Effects and mechanism of annexin A1-overexpressing human adipose-derived mesenchymal stem cells in the treatment of mice with acute respiratory distress syndrome
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摘要:
目的 探索过表达膜联蛋白A1（ ANXA1）的人脂肪间充质干细胞（AMSC）治疗急性呼吸窘迫综合征（ARDS）小鼠的效果及其机制。 方法 采用实验研究方法。采用流式细胞术鉴定成人AMSC后，取第3代细胞进行后续实验。采用随机数字表法（分组方法下同），将细胞分为转染含 ANXA1基因RNA序列的质粒的过表达ANXA1组、转染对应空载质粒的空载对照组，另取细胞分为转染含 ANXA1基因小干扰RNA序列的质粒的敲减ANXA1组、转染对应空载质粒的空载对照组，转染后72 h，于荧光显微镜成像系统下观察荧光表达情况，分别采用蛋白质印迹法和实时荧光定量反转录PCR法检测ANXA1的蛋白和mRNA表达（样本数均为3）。取50只6~8周龄雄性C57BL/6J小鼠，分为假伤组、单纯ARDS组、正常细胞组、过表达ANXA1组、敲减ANXA1组，每组10只。将后4组小鼠均用内毒素/脂多糖制成ARDS肺损伤模型，假伤组小鼠模拟致假伤。伤后即刻，假伤组、单纯ARDS组小鼠均经尾静脉注射生理盐水，正常细胞组、过表达ANXA1组、敲减ANXA1组小鼠对应注射正常AMSC、过表达 ANXA1的AMSC、敲减 ANXA1的AMSC。注射后24 h，取每组5只小鼠，行伊文思蓝染色观察右肺组织大体染色情况，采用酶标仪检测左肺支气管肺泡灌洗液（BALF）上清液的吸光度值，了解肺血管通透性。注射后3 d，取各组剩余5只小鼠，取右肺组织，行苏木精-伊红染色观察病理学变化，行免疫组织化学染色观察CD11b和F4/80阳性巨噬细胞情况；采用酶联免疫吸附测定法检测左肺BALF上清液中肿瘤坏死因子α（TNF-α）、白细胞介素6（IL-6）和IL-1β水平。对数据行配对样本 t检验、单因素方差分析与LSD检验。 结果 转染后72 h，过表达ANXA1组与敲减ANXA1组AMSC均分别较对应空载对照组AMSC表达更高强度的荧光。转染后72 h，与对应空载对照组比较，过表达ANXA1组AMSC中ANXA1蛋白与mRNA表达均明显增多（ t值分别为249.80、6.56， P<0.05），敲减ANXA1组AMSC中ANXA1蛋白与mRNA表达均明显减少（ t值分别为176.50、18.18， P<0.05）。注射后24 h，与假伤组（BALF上清液的吸光度值为0.041±0.009）比较，单纯ARDS组小鼠肺组织明显蓝染且BALF上清液的吸光度值（0.126±0.022）明显升高（ P<0.05）；与单纯ARDS组比较，正常细胞组、过表达ANXA1组小鼠肺组织蓝染程度明显减轻且BALF上清液的吸光度值（0.095±0.020、0.069±0.015）明显降低（ P<0.05），敲减ANXA1组小鼠肺组织蓝染程度与BALF上清液的吸光度值（0.109±0.016， P>0.05）无明显变化；与正常细胞组比较，过表达ANXA1组小鼠BALF上清液的吸光度值明显降低（ P<0.05）。注射后3 d，单纯ARDS组小鼠肺组织结构较假伤组明显损伤；与单纯ARDS组比较，正常细胞组和过表达ANXA1组小鼠肺组织出血、炎症细胞浸润、肺泡萎陷及间质增宽程度等改变均明显减轻，敲减ANXA1组小鼠前述肺组织表现未明显改善。注射后3 d，单纯ARDS组小鼠肺组织中CD11b和F4/80阳性巨噬细胞数量均较假伤组明显增多；与单纯ARDS组比较，正常细胞组、过表达ANXA1组、敲减ANXA1组小鼠肺组织中CD11b和F4/80阳性巨噬细胞数量均减少，以过表达ANXA1组减少最明显。注射后3 d，与假伤组比较，单纯ARDS组小鼠BALF上清液中TNF-α、IL-6、IL-1β水平均显著升高（ P<0.05）；与单纯ARDS组比较，正常细胞组、过表达ANXA1组小鼠BALF上清液中TNF-α、IL-6、IL-1β水平以及敲减ANXA1组小鼠BALF上清液中IL-1β水平均明显降低（ P<0.05）；与正常细胞组比较，过表达ANXA1组小鼠BALF上清液中TNF-α水平明显降低（ P<0.05），敲减ANXA1组小鼠BALF上清液中TNF-α水平明显升高（ P<0.05）。 结论 过表达 ANXA1可以优化AMSC在ARDS治疗中的效果，增强该类细胞抑制炎症反应和改善肺血管通透性的作用，进而缓解ARDS小鼠的肺损伤。 Abstract:Objective To explore the effects and mechanism of annexin A1 ( ANXA1)-overexpressing human adipose-derived mesenchymal stem cells (AMSCs) in the treatment of mice with acute respiratory distress syndrome (ARDS). Methods The experimental study method was adopted. After the adult AMSCs were identified by flow cytometry, the 3 rd passage cells were selected for the follow-up experiments. According to the random number table (the same grouping method below), the cells were divided into ANXA1-overexpressing group transfected with plasmid