Effects of exosomes from human adipose-derived mesenchymal stem cells on pulmonary vascular endothelial cells injury in septic mice and its mechanism
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摘要:
目的 探讨人脂肪间充质干细胞（ADSC）来源外泌体对脓毒症小鼠肺血管内皮细胞（PMVEC）损伤的影响及其机制。 方法 采用实验研究方法。取空军军医大学第一附属医院收治的3例行腹部手术切除患者（均为女性，年龄10~25岁）遗弃的新鲜脂肪组织进行原代人ADSC的分离培养，于第5天采用倒置相差显微镜观察细胞形态。取第3代ADSC，采用流式细胞术检测ADSC中CD29、CD34、CD44、CD45、CD73和CD90的表达情况。选取第3~5代ADSC，采用差速超高速离心法获取其上清液中的外泌体，采用透射电镜和纳米颗粒跟踪分析法及蛋白质印迹法分别检测外泌体形状、粒径大小及CD9、CD63、肿瘤易感基因101（TSG101）和β肌动蛋白的蛋白表达。取24只成年雄性BALB/c小鼠，按照随机数字表法（分组方法下同）分成正常对照组、单纯盲肠结扎穿孔（CLP）组和CLP+ADSC外泌体组，每组8只，分别进行相应处理。术后24 h，采用酶联免疫吸附测定法检测小鼠血清中肿瘤坏死因子α（TNF-α）和白细胞介素1β（IL-1β）水平，采用苏木精-伊红染色和髓过氧化物酶染色检测小鼠肺组织形态，采用免疫荧光法检测小鼠肺组织细胞中8-羟基脱氧鸟苷（8-OHdG）的表达。取1个月龄雌雄不拘C57小鼠，采用组织块法获取原代PMVEC。取第3代PMVEC，采用免疫荧光法和流式细胞术检测PMVEC中CD31的表达。取第3代PMVEC，与ADSC来源外泌体共培养12 h，采用PKH26试剂盒检测PMVEC吞噬外泌体的情况。取第3代PMVEC，将细胞分为空白对照组、单纯巨噬细胞上清液组和巨噬细胞上清液+ADSC外泌体组，每组3孔，分别进行相应处理。24 h后，采用流式细胞术检测细胞中活性氧含量，采用免疫荧光法检测细胞中8-OHdG表达，采用Transwell实验测定细胞单分子层通透性。以上样本数均为3。对数据行单因素方差分析和LSD-t检验。 结果 分离的原代ADSC培养至第5天，呈梭形密集生长，呈典型的漩涡样排列。第3代ADSC中CD29、CD44、CD73和CD90阳性细胞百分比均>90%，CD34和CD45阳性细胞百分比均<5%。第3~5代ADSC来源外泌体呈典型的双凹盘状结构，外泌体平均粒径为103 nm，外泌体中CD9、CD63和TSG101的蛋白表达均呈阳性，β肌动蛋白的蛋白表达呈阴性。术后24 h，与正常对照组比较，单纯CLP组小鼠血清中TNF-α和IL-1β的含量均明显增加（t值分别为28.76、29.69，P<0.01）；与单纯CLP组比较，CLP+ADSC外泌体组小鼠血清中TNF-α和IL-1β的含量均明显降低（t值分别为9.90、4.76，P<0.05或P<0.01）。术后24 h，正常对照组小鼠肺组织结构清晰完整、无炎症细胞浸润，单纯CLP组小鼠的肺组织较正常对照组水肿明显、炎症细胞浸润现象明显，CLP+ADSC外泌体组小鼠肺组织较单纯CLP组水肿症状明显减轻、炎症细胞浸润明显减少。术后24 h，3组小鼠肺组织中CD31呈阳性表达；单纯CLP组小鼠肺组织中8-OHdG阳性细胞荧光强度较正常对照组显著增大，CLP+ADSC外泌体组小鼠肺组织中8-OHdG阳性细胞荧光强度较单纯CLP组明显下降。第3代PMVEC的免疫荧光结果显示CD31呈阳性表达，流式细胞术鉴定结果显示CD31阳性细胞占比为99.5%。共培养12 h，ADSC来源外泌体成功被PMVEC吞噬，进入胞质。第3代PMVEC培养24 h后，与空白对照组比较，单纯巨噬细胞上清液组PMVEC的活性氧荧光强度明显增加（t=15.73，P<0.01）；与单纯巨噬细胞上清液组比较，巨噬细胞上清液+ADSC外泌体组PMVEC的活性氧荧光强度明显降低（t=4.72，P<0.01）。第3代PMVEC培养24 h，与空白对照组相比，单纯巨噬细胞上清液组PMVEC的8-OHdG阳性荧光强度明显增强，巨噬细胞上清液+ADSC外泌体组PMVEC的8-OHdG阳性荧光强度介于空白对照组和单纯巨噬细胞上清液组之间。第3代PMVEC培养24 h，与空白对照组比较，单纯巨噬细胞上清液组的PMVEC单分子层细胞通透性明显增加（t=6.34，P<0.01）；与单纯巨噬细胞上清液组比较，巨噬细胞上清+ADSC外泌体组的PMVEC单分子层细胞通透性明显降低（t=2.93，P<0.05）。 结论 ADSC来源外泌体可改善小鼠肺组织氧化性损伤，降低由巨噬细胞上清液诱导的PMVEC的活性氧含量、8-OHdG表达以及细胞通透性。 Abstract:Objective To investigate the effects of exosomes from human adipose-derived mesenchymal stem cells (ADSCs) on pulmonary vascular endothelial cells (PMVECs) injury in septic mice and its mechanism. Methods The experimental research method was adopted. The primary ADSCs were isolated and cultured from the discarded fresh adipose tissue of 3 patients (female, 10-25 years old), who were admitted to the First Affiliated Hospital of Air Force Medical University undergoing abdominal surgery, and the cell morphology was observed by inverted phase contrast microscope on the 5th day. The expressions of CD29, CD34, CD44, CD45, CD73, and CD90 of ADSCs in the