骨髓间充质干细胞向肺泡细胞转化的实验研究  

Bone Marrow Mesenchymal Stem Cells Transform to Alveolar Epithelial Cells in Bleomycin Induced Lung Injury

作　　者：黄坤[1] 吴晓梅[1] 王欣燕[1] 康小文[1] 肖金玲[1] 李兆国[1] 

机构地区：[1]哈尔滨医科大学第二附属医院呼吸内科,黑龙江哈尔滨150086

出　　处：《中国呼吸与危重监护杂志》2011年第6期572-576,共5页Chinese Journal of Respiratory and Critical Care Medicine

基　　金：黑龙江省科技局攻关课题资助项目(编号:GC10C303-3)

摘　　要：目的通过观察外源性骨髓间充质干细胞(MSC)的肺脏迁移及分化情况,探讨MSC治疗肺纤维化的细胞学机制。方法体外分离、培养雄性Wistar大鼠的MSC。将60只雌性Wistar大鼠随机分为4组,A组制备博莱霉素致肺纤维化模型;B组和C组分别于博莱霉素注射后,立即和7 d后通过尾静脉注入5-溴-2-脱氧尿苷(BrdU)标记的雄鼠MSC液0.5 mL(含细胞数2.5×106个);D组造模后,立即注入未标记的等量雄鼠MSC液,作为阴性对照。提取肺组织的DNA,通过聚合酶链反应(PCR)检测雄性鼠的性别决定基因(sry基因),以判断外源性MSC是否存在于雌性鼠肺组织中;留取肺组织、新鲜提取的骨髓干细胞和培养至第5代的MSC行BrdU和肺表面活性蛋白C(SP-C)的双重免疫荧光染色,通过逆转录PCR(RT-PCR)检测其是否表达SP-C mRNA和水通道蛋白5(AQP-5)的mRNA,观察MSC移植前后的分化情况。结果造模后立即和7 d后移入雄性MSC的雌性鼠肺组织均可表达sry基因,且肺组织中的MSC可转化为Ⅱ型肺泡上皮细胞(AECⅡ),并具备AECⅡ的形态学特征和分子生物学表型,但立即移入组明显好于7 d后移入组(P<0.01)。新提取的骨髓干细胞和培养至第5代的MSC表达SP-C mRNA,而不表达AQP-5 mRNA,且SP-C的免疫荧光染色阴性。结论外源性MSC可移入到损伤的肺组织内并转化为AECⅡ,而且早期移入更显著。这种分化潜能在骨髓阶段已具备,但要到损伤的微环境下才能发生转化。Objective To explore the migration and differentiation of bone marrow mesenchymal stem cells（MSCs） in lung.Methods MSCs were harvested from a male Wistar rat.Sixty female Wistar rats were randomly divided into four groups.The pulmonary fibrosis model was established by intratracheal instillation of bleomycin in group A-D.Immediately and 7 days after bleomycin administration respectively,the rats in group B and C received infusion with 5-bromodeoxynridine（BrdU） labeled MSCs via tail vein.And the rats in group D were infused MSCs without BrdU labeling serving as a negative control.The sry gene of Y chromosome was detected by polymerase chain reaction（PCR）.Double immunofluorescence staining was used to detected BrdU and surfactant associated protein-C（SP-C） expression in lung tissue,fresh bone marrow,and the 5th generation MSCs.Reverse transcriptipon-PCR was used to detect the expressions of SP-C mRNA and AQP-5 mRNA.Results The sry gene was detected in bleomycin induced lung injury tissues of the rats after MSCs infusion immediately and on the 7th day.The MSCs in lung tissue could transformed into cells with ACEⅡ morphological features and molecular phenotype.The transformation rate was higher in the rats received MSCs infusion immediately than the rats received on 7th day.The 5th generation MSCs and fresh bone marrow expressed SP-C mRNA,without AQP-5 mRNA and SP-C expression.Conclusions Exogenous MSCs can be transplanted into injured lung tissues and transform into AECⅡ,especially in early stage of lung injury.The differentiation potential of MSCs can be activated in injury micro-environment.

关 键 词：肺纤维化 肺损伤 间充质干细胞 博莱霉素 Ⅱ型肺泡上皮细胞 肺表面活性蛋白C 

分 类 号：R563.9[医药卫生—呼吸系统]