containing RNA sequences of ANXA1 gene and no-load control group transfected with the corresponding no-load plasmid. The other cells were divided into ANXA1-knockdown group transfected with plasmid containing small interfering RNA sequences of ANXA1gene and no-load control group transfected with the corresponding no-load plasmid. At post transfection hour (PTH) 72, the fluorescence expression was observed under a fluorescence microscope imaging system, and the protein and mRNA expressions of ANXA1 were detected by Western blotting and real-time fluorescence quantitative reverse transcription polymerase chain reaction respectively (with the sample numbers being 3). Fifty male C57BL/6J mice aged 6-8 weeks were divided into sham injury group, ARDS alone group, normal cell group, ANXA1-overexpressing group, and ANXA1-knockdown group, with 10 mice in each group. Mice in the last 4 groups were treated with endotoxin/lipopolysaccharide to make ARDS lung injury model, and mice in sham injury group were simulated to cause false injury. Immediately after injury, mice in sham injury group and ARDS alone group were injected with normal saline through the tail vein, while mice in normal cell group, ANXA1-overexpressing group, and ANXA1-knockdown group were injected with normal AMSCs, ANXA1-overexpressing AMSCs, and ANXA1-knockdown AMSCs, correspondingly. At post injection hour (PIH) 24, 5 mice in each group were selected, the Evans blue staining was performed to observe the gross staining of the right lung tissue, and the absorbance value of bronchoalveolar lavage fluid (BALF) supernatant of left lung was detected by microplate reader to evaluate the pulmonary vascular permeability. Three days after injection, the remaining 5 mice in each group were taken, the right lung tissue was collected for hematoxylin-eosin staining to observe the pathological changes and immunohistochemical staining to observe the CD11b and F4/80 positive macrophages, and the levels of tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), and IL-1β in BALF supernatant of left lung were determined by enzyme-linked immunosorbent assay. Data were statistically analyzed with paired sample t test, one-way analysis of variance, and least significant difference test. Results At PTH 72, AMSCs in both ANXA1-overexpressing group and ANXA1-knockdown group expressed higher fluorescence intensity than AMSCs in corresponding no-load control group, respectively. At PTH 72, compared with those in corresponding no-load control group, the protein and mRNA expressions of