循环纤维细胞在ALI/ARDS损伤修复中的作用及预后价值研究

Acute lung injury (ALI) and its extreme form, acute respiratory distress syndrome (ARDS) remain has high mortality, poor prognosis. Poor outcome of ALI/ARDS is correlates with excessive fibroproliferation . Circulating fibrocytes (CF)are a newly identified leukocyte subpopulation that come from bone marrow displaying fibroblast-like property, coexpressing CD45+Col-1+. Circulating fibrocytes rapidly enter sites of tissue injury, play an important role in fibroproliferative repair. We hypothesized that circulating fibrocytes are recruited at the site of lung injury and that their presence could be associated with a poor outcome. Our research include two parts.The first one is going to establish ALI mice models by intratracheal instillation of LPS. GFP transgenic mice bone marrow cells will be injected into ALI mice. CF engraftment in response to CXCL12/CXCR4 and differentiation in the lung of WT mice will be observed under confocal fluorescence microscope, CF and differentiated CF will be quantified by flow cytometry. Analyze the relationship of CF counts with ALI development,lung injury scores,survival rate.Clarifying that CF contribute to ALI fibroproliferation stage and poor prognosis.The second one is to choose ALI/ARDS patients to quantify fibrocytes in periblood and BALF, compare CF counts with disease progress,clinical parameters,mortality,evaluate the prognostic value of CF . The counts of circulating fibrocyte from patients with ALI /ARDS or without ALI/ARDS will be compared . A multivariate analysis using a Cox model will be performed to study CF be an independent predictors of survival.Receiver operating curve (ROC) will be used to identify a threshold value in patients with ALI/ARDS.The research may illuminate that CF are a novel reliable indicator of poor prognosis in ALI/ARDS.

急性肺损伤/急性呼吸窘迫综合症（ALI/ARDS）死亡率高,预后差，肺损伤后纤维增生修复失控是影响其预后的主要原因之一。循环纤维细胞（CF）是一种新近发现来源于骨髓、具有成纤维细胞特性、表达CD45+Col-1+的白细胞亚群。CF具有迁移到损伤组织、多能转化特性,它可能参与了ALI修复,与ALI预后相关。本项目拟在LPS致ALI小鼠模型上采用GFP转基因小鼠骨髓移植示踪肺损伤后CF在CXCL12/CXCR4作用下由骨髓释放至外周血液迁移至肺部，研究CF数量与肺组织胶原沉积量、转化为肌成纤维细胞数量以及生存率之间的关系，阐明CF是参与ALI修复、影响ALI预后的主要细胞。临床实验拟选择ALI/ARDS患者探讨验证CF数量与疾病进展、病情严重程度、肺组织纤维细胞数量以及生存率之间的关系，确定CF对ALI/ARDS的最佳预测值，评价CF的预后价值，为临床探索发现一种新的ALI/ARDS预后指标。

【研究背景】 循环纤维细胞（circulating fibrocytes,CF）是迁移至损伤组织转化为肌成纤维细胞的主要来源之一，在肺纤维化形成中作用已被证明。据此推测CF细胞在ALI/ARDS损伤修复和重建及预后评价起着重要作用。本研究旨在探讨CF细胞在LPS诱导ALI中修复损伤的作用和对ALI/ ARDS 病人预后评价的作用，确定CF是ALI/ ARDS 病人预后评价的一个可靠、有价值的指标。【研究内容】1）流式细胞术检LPS诱导ALI发生后不同时间（第1d、3d、5d、7d、14d、21d和28d）外周血循环纤维细胞数量变化； Gustavo Matute-Bello和Hubner病理学评分方法在光镜下观察肺组织损伤和肺纤维化程度；免疫组化法观察肺组织中COL-1蛋白表达。2）酶联免疫夹心法(ELISA)检测不同时间（第1d、3d、5d、7d、14d、21d和28d）小鼠外周血外周血中CXCL12含量。.【结 果】损伤组外周血CF数量与肺损伤程度呈现正相关性（r =0.815，P＜0.01）;损伤组外周血CF数量与肺损伤后纤维化评分呈现正相关性（r =0.904, P<0.01）。对照组小鼠肺组织内Ⅰ型胶原蛋白（COL-1）有少量的表达；损伤组第7、14、28天时肺组织标内Ⅰ型胶原（COL-1）蛋白表达均明显高于对照组（P＜0.05）。损伤组小鼠外周血液中CXCL12含量明显高于对照组（5843.23 vs 1826.49，P＜0.05）；阻断剂组外周血液中CXCL12含量明显低于损伤组降低（2913.70 vs 5843.23，P＜0.05），但仍高于对照组（5843.23 vs1826.49，P＜0.05）。 ALI/ARDS患者外周血CF>5.85%预测ALI/ARDS 病人28 天死亡率具有最佳的预测能力（敏感度:0.889，特异性:0.928）。【结 论】1.循环纤维细胞由骨髓释放入外周血液，迁移至急性肺损伤的受损肺组织中，CXCL12/CXCR4起着重要作用。2.循环纤维细胞数量与肺组织损伤程度及肺纤维化评分呈正相关性，提示循环纤维细胞参与急性肺损伤后纤维化修复和重建过程。3、 ALI/ARDS病人外周血循环纤维细胞数量与肺功能指标和肺损伤纤维化程度密切相关，可作为对ALI/ ARDS 病人预后评价可靠、有价值的指标