间充质干细胞对大鼠急性肺损伤模型的干预研究

Study on the mesenchymal stem cell in treatment of acute lung injury rat induced by bleomycin

目的通过博来霉素诱导大鼠急性肺损伤为研究模型,观察携带ACE2基因的人脐带间充质干细胞对大鼠急性肺损伤的影响。方法取正常足月剖宫产健康婴儿脐带分离人脐带间充质干细胞,经流式细胞术鉴定细胞表面抗原;以三质粒共转染法在293T细胞中制备病毒颗粒Lenti-ACE2,并转染HUMSCs;采用一次性气管内滴注BLM(5 mg/kg)建立肺损伤的动物模型,收集损伤前和滴注BLM手术后3、7、14 d肺泡灌洗液及肺组织进行相关指标检测。计数法测定肺泡灌洗液中中性粒细胞数量;BCA法测定肺泡灌洗液中总蛋白含量;化学法测定MPO活性;HE染色法观察肺组织病理学形态;RT-PCR测定肺组织中细胞因子INF-γ、IL-4的mRNA 变化,ELISA测定肺组织中细胞因子TNF-α的含量,Western-blotting测定肺组织ACE2蛋白的表达。结果 HUMSCs可成功分离提纯,转染Lenti-ACE2前后,HUMSCs细胞表型不受影响。携带目的基因ACE2的HUMSCs在输入大鼠体内后,ACE2与HUMSCs发挥协同作用,能有效降低ALI大鼠肺部中性粒细胞浸润和总蛋白渗出,改善血管通透性程度,降低肺组织中促炎因子INF-γ基因水平,并能提高抗炎因子IL-4以及ACE2的表达,从而减轻ALI大鼠病理损伤程度。结论人脐带间充质干细胞携带ACE2基因可以作为治疗急性肺损伤的一种有效方法。

Objective To find a new and effective therapeutic method by induced bleomycin (BLM) and set up the rat model of mesenchymal stem cell-based angiotensin-converting enzyme 2 in treatment of acute lung injury (ALI). Methods ALI was a high incidence disease with no effective therapeutic method (mortality rate > 40%). It was transfected the human umbilical cord mesenchymal stem cells (HUMSCs) with Lenti- angiotensin-converting enzyme 2 (ACE2) viral particles, after the isolation of HUMSCs from cesarean fetus. Then evaluated the therapeutic effect of HUMSCs harboring ACE2 on ALI, which induced by BLM in rat model. Results The results showed that HUMSCs harboring ACE2 could attenuate ALI degree through reducing pulmonary inflammatory infiltration and degree of vascular permeability, repressing the mRNA level of pro-inflammatory cytokines, activating the mRNA level of anti-inflammatory cytokines and ACE2. Besides, results also demonstrated that HUMSCs harboring ACE2 gene had higher therapeutic effects to ALI than the single factor of HUMSCs or ACE2. Conclusion The research provides clues for the development of effective therapeutic methods to ALI using stem cell transplantation and ACE2 gene therapy.