ANXA1 in ANXA1-overexpressing group were significantly increased (wth t values of 249.80 and 6.56, respectively, P<0.05), while the protein and mRNA expressions of ANXA1 in ANXA1-knockdown group were significantly decreased (wth t values of 176.50 and 18.18, respectively, P<0.05). At PIH 24, compared with those in sham injury group (with the absorbance value of BALF supernatant being 0.041±0.009), the lung tissue of mice in ARDS alone group was obviously blue-stained and the absorbance value of BALF supernatant (0.126±0.022) was significantly increased ( P<0.05). Compared with those in ARDS alone group, the degree of blue-staining in lung tissue of mice was significantly reduced in normal cell group or ANXA1-overexpressing group, and the absorbance values of BALF supernatant (0.095±0.020 and 0.069±0.015) were significantly decreased ( P<0.05), but the degree of blue-staining in lung tissue and the absorbance value of BALF supernatant (0.109±0.016, P>0.05) of mice in ANXA1-knockdown group had no significant change. Compared with that in normal cell group, the absorbance value of BALF supernatant of mice in ANXA1-overexpressing group was significantly decreased ( P<0.05). Three days after injection, the lung tissue structure of mice in ARDS alone group was significantly damaged compared with that in sham injury group. Compared with those in ARDS alone group, hemorrhage, infiltration of inflammatory cells, alveolar collapse, and interstitial widening in the lung tissue of mice were significantly alleviated in normal cell group and ANXA1-overexpressing group, while no significant improvement of above-mentioned lung tissue manifestation was observed in ANXA1-knockdown group. Three days after injection, the numbers of CD11b and F4/80 positive macrophages in the lung tissue of mice in ARDS alone group were significantly increased compared with those in sham injury group. Compared with those in ARDS alone group, the numbers of CD11b and F4/80 positive macrophages in lung tissue of mice in normal cell group, ANXA1-overexpressing group, and ANXA1-knockdown group reduced, with the most significant reduction in ANXA1-overexpressing group. Three days after injection, compared with those in sham injury group, the levels of TNF-α, IL-6, and IL-1β in BALF supernatant of mice in ARDS alone group were significantly increased ( P<0.05). Compared with those in ARDS alone group, the levels of TNF-α, IL-6, and IL-1β in BALF supernatant of mice in normal cell group and ANXA1-overexpressing group, as well as the level of IL-1β in BALF