甘草酸二铵联合骨髓间充质干细胞治疗大鼠肺纤维化急性加重的实验研究  

Study on the treatment of acute exacerbation of pulmonary fibrosis in rats by diammonium glycyrrhizinate combined with bone marrow mesenchymal stem cells

作　　者：黄坤[1] 周勇 刘美芳[1] 王欣燕[1] 康小文[1] 肖金玲[1] HUANG Kun;ZHOU Yong;LIU Meifang;WANG Xinyan;KANG Xiaowen;XIAO Jinling(Department of Respiratory and Critical Care Medicine,The Second Affiliated Hospital of Harbin Medical University,Harbin,Heilongjiang 150086,P.R.China;Department of Respiratory and Critical Care Medicine,People’s Hospital of Aletai Region,Aletai,Xinjiang 836500,P.R.China)

机构地区：[1]哈尔滨医科大学附属第二医院呼吸与危重症科,黑龙江哈尔滨150086 [2]新疆维吾尔自治区伊犁哈萨克自治州阿勒泰地区人民医院呼吸与危重症科,新疆阿勒泰836500

出　　处：《中国呼吸与危重监护杂志》2020年第1期64-69,共6页Chinese Journal of Respiratory and Critical Care Medicine

基　　金：黑龙江省教育厅科学技术研究项目(12541517)。

摘　　要：目的探讨甘草酸二铵对骨髓间充质干细胞(MSC)移植治疗博来霉素(BLM)所致大鼠肺纤维化急性加重的干预作用。方法体外分离、培养雄性Wistar大鼠的MSC细胞,将24只雌性Wistar大鼠随机分为4组:NC组,气管内注入生理盐水;BLM组,气管内注入BLM;MSC组,气管内注入BLM 7 d后,经尾静脉注入雄性大鼠MSC液0.5 mL(细胞数为2.5×106个);DGMSC组,在MSC组基础上,按150 mg·kg^-1·d^-1剂量连续腹腔注射甘草酸二铵21 d。于第28 d各组均处死6只大鼠,提取肺组织,行病理学检查评估肺泡炎及肺纤维化的程度,免疫荧光法检测Ⅱ型肺泡上皮细胞数量及分布。碱水解法测定肺组织羟脯氨酸(HYP)含量,硫代巴比妥酸法测定肺组织丙二醛(MDA)含量,比色法测定肺组织超氧化物歧化酶(SOD)的活性和总抗氧化能力(T-AOC),酶联免疫吸附试验检测肺组织匀浆中肿瘤坏死因子-α(TNF-α)和转化生长因子-β1(TGF-β1)表达水平。结果甘草酸二铵联合MSC 7 d后移入可以减轻造模大鼠肺泡炎及肺纤维化的程度,其中肺组织匀浆中的HYP及TGF-β1含量较MSC组明显下降,差异有统计学意义(P<0.05)。甘草酸二铵联合MSC 7 d后移入可明显增加造模大鼠的抗氧化能力,与单纯MSC 7 d后移入比较,MDA含量下降,SOD活性及T-AOC能力改善明显,差异有统计学意义(P<0.05);Ⅱ型肺泡上皮细胞较单独MSC移入明显增多,且细胞形态完整。结论甘草酸二铵对MSC治疗肺纤维化急性加重有协同作用。其机制可能与抑制肺纤维化过程中的炎性因子、减弱氧化应激反应以及促进MSC向肺组织移入并向Ⅱ型肺泡上皮细胞转化有关。Objective To investigate the effect of diammonium glycyrrhizinate(DG) plus bone marrow mesenchymal stem cells(MSCs) transplantation in the treatment of acute exacerbation of pulmonary fibrosis induced by bleomycin(BLM) in rats. Methods MSCs were isolated from male Wistar rats and cultured in vitro. Twenty-four female Wistar rats were randomly divided into 4 groups. The NC group was intratracheally injected with normal saline;the BLM group, the MSC group and the DGMSC group were intratracheally injected with BLM for 7 days;then the MSC group was injected with 0.5 mL of MSCs solution(2.5×106 cells) into the tail vein;the DGMSC group was intraperitoneally injected with DG for 21 days in a dose of 150 mg·kg^-1·d^-1 on the base of the MSCs injection. The rats were sacrificed on the 28 th day and the lung tissue was extracted. Pathological examination was performed to determine the degree of alveolitis and pulmonary fibrosis. Immunofluorescence was used to detect the number and distribution of alveolar type Ⅱ epithelial cells. Alkali hydrolysis method was used to determine the content of hydroxyproline(HYP) in lung tissue;thiobarbituric acid method was used to measure the content of malondialdehyde(MDA) in lung tissue;colorimetric method was used to determine the superoxide dismutase activity(SOD) and total antioxidant capacity(T-AOC);enzyme linked immunosorbent assay was used to detect the expression levels of tumor necrosis factor-α(TNF-α) and transforming growth factor-β1(TGF-β1) in lung tissue homogenates. Results The DG combined with MSCs injection can reduce the degree of alveolitis and pulmonary fibrosis in BLM model rats. The content of HYP and TGF-β1 in lung tissue homogenate of the DGMSC group were significantly lower than those in the MSC group(P<0.05). Meanwhile, DG combined with MSCs injection significantly increased the antioxidant capacity of the BLM model rats. MDA content decreased, SOD activity and T-AOC ability improved significantly in the DGMSC group compared with the MSC group(P<0.05). T

关 键 词：肺纤维化 急性加重 骨髓间充质干细胞 甘草酸二铵 转化生长因子-Β1 氧化应激 

分 类 号：R563[医药卫生—呼吸系统]