小鼠同种异体内皮祖细胞移植治疗急性肺损伤的相关研究-东南大学学报医学版

小鼠同种异体内皮祖细胞移植治疗急性肺损伤的相关研究

单位：南京大学临床学院, 南京军区南京总医院心胸外科, 江苏南京 210002

分类号：R459.9

出版年·卷·期（页码）：2014·33·第四期（422-428）

摘要：

目的：观察外源性内皮祖细胞（endothelial progenitor cells，EPCs）移植对急性肺损伤（ALI）小鼠受损肺组织的保护作用并探讨其机制。方法：选用转基因EGFP-BALB/c小鼠作为细胞移植供体，获得骨髓单核细胞，体外培养扩增获得足够数量的表达绿色荧光蛋白的EPCs（EGFP-EPCs）。选用野生型BALB/c小鼠18只，随机分为假手术组、ALI组及EPC治疗组，每组6只。假手术组气管内滴注PBS，其后经尾静脉注射PBS；ALI组气管内滴注脂多糖溶液，其后经尾静脉注射PBS；EPC治疗组气管内滴注LPS，其后经尾静脉注射EGFP-EPCs。各组小鼠术后3 d处死，留取肺组织标本。荧光显微镜下观察各组肺组织中EGFP-EPCs表达情况，HE染色评价各组肺组织损伤程度并进行肺损伤指数评分，比较各组肺组织湿干重比，行肺组织CD34免疫组化染色并测定肺微血管密度（MVD）。结果：各组小鼠建模过程顺利，无围手术期死亡。EPC治疗组小鼠肺组织冰冻切片中可观察到均匀附着于血管内皮的绿色荧光表达，而假手术组及ALI组则未见明显的绿色荧光。HE切片显示：LPS可致严重的肺组织损伤，而EPC治疗组肺损伤程度较ALI组明显减轻，肺损伤指数明显低于ALI对照组（P<0.001）。肺组织湿干重比ALI组明显高于假手术组（P<0.001），而EPC治疗组较ALI组明显降低（P<0.05）。免疫组化染色显示：EPC治疗组肺组织切片中CD34的表达明显增加，MVD值均明显高于假手术组及ALI组（P<0.001）。结论：对ALI小鼠行外源性EPC移植治疗，输入体内的EPC可定向迁移并黏附于受损的肺毛细血管内皮，直接参与肺毛细血管内皮修复及重建，维持肺毛细血管内皮完整性，减少炎症细胞向肺间质、肺泡内的浸润及对肺组织的炎性损伤。同时，外源性EPC治疗提高了EPC向受损肺组织归巢及受损肺组织内的血管新生。

Objective: To observe the protective effects of transplanted exogenous endothelial progenitor cells (EPCs) on damaged lung tissue of acute lung injury (ALI) in mice. Methods: EGFP-BALB/c mice were selected as cell donors and obtained bone marrow derived mononuclear cells which were later cultured as EGFP-EPCs to a required amount. Eighteen wild type BALB/c mice were randomly divided into 3 groups (6 mice in each group): Sham group (tracheal instillation of PBS and PBS iv. afterwards), ALI group (tracheal instillation of LPS and PBS iv. afterwards), and EPC group(tracheal instillation of LPS followed by EGFP-EPCs iv). Three days after surgery, the mice were killed and the lung tissues were harvested for the following experiments. Lung tissues were observed immediately under fluorescence microscope to detect EGFP-EPCs after frozen section. Paraffin sections were stained with HE for histopathological analysis and determined lung injury score of each group. Lung wet to dry ratio of each group were also determined. Additionally, we did immunohistochemical analysis of CD34 of lung tissues and measured the CD34 positive micro vessel density (MVD) in lung tissues of each group. Results: Surgical procedures were well proceeded and none of the experimental mice die during the perioperative period. By observing the lung tissue cryosections through the fluorescence microscope, we found the engrafted EGFP-EPCs were adherent to the lumen of pulmonary vessels while no EGFP signals were detected in the neither Sham group nor ALI group. HE staining results showed that LPS induced typical and severe ALI pathological traits while the severity of lung injury was evidently attenuated in EPC treatment group and the lung injury score was also significantly alleviated (P<0.001). Compared with ALI group, the wet to dry ratio of EPC group were significantly reduced (P<0.05). CD34 staining revealed that the positive staining area was remarkably enhanced by EPC transplantation, and CD34 positive MVD were significantly increased by EPC transplantation(P<0.001). Conclusion: The transplanted EPCs directly home to the injury sites of pulmonary endothelium and participate in reendothelizaiton which may maintain the integrity of alveolar-capillary barriers and inhibited the inflammatory cell infiltration to the alveolar compartments and pulmonary interstitial tissues. Moreover, the EPC engraftment increases EPCs homing to the injured lung tissues and stimulates pulmonary angiogenesis.

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