supernatant of mice in ANXA1-knockdown group were significantly decreased ( P<0.05). Compared with that in normal cell group, the level of TNF-α in BALF supernatant of mice was significantly decreased in ANXA1-overexpressing group ( P<0.05) but significantly increased in ANXA1-knockdown group ( P<0.05). Conclusions Overexpression of ANXA1 can optimize the efficacy of AMSCs in treating ARDS and enhance the effects of these cells in inhibiting inflammatory response and improving pulmonary vascular permeability, thereby alleviating lung injury of mice with ARDS. -
参考文献
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1 4组成人脂肪间充质干细胞转染后72 h荧光（绿色）表达情况 绿色荧光蛋白×40。1A.转染pCDH-CMV-MCS-EF1-GFP-Puro质粒的空载对照组细胞荧光强度较弱；1B.转染pCDH-CMV-ANXA1-EF1-GFP-Puro质粒的过表达膜联蛋白A1（ANXA1）组细胞荧光强度明显强于图1A；1C.转染pLKO.1-EGFP-puro-human scramble质粒的空载对照组细胞荧光强度较弱；1D.转染pLKO.1-EGFP-puro-shANXA1质粒的敲减ANXA1组细胞荧光强度明显强于图1C
2 蛋白质印迹法检测的4组成人脂肪间充质干细胞转染后72 h ANXA1蛋白表达
注：ANXA1为膜联蛋白A1，GAPDH为3-磷酸甘油醛脱氢酶；条带上1、2、3、4分别指转染pCDH-CMV-MCS-EF1-GFP-Puro质粒的空载对照组、转染pCDH-CMV-ANXA1-EF1-GFP-Puro质粒的过表达ANXA1组、转染pLKO.1-EGFP-puro-human scramble质粒的空载对照组、转染pLKO.1-EGFP-puro-shANXA1质粒的敲减ANXA1组
3 假伤组与4组ARDS小鼠注射后24 h进行肺组织伊文思蓝染色（阳性染色为蓝色）观察到的情况。3A.假伤组正常肺组织蓝染不明显；3B.单纯ARDS组肺组织较图3A明显蓝染；3C、3D.分别为正常细胞组、过表达ANXA1组，肺组织蓝染程度均较图3B明显减轻且图3D减轻程度更明显；3E.敲减ANXA1组肺组织蓝染程度与图3B相近
注：假伤组、单纯急性呼吸窘迫综合征（ARDS）组小鼠均注射生理盐水，正常细胞组、过表达膜联蛋白A1（ANXA1）组、敲减ANXA1组小鼠对应注射正常成人脂肪间充质干细胞（AMSC）、过表达ANXA1的成人AMSC、敲减ANXA1的成人AMSC
4 假伤组与4组ARDS小鼠注射后3 d肺组织CD11b和F4/80阳性巨噬细胞（均为棕色）情况 辣根过氧化物酶-二氨基联苯胺×40。4A、4B、4C、4D、4E.分别为假伤组、单纯ARDS组、正常细胞组、过表达ANXA1组、敲减ANXA1组CD11b阳性巨噬细胞情况，图4A、4C、4D、4E阳性染色细胞数量均明显少于图4B，且图4D阳性染色细胞数量减少最明显；4F、4G、4H、4I、4J.分别为假伤组、单纯ARDS组、正常细胞组、过表达ANXA1组、敲减ANXA1组F4/80阳性巨噬细胞情况，图4F、4H、4I、4J阳性染色细胞数量均明显少于图4G，且图4I阳性染色细胞数量减少最明显
注：假伤组、单纯急性呼吸窘迫综合征（ARDS）组小鼠均注射生理盐水，正常细胞组、过表达膜联蛋白A1（ANXA1）组、敲减ANXA1组小鼠对应注射正常成人脂肪间充质干细胞（AMSC）、过表达ANXA1的成人AMSC、敲减ANXA1的成人AMSC
表1 假伤组与4组ARDS小鼠注射后3 d支气管肺泡灌洗液上清液中炎症因子水平比较（pg/mL，
组别 样本数 TNF-α IL-6 IL-1β 假伤组 5 59±6 81±17 125±13 单纯ARDS组 5 205±54 166±9 293±31 正常细胞组 5 136±11 133±16 184±17 过表达ANXA1组 5 113±15 129±10 187±30 敲减ANXA1组 5 171±24 153±12 185±34 F值 15.87 23.78 21.34 P值 <0.001 <0.001 <0.001 P 1值 0.007 <0.001 <0.001 P 2值 0.036 0.007 <0.001 P 3值 0.011 <0.001 0.001 P 4值 0.285 0.121 0.002 P 5值 0.042 0.695 0.871 P 6值 0.028 0.080 0.970 注：假伤组、单纯急性呼吸窘迫综合征（ARDS）组小鼠均注射生理盐水，正常细胞组、过表达膜联蛋白A1（ANXA1）组、敲减ANXA1组小鼠对应注射正常成人脂肪间充质干细胞（AMSC）、过表达 ANXA1的成人AMSC、敲减 ANXA1的成人AMSC；TNF-α为肿瘤坏死因子α，IL为白细胞介素； P 1值为假伤组与单纯ARDS组比较所得， P 2值、 P 3值、 P 4值分别为正常细胞组、过表达ANXA1组、敲减ANXA1组与单纯ARDS组比较所得， P 5值、 P 6值分别为过表达ANXA1组、敲减ANXA1组与正常细胞组比较所得 
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