骨髓间充质干细胞移植对大鼠肺纤维化的影响

The effect of marrow mesenchymal stem cell transplantation on pulmonary fibrosis in rats

目的研究骨髓间充质干细胞（MSC）对博来霉素（BLM）所致大鼠肺间质纤维化治疗作用的可能机制。方法将54只雌性Wister大鼠按随机数字表法分为对照组、BLM组和MSC组，每组18只。对照组气管内注人生理盐水，BLM组气管内注入博来霉素，MSC组气管内注入博来霉素后，立即经尾静脉注入雄性大鼠MSC液0．5ml（细胞数为2．5×10^6个），分别于第7、14、28天各组均处死6只大鼠。检测MSC移植前的5-溴-2-脱氧尿苷（BrdU）标记率，留取肺组织行病理学检查，测定羟脯氨酸含量，采用双重免疫荧光法检测BrdU标记的MSC是否表达Ⅱ型肺泡细胞（ATⅡ）特异性标志物肺表面活性蛋白-C（SP-C）；RT-PCR法检测不同时期肺组织和骨髓中SP-CmRNA表达量，观察肺损伤后自身骨髓动员是否参与Ⅱ型肺泡细胞的修复。结果BrdU标记的MSC于48h终浓度为10μmoL／L时标记率〉98％，且传代细胞可连续标记。MSC组7、14、28d的肺组织冰冻切片中均可见到BrdU标记的MSC定位于肺组织并同时表达SP-C。28d时MSC组与BLM组肺纤维化程度评分分别为（2．17±0．26）分和（2．83±0．24）分，肺组织羟脯氨酸含量分别为（138±21）mg／g和（184±19）mg／g。肺组织中SP-CmRNA的表达量分别为0．98±0．15和0．59±0．14，且两组在不同时期骨髓中SP-CmRNA表达量均较对照组明显增加。结论骨髓间充质干细胞可定植于肺组织，并转化为Ⅱ型肺泡细胞，可阻止肺纤维化的进展，同时损伤诱导自身骨髓动员增强也参与修复过程。

Objective To study the possible mechanisms of marrow mesenchymal stem cells （MSC） in therapy of bleomycin （BLM）-induced pulmonary fibrosis in rats. Methods Fifty-four female Wistar rats were randomly divided into a control group, a BLM group and a MSC group. The control group received intratracheal normal saline, the BLM group received intratracheal instillation of bleomyein, and the MSC group was injected with male rat MSC solution of 0. 5 ml （2.5 ×10^6 cells） via the tail vein after intratraeheal instillation of bleomycin. Six rats from each group were killed on day 7, 14 and 28 of the experiments. BrdU labeling rate was measured before MSC transplantation. Lung tissue specimens were obtained for pathological examination, hydroxyproline content measurement, and detection of the expression of type Ⅱ alveolar cell （ ATⅡ ） specific marker-pulmonary surfaetant protein-C （ SP-C ） in BrdU labeled MSC using dual immunofluorescence method. RT-PCR method was used to detect SP-C mRNA expression in the lung tissue and the bone marrow at different stages. The bone marrow mobilization involved in repair of type Ⅱ alveolar cells after lung injury was observed. Results The final concentration of BrdU labeled MSC at 48 h was 10 μmol/L, while the labeling efficiency was〉98%, and the passage cells could be continuously labeled. In the MSC group, BrdU labeled MSCs with expression of SP-C were observed in all frozen sections of lung tissue at day 7, 14, and 28. By day 28, the lung fibrosis scores of the MSC group and the BLM group were （ 2. 17 ±0. 26 ） and （ 2. 83 ±0. 24 ） , respectively, the lung tissue hydroxyproline contents were （ 138 ± 21 ） mg/g and （ 184 ± 19） mg/g, respectively, and the lung tissue SP-C mRNA expressions were （0. 98±0. 15 ） and （0. 59 ± 0. 14）, respeetively. For both groups the SP-C mRNA expressions in the bone marrow at different stages were significantly inereased as compared to the controlgroup. Conclusions Marrow mesenchymal stem cells could be transplanted into lung tissues of rats, and transformed into type Ⅱ alveolar cells and was shown to prevent the development of pulmonaFy fibrosis. The damage-induced enhancement of host bone marrow mobilization was also involved in the repair process.