third passage were detected by flow cytometry. The third to the fifth passage of ADSCs were collected, and their exosomes from the cell supernatant were obtained by differential ultracentrifugation, and the shape, particle size, and the protein expressions of CD9, CD63, tumor susceptibility gene 101 (TSG101), and β-actin of exosomes were detected, respectively, by transmission electron microscopy, nano-particle tracking analysis and Western blotting. Twenty-four adult male BALB/c mice were adopted and were divided into normal control group, caecal ligation perforation (CLP) alone group, and CLP+ADSC-exosome group with each group of 8 according to random number table (the same grouping method below) and were treated accordingly. At 24 h after operation, tumor necrosis factor (TNF-α) and interleukin 1β (IL-1β) levels of mice serum were detected by enzyme-linked immunosorbent assay, and lung tissue morphology of mice was detected by hematoxylin-eosin and myeloperoxidase staining, and the expression of 8-hydroxy-deoxyguanosine (8-OHdG) of mouse lung cells was detected by immunofluorescence method. Primary PMVECs were obtained from 1-month-old C57 mice regardless gender by tissue block method. The expression of CD31 of PMVECs was detected by immunofluorescence and flow cytometry. The third passage of PMVECs was co-cultured with ADSCs derived exosomes for 12 h, and the phagocytosis of exosomes by PMVECs was detected by PKH26 kit. The third passage of PMVECs were adopted and were divided into blank control group, macrophage supernatant alone group, and macrophage supernatant+ADSC-exosome group, with 3 wells in each group, which were treated accordingly. After 24 h, the content of reactive oxygen species in cells was detected by flow cytometry, the expression of 8-OHdG in cells was detected by immunofluorescence, and Transwell assay was used to determine the permeability of cell monolayer. The number of samples in above were all 3. Data were statistically analyzed with one-way analysis of variance and least significant difference t test. Results The primary ADSCs were isolated and cultured to day 5, growing densely in a spindle shape with a typical swirl-like. The percentages of CD29, CD44, CD73 and CD90 positive cells of ADSCs in the third passage were all >90%, and the percentages of CD34 and CD45 positive cells were <5%. Exosomes derived from ADSCs of the third to fifth passages showed a typical double-cavity disc-like structure with an average particle size of 103 nm, and the protein expressions of CD9, CD63 and TSG101 of exosomes were positive, while the protein expression of β-actin of exosomes was negative. At 24 h after operation, compared with those in normal control group, both the levels of TNF-α and IL-1β of