Effects and underlying mechanism of human adipose mesenchymal stem cells-derived exosomes on acute lung injury in septic mice
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摘要:
目的 探讨人脂肪间充质干细胞（ADSC）外泌体对脓毒症小鼠急性肺损伤的影响及其机制。 方法 该研究为实验研究。选取第4~5代人ADSC,采用差速超高速离心法分离并提取其上清液中的外泌体,对外泌体进行鉴定后使用。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,对单纯CLP组小鼠行CLP（构建脓毒症小鼠急性肺损伤动物模型）后注射磷酸盐缓冲液,对CLP+ADSC外泌体组小鼠进行组名相应的处理,对正常对照组小鼠仅注射磷酸盐缓冲液,每组8只。术后24 h,采用苏木精-伊红染色观测小鼠肺组织形态,采用原位末端标记法检测肺组织细胞凋亡情况,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）含量,使用酶标仪检测肺组织中丙二醛和超氧化物歧化酶（SOD）的含量,采用免疫荧光法检测小鼠肺组织细胞中CD86、CD206的表达。取小鼠巨噬细胞RAW264.7,分为空白对照组、单纯内毒素/脂多糖（LPS）组和LPS+ADSC外泌体组,在LPS+ADSC外泌体组和单纯LPS组细胞中分别加入LPS+ADSC外泌体、LPS进行培养,对空白对照组细胞进行常规培养。培养12 h 后,采用相关试剂盒检测细胞中ATP含量、线粒体活性氧的阳性细胞百分比、线粒体膜电位情况,采用实时荧光定量反转录PCR法检测细胞中M1型极化标志因子诱导型一氧化氮合酶（iNOS）、M2