【作者】 徐金富；

【导师】 瞿介明； 余龙； 李惠萍；

【作者基本信息】 复旦大学 ， 内科学， 2007， 博士

【摘要】 研究背景及目的：以急性肺部感染为主的感染所致的肺损伤仍然是急性肺损伤发病的最重要，最常见因素。其中以脂多糖所致炎症性肺损伤为多见，目前尚缺乏十分有效的辅助治疗手段。我国目前缺乏大样本的相关的流行病学资料，上海市12家大学附属医院的15个ICU的为期1年的前瞻性研究显示，ALI／ARDS的最常见患病因素为肺炎(34.3％)和肺外败血症(30.6％)，且样本中患者90天死亡率达到70.4％，足见感染性肺损伤的多发性和严重性。本课题设想将间充质干细胞(Mesenchymal stem cells，MSC)载体，利用基因转染技术使其携带促血管生成素1(Agiopoietin-1，Ang1)基因，回输体内进行干预脂多糖(LPS)所致的肺损伤模型，充分利用基因治疗与干细胞治疗的优点，有效干预LPS所致肺损伤。方法：采用MSCs分离、培养和扩增至第4代，经流式细胞仪鉴定，得到纯度较高的MSCs。同时以三质粒共转染法在293T细胞中制备病毒颗粒Lenti-Ang1和Lenti-EGFP，并转染MSCs，通过试验确定转染的最佳M.O.I.和最佳时间，并通过RT-PCR和western-blot检测转染后MSCs中Ang1的基因和蛋白表达，确定转染成功。以脂多糖雾化吸入的方式诱导小鼠炎症性肺损伤模型，设未处理组为对照组，设三组干预组，包括：携带Ang1的MSCs组(MSC-Ang1组)，单纯Ang1组(Ang1组)和单纯MSCs组(MSC组)。观察并记录生存天数，计算生存率；提取肺组织总蛋白并用western blot方法检测Ang1蛋白的表达；测定肺部炎症和损伤指标包括肺组织髓过氧化物酶(MPO)活性和肺湿干比，实时荧光定量RT-PCR检测肺组织中TNF-.的mRNA含量；光镜观察肺组织病理学改变并进行评估；荧光显微镜检测肺组织中外源MSCs携带的eGFP所发荧光，免疫组化双染色进行外源细胞的定位，确定外源MSCs源性细胞在肺部的表现。结果：1．改良的Peister法能成功分离培养到符合实验需要的较高纯度的MSCs。2．通过Lentiviral载体系统，Ang1可转染MSCs并在其中稳定表达。其最佳MOI为20，最佳时间为第5天，转染效率在90％以上。3．CD44，Sca-1，CD31和CD45等MSCs的重要表面标记，在转染前后未见明显改变，提示转染Ang1不影响MSCs的细胞表型及特性。4．MSCs本身微量表达Ang1，而转染外源基因Ang1后，MSCs中Ang1的mRNA和蛋白表达量明显上升。经多重纯化后，流式细胞仪鉴定获得的干细胞为CD44(+)，Sca-1(+)，CD31(-)和CD45(-)的MSCs，纯度在90％以上。5．在第14天可见肺组织受损处多处表达GFP的细胞出现，根据其形态可判定为肺泡上皮细胞和肺血管内皮细胞，在肺损伤较重的部位表达GFP的细胞数量相对较多。6．对Ang1在体内表达的检测显示对照组Ang1蛋白在脂多糖诱导肺损伤后呈下降趋势，直至14d仍然低于肺损伤前，而MSC-Ang1组第3天下降明显，但随后回升，至第14天已恢复并高于损伤前水平。7．MSC-Ang1组在第14天的MPO活性和肺湿干比较其它三组明显降低，与对照组比较有统计学差异，提示Ang1的转入有效减低肺组织MPO的激活和肺水肿。8．肺组织病理学及其评分显示，与对照组比较，MSC-Ang1组在改善肺组织病理损伤总评分上差异有统计学意义，而另二组无统计学差异。9．MSC-Ang1生存率高于对照组，生存率分析显示存在一定优势，但尚未达到统计学差异(P=0.066)。结论：1．MSCs可成功分离提纯，并可经由Lentivirus系统高效率、长时间稳定转染，使其携带Ang1基因，且MSCs本身主要表面标志不受影响。MSCs是良好的基因治疗运载工具。2．携带目的基因Ang1的MSCs在输入体内后，Ang1与MSCs发挥协同作用，Ang1肺内高表达，发挥其抗炎和稳定血管减轻渗漏的作用，同时MSCs通过转化或融合，修复肺部上皮或内皮细胞。MSCs细胞介导的Ang1基因治疗不失为一种可行性的干预炎症性肺损伤的方法。更多还原

【Abstract】 RationaleLipopolysaccharide (LPS), a major component of the cell wall of gram-negative bacteria, plays an important role in the pathogenesis of ALI/ARDS. LPS induced lung injury is characterized by microvascular protein leakage, neutrophil influx, and expression of pro inflammatory mediators, followed by severe lung damage. It is still remain little effective treatment to attenuate severe lung injury caused by LPS. Recent literature suggests that bone marrow-derived mesenchymal stem cells(MSCs) can serve as vehicle of gene therapy. We speculated that MSCs therapy combined with gene therapy might be a potential therapeutic approach for lung injury.ObjectiveTo determine whether MSCs carried with agiopoietin-1 (Ang1) could attenuate injuries of lung epithelium and endothelium in lipopolysaccharide induced lung injury.MethodsMSCs were obtained from adult male inbred mice and then were transduced with Ang1 gene efficiently through lentivirus vector. Phenotypes of MSCs were analyzed by fluorescence-activated cell sorting (FACS) before and after Ang1 transduced. Two hours after LPS inhalation, mice were infused by normal saline (NS group), lentiviral vector carried Ang1 (Ang1 group), lentiviral vector carried eGFP(MSCs group) and lentiviral vector carried Ang1/eGFP (MSCs-Ang1 group) by jugular vein injection. Ectogenic MSCs origined lung cells and Ang1 expressions in lungs were detected in receipt mice. Indexes of lung injury were evaluated at different time points. Lung myeloperoxidase activity, lung wet to dry ratio and TNF. were quantified in lung tissues of four groups.ResultsPhenotypes of MSCs were not change significantly after they were transduced with lentivirus vector. LPS-induced lung damages were attenuated and MSCs original epithelial and endothelial cells were detected in injuried lung areas 14 days after MSCs administration. Expressions of Ang1 protein in lungs were enhanced significantly 7days after MSCs-Ang1 administration. Lung MPO activities, lung W/D ratios and TNF. mRNA in lungs were attenuated 7 days after MSCs-Ang1 infusion, compared with NS group. Kaplan-Meier survival assay showed that, although did not reach statistically significance, MSCs-Ang1 infusion appeared predominance in improving survival rates of LPS-induced lung injury (p=0.066), compared with control.ConclusionsMSCs played roles of not only a vehicle but also a therapeutic cell in this study. Infusion of MSCs-Ang1 improved histopathology and Ang1 expression of injured lung, attenuated the severity of lung injury. MSCs based gene transfer of Ang1 provided a feasible, if imperfect, approach on ameliorating lung injury caused by LPS更多还原