mice serum in CLP alone group were significantly increased (with t values of 28.76 and 29.69, respectively, P<0.01); compared with those in CLP alone group, both the content of TNF-α and IL-1β of mice serum in CLP+ADSC-exosome group was significantly decreased (with t values of 9.90 and 4.76, respectively, P<0.05 or P<0.01). At 24 h after surgery, the pulmonary tissue structure of mice in normal control group was clear and complete without inflammatory cell infiltration; compared with those in normal control group, the pulmonary tissue edema and inflammatory cell infiltration of mice in CLP alone group were more obvious; compared with those in CLP alone group, the pulmonary tissue edema and inflammatory cell infiltration of mice in CLP+ADSC-exosome group were significantly reduced. At 24 h after operation, endothelial cells in lung tissues of mice in 3 groups showed positive expression of CD31; compared with that in normal control group, the fluorescence intensity of 8-OHdG positive cells of the lung tissues of mice in CLP alone group was significantly increased, and compared with that in CLP alone group, the fluorescence intensity of 8-OHdG positive cells in the lung tissues of mice in CLP+ADSC-exosome group was significantly decreased. The PMVECs in the 3rd passage showed CD31 positive expression by immunofluorescence, and the result of flow cytometry showed that CD31 positive cells accounted for 99.5%. At 12 h after co-culture, ADSC-derived exosomes were successfully phagocytose by PMVECs and entered its cytoplasm. At 12 h after culture of the third passage of PMVECs, compared with that in blank control group, the fluorescence intensity of reactive oxygen species of PMVECs in macrophage supernatant alone group was significantly increased (t=15.73, P<0.01); compared with that in macrophage supernatant alone group, the fluorescence intensity of reactive oxygen species of PMVECs in macrophage supernatant+ADSC-exosome group was significantly decreased (t=4.72, P<0.01). At 12 h after culture of the third passage of PMVECs, and the 8-OHdG positive fluorescence intensity of PMVECs in macrophage supernatant alone group was significantly increased; and compared with that in blank control group, the 8-OHdG positive fluorescence intensity of PMVECs in macrophage+ADSC-exosome supernatant group was between blank control group and macrophage supernatant alone group. At 12 h after culture of the third passage PMVECs, compared with that in blank control group, the permeability of PMVECs monolayer in macrophage supernatant alone group was significantly increased (t=6.34, P<0.01); compared with that in macrophage supernatant alone group, the permeability of PMVECs monolayer cells in macrophage supernatant+ADSC-exosome group was significantly decreased (t=2.93, P<0.05). Conclusions Exosomes derived from ADSCs can ameliorate oxidative damage in mouse lung tissue, decrease the level of reactive oxygen species, 8-OHdG expression, and permeability of PMVECs induced by macrophage supernatant. -
