携带血管紧张素转换酶2基因的人脐带间充质干细胞尾静脉注射移植可减轻博来霉素诱导的大鼠急性肺损伤  

Intravenous human umbilical mesenchymal stem cells carrying angiotensin converting enzyme 2 gene in alleviating acute lung injury induced by bleomycin in rats

作　　者：高风英 金文强 王星海 刘振威[2] GAO Feng-ying;JIN Wen-qiang;WANG Xing-hai;LIU Zhen-wei(Department of Respiratory Medicine,Shanghai Jiangong Hospital,Shanghai 200083,China;Department of Respiratory Medicine,Shanghai General Hospital,Shanghai Jiao Tong University,Shanghai 200080,China)

机构地区：[1]上海市建工医院呼吸科,上海200083 [2]上海市第一人民医院呼吸科,上海200080

出　　处：《中国临床医学》2018年第2期230-238,共9页Chinese Journal of Clinical Medicine

基　　金：上海市卫生和计划生育委员会科研课题(201740236)~~

摘　　要：目的:探讨携带血管紧张素转换酶2(angiotensin converting enzyme 2,ACE2)基因的人脐带间充质干细胞(human umbilical mesenchymal stem cells,HUMSCs)尾静脉注射移植对博来霉素(bleomycin,BLM)诱导的大鼠急性肺损伤(ALI)的可能作用。方法:取正常足月剖宫产健康婴儿脐带分离HUMSCs,流式细胞术鉴定细胞表面抗原;三质粒共转染法在293T细胞中制备病毒颗粒Lenti-ACE2,并转染HUMSCs。大鼠随机分为正常对照组、BLM组、ACE2组、HUMSCs组、HUMSCsACE2组(n=24)。后4组大鼠采用一次性气管内滴注BLM(5mg/kg)建立肺损伤大鼠模型,收集各组大鼠损伤前和滴注BLM后第3、7、14天肺泡灌洗液(BALF)及肺组织进行相关指标检测。计数法测定BALF中性粒细胞数量;BCA法测定BALF总蛋白含量;化学法测定髓过氧化物酶(myeloperoxidase,MPO)活性;H-E染色法观察肺组织病理学形态;RT-PCR测定肺组织中INF-γ、IL-4mRNA表达的变化;ELISA法测定肺组织TNF-α含量;Western印迹法测定肺组织ACE2蛋白表达。结果:HUMSCs组、ACE2组及HUMSCs-ACE2组大鼠BALF中性粒细胞计数在第3、7、14天较同期BLM组呈现下降趋势,其中以HUMSCs-ACE2组降低量最明显,差异有统计学意义(P<0.05)。与同期BLM组相比,HUMSCs组、ACE2组及HUMSCsACE2组BALF总蛋白量降低,以HUMSCs-ACE2组降低量明显,差异有统计学意义(P<0.05)。肺损伤严重度病理评分结果显示:与BLM相比,ACE2、HUMSCs、HUMSCs-ACE2组大鼠肺组织损伤得到不同程度的改善,尤以HUMSCs-ACE2组表现最佳,在损伤后第3、7、14天差异有统计学意义(P<0.05)。与同期BLM组相比,HUMSCs-ACE2组总蛋白中ACE2表达增量最明显,第14天达高峰,接近BLM刺激前水平,差异有统计学意义(P<0.05)。与同期BLM组相比,HUMSCs组、ACE2组及HUMSCs-ACE2组MPO活性降低,以HUMSCs-ACE2组降低量最明显(P<0.05)。与同期BLM组相比,HUMSCs组、ACE2组及HUMSCs-ACE2组INF-γmRNA水平降低,以HUMSCs-ACE2组降低量最明显(P<0.05)。�Objective:To explore the effect of transplantation of human umbilical mesenchymal stem cells(HUMSCs)carrying angiotensin converting enzyme 2(ACE2)gene in treatment of acute lung injury(ALI)induced by bleomycin in rats.Methods:HUMSCs were isolated from the umbilical cord of normal mature infants delivered by caesarean section,and flow cytometry was used to identify the cell surface antigen.The virus granules Lenti-ACE2 were prepared by three-plasmid co-transfection method in 293T cells,and then were transfected into HUMSCs.The rats were randomly divided into normal control group,BLM group,ACE2 group,HUMSCs group,and HUMSCs-ACE2 group(n=24).The rats were dripped by BLM(5 mg/kg)in endotracheal to establish ALI rat model except normal control group.The broncho-alveolar lavage fluid(BALF)and lung tissue were collected to detect the related indicators before and 3,7,14 days after BLM dripping.The number of neutrophils and total protein content in alveolar lavage were determined by counting method and BCA method,respectively.The histopathological morphology of lung was observed by H-E staining,the activity of myeloperoxidase(MPO),the levels of INF-γand IL-4 mRNA,the level of TNF-αand the expression of ACE2 protein in lung tissue were respectively determined by chemical method,RT-PCR,ELISA method and Western blotting.Results:The neutrophil count and total protein content in BALF in HUMSCs group,ACE2 group and HUMSCs-ACE2 group showed a trend of decline compared with that in BLM group at 3,7,14 days time points,especially in HUMSCs-ACE2 group(P<0.05).Pathological grading of lung injury severity showed that the lung tissue injury was significantly improved in ACE2,HUMSCs,HUMSCs-ACE2 group rats compared with BLM group,especially in HUMSCs-ACE2 group(P<0.05)3,7,and 14 days after injury.Compared with BLM group at the same time points,the expression of ACE2 in HUMSCs-ACE2 group had the most obvious increment,reaching the peak at the 14 th day(P<0.05).Compared with BLM group at the same time points,the MPO activity and INF-γ

关 键 词：血管紧张素转换酶2 人脐带间充质干细胞 博来霉素 急性肺损伤 

分 类 号：R-332[医药卫生]