绿色荧光蛋白标志的同种异体骨髓间充质干细胞对博来霉素致大鼠肺组织纤维化损伤的影响  

Effects of allogeneic bone marrow mesenchymal stem cells marked with green fluorescence proteins on Bleomycin⁃induced pulmonary fibrosis injuries in rats

作　　者：左万里 邓冏睿 黄积雄 赵洁敏 周伟 雷泽洪 黄炎明 钟慧霖 邹清雁 Zuo Wanli;Deng Jiongrui;Huang Jixiong;Zhao Jiemin;Zhou Wei;Lei Zehong;Huang Yanming;Zhong Huilin;Zou Qingyan(Department of Respiratory Medicine,Wuyi Traditional Chinese Medicine Hospital,Jiangmen Traditional Chinese Medicine Hospital Affiliated to Jinan University,Jiangmen,Guangdong 529000,China;不详)

机构地区：[1]暨南大学附属江门中医院江门市五邑中医院呼吸科,广东江门529000 [2]中山大学附属江门医院江门市中心医院呼吸科 [3]中山大学附属江门医院江门市中心医院病理科 [4]中山大学附属江门医院江门市中心医院检验科 [5]广州吉帝生物科技有限公司

出　　处：《中华生物医学工程杂志》2018年第4期242-248,共7页Chinese Journal of Biomedical Engineering

基　　金：广东省省科技计划资助项目(2013B091500041);国家科技型中小企业技术创新资助项目(14C26214402590)。

摘　　要：目的制备并用绿色荧光标志骨髓间充质干细胞(BM⁃MSC),观察其对博来霉素所致大鼠肺纤维化损伤的影响。方法60只雄性SD大鼠随机分为5组,A组于气管内注入生理盐水,B、C、D、E组于气管内注入5 mg/kg博来霉素0.1 ml。建模后1 d,A、B组经尾静脉注射生理盐水1.0 ml,C组经尾静脉注射1.0 ml BM⁃MSC(1.0×106细胞),D组尾静脉注射1.0 ml BM⁃MSC(5.0×10^(5)细胞),建模后8 d,再给1.0 ml BM⁃MSC(5.0×10^(5)细胞)。E组尾静脉注射1.0 ml BM⁃MSC(1.0×107细胞)。建模后28、42 d处死各组一半大鼠,留取肺组织。行病理学检查,对HE染色切片评定肺组织肺泡炎程度,对Masson染色切片评定肺组织纤维化程度;测定肺组织的转化生长因子⁃β1(TGF⁃β1)、羟脯氨酸(HYP)、基质金属蛋白酶⁃2(MMP⁃2)和组织金属蛋白酶抑制剂⁃1(TIMP⁃1)的表达水平,分析TGF⁃β1、HYP、MMP⁃2/TIMP⁃1与肺泡炎程度及肺纤维化程度评分的相关性。结果A组肺泡结构正常,无明显的炎症或纤维化改变,B组博来霉素可致肺泡结构严重紊乱,大量胶原纤维沉积,与B组比较,C、D、E组注射BM⁃MSC后肺泡炎性和肺纤维化程度较轻。与A组比较,B组肺泡炎程度评分、肺纤维化程度评分、TGF⁃β1、HYP、MMP⁃2/TIMP⁃1水平增高(均P<0.05),MSC治疗后的C、D、E 3组可使上述指标下降(均P<0.05)。肺组织TGF⁃β1与肺泡炎程度评分、肺纤维化程度评分相关系数r=0.82、0.89,HYP与肺泡炎程度评分、肺纤维化程度评分相关系数r=0.86、0.92,MMP⁃2/TIMP⁃1与肺泡炎程度评分、肺纤维化程度评分相关系数r=0.83、0.92,均存在正相关(均P<0.05)。结论BM⁃MSC可减低博来霉素致肺纤维化损伤,较高剂量和分次移植效果较好,其机制与移植细胞长期存活无关,可能与下调TGF⁃β1水平、改善MMP/TIMP失衡有关。Objective To prepare bone marrow mesenchymal stem cells(BM⁃MSCs)marked with green fluorescence proteins(GFP),and to determine its effects on Bleomycin⁃induced pulmonary fibrosis injuries in rats.Methods Sixty male SD rats were randomly divided into 5 groups.Group A received intratracheal instillation with normal saline(NS),and groups B,C and D received intratracheal instillation with Bleomycin(5 mg/kg in 0.1 ml NS).At 1 d after the modeling,groups A and B were injected with 1.0 ml NS via the tail vein;group C was injected with 1.0 ml BM⁃MSCs solution(1.0×106 cells)via the tail vein;group D was injected with 1.0 ml BM⁃MSCs solution(5×105 cells)via the tail vein and injected with the same dose again at 8 d;and group E was injected with 1.0 ml BM⁃MSCs solution(1.0×107 cells)via the tail vein,respectively.Half of the rats in each group were sacrificed at 28 and 42 d after the modeling,respectively,and lung tissue was obtained.The pathological examination was performed.The degree of alveolitis in lung tissues was evaluated by HE staining,and the degree of fibrosis in lung tissues was evaluated by Masson staining.The expression levels of transforming growth factor⁃β1(TGF⁃β1),hydroxyproline(HYP),matrix metalloproteinase⁃2(MMP⁃2)and tissue inhibitor of metalloproteinase⁃1(TIMP⁃1)in the lung tissues were determined.The correlation between TGF⁃β1,HYP and MMP⁃2/TIMP⁃1 and the scores of alveolitis and pulmonary fibrosis degrees was analyzed.Results The alveolar structure of group A was normal,and no significant inflammation or fibrotic changes were found.In group B,Bleomycin caused severe disorder of alveolar structure,and a large amount of collagen fibers were deposited.Compared with group B,the alveolitis and pulmonary fibrosis were less severe after injection of BM⁃MSCs in groups C,D,and E.Compared with group A,the alveolitis degree score,pulmonary fibrosis degree score,and the levels of TGF⁃β1,HYP and MMP⁃2/TIMP⁃1 increased in group B(all P<0.05).The indexes decreased in groups

关 键 词：肺纤维化 博来霉素 间质干细胞 绿色荧光蛋白质类 

分 类 号：R563[医药卫生—呼吸系统]