参考文献
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1 术后24 h 3组脓毒症小鼠肺组织情况 1A、1B、1C:苏木精-伊红×20，图中标尺为100 μm；1D、1E、1F:髓过氧化物酶×20，图中标尺为100 μm。1A.正常对照组小鼠肺组织清晰完整；1B.单纯盲肠结扎穿孔（CLP）组小鼠肺组织较图1A水肿明显，炎症细胞浸润明显增加，细胞坏死明显增多；1C.CLP+脂肪间充质干细胞（ADSC）外泌体组小鼠肺组织水肿症状较图1B明显减轻，炎症细胞和红细胞浸润明显减少，细胞坏死情况明显改善；1D.正常对照组小鼠肺组织无炎症细胞浸润，肺间质较薄；1E.单纯CLP组小鼠肺组织炎症细胞浸润较图1D明显增加；1F.CLP+ADSC外泌体组小鼠肺组织炎症细胞和红细胞浸润较图1E明显减少
2 术后24 h 3组脓毒症小鼠的肺组织中CD31和8-羟基脱氧鸟苷（8-OHdG）荧光共染检测其氧化性损伤情况 激光扫描共聚集显微镜×40，图中标尺为50 μm。2A.正常对照组小鼠肺组织中CD31呈阳性表达，8-OHdG阳性细胞少；2B.单纯CLP组小鼠肺组织中CD31呈阳性表达，8-OHdG阳性细胞较图2A明显增加；2C.CLP+脂肪间充质干细胞外泌体组小鼠肺组织中CD31呈阳性表达，8-OHdG阳性细胞较图2B明显减少
注：CLP为盲肠结扎穿孔；绿色荧光为CD31阳性表达，红色荧光为8-OHdG阳性表达，蓝色荧光为细胞核阳性表达
3 第3代肺血管内皮细胞（PMVEC）鉴定及吞噬人脂肪间充质干细胞（ADSC）来源外泌体情况。 3A.内皮细胞特异性标志物CD31呈阳性表达 FITC-4',6-二脒基-2-苯基吲哚×40，图中标尺为150 μm；3B.流式细胞术检测显示CD31阳性细胞占比为99.5%；3C.ADSC来源外泌体被PMVEC吞噬，分布于细胞质中 PKH26-4',6-二脒基-2-苯基吲哚×200，图中标尺为100 μm
注：图3A中绿色为CD31阳性表达，蓝色为细胞核阳性表达；图3B中FITC为异硫氰酸荧光素，图中线段指平均荧光强度，P2为流式细胞仪设定的门，即在FITC的P2门里阳性细胞占总细胞数的99.5%；图3C中蓝色为细胞核阳性表达，红色为外泌体阳性表达
4 免疫荧光法检测第3代肺血管内皮细胞（PMVEC）的8-羟基脱氧鸟苷（8-OHdG）表达情况 Alexa Fluor-4',6-二脒基-2-苯基吲哚×40，图中标尺为125 μm。4A.空白对照组细胞8-OHdG低表达；4B.单纯巨噬细胞上清液组8-OHdG表达较图4A明显增加；4C.巨噬细胞上清液+脂肪间充质干细胞外泌体组细胞8-OHdG表达介于图4A与图4B之间
表1 3组第3代肺血管内皮细胞培养24 h后活性氧含量和通透性比较（
组别 样本数 活性氧 通透性（ng/mL） 空白对照组 3 2 227±90 70±10 单纯巨噬细胞上清液组 3 3 650±128 193±32 巨噬细胞上清液+ADSC外泌体组 3 2 808±281 121±28 F值 44.54 18.25 P值 <0.001 <0.005 t1值 15.73 6.34 P1值 <0.001 0.003 t2值 4.72 2.93 P2值 0.009 0.043 注：ADSC为脂肪间充质干细胞；活性氧含量以荧光强度反映，通透性以牛血清白蛋白质量浓度反映；F值、P值为3组间各指标总体比较所得；t1值、P1值为单纯巨噬细胞上清液组与空白对照组各指标比较所得；t2值、P2值为巨噬细胞上清液+ADSC外泌体组与单纯巨噬细胞上清液组各指标比较所得 
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《中华烧伤与创面修复杂志》第六届编辑委员会编辑委员名单
终身顾问 盛志勇 程天民 王正国 樊代明 付小兵 夏照帆 卞修武 顾晓松 李校堃 顾 问 肖光夏 杨宗城 汪仕良 孙永华 柴家科 黄跃生 岑瑛 王旭 名誉总编辑 彭毅志 总编辑 罗高兴 以下按姓氏拼音排序 副总编辑 郭光华 韩春茂 胡大海 郇京宁 梁光萍 刘毅 吕国忠 吴军 谢卫国 姚咏明 常务编辑委员 官浩 贺伟峰 李孝建 李宗瑜 刘琰 陆树良 马显杰 申传安 沈余明 孙炳伟 谭 谦 王达利 王一兵 夏成德 肖仕初 徐庆连 于家傲 袁志强 张丕红 张庆富 张逸 章一新 编辑委员 巴特 陈国贤 陈炯 陈俊杰 陈欣 陈旭 陈旭林 陈昭宏 程飚 崔正军 邓 君 范锟铻 方勇 冯世海 冯正直 官浩 郭光华 韩春茂 韩军涛 郝岱峰 贺伟峰 胡大海 郇京宁 黄沙 霍然 姜笃银 金培生 赖文 雷晋 李德绘 李小兵 李晓亮 李孝建 李学拥 李 毅 李智 李宗瑜 梁光萍 刘文军 刘小龙 刘旭盛 刘琰 刘毅 陆树良 罗高兴 吕大伦 吕国忠 马朋林 马显杰 潘云川 彭曦 齐鸿燕 邱林 荣新洲 申传安 沈余明 沈运彪 史春梦 宋保强 宋国栋 宋华培 孙炳伟 孙天骏 谭谦 唐洪泰 陶克 童亚林 王达利 王德运 王光毅 王凌峰 王新刚 王杨 王一兵 魏在荣 吴健 吴军 吴银生 夏成德 肖厚安 肖健 肖仕初 谢挺 谢卫国 徐庆连 颜洪 杨磊 姚咏明 于家傲 袁志强 曾元临 詹剑华 张恒术 张家平 张建祥 张明华 张丕红 张 勤 张庆富 张逸 章一新 赵耀华 赵永健 朱世辉 以下按英文首字母排序 Chong Si Jack（新加坡） David N. Herndon（美国） Fiona Wood（澳大利亚） Malcolm Xing（邢孟秋，加拿大） Naiem S. Moiemen（英国） Ronald G. Tompkins（美国） Steven E. Wolf（美国） Tina L. Palmieri（美国） Yong-Ming Yu（尤永明，美国）
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