织修复能力及综合治疗疗效，为抗菌型MSCs进入临床转化研究奠定基础，将为干细胞移植在脓毒症治疗中的应用探索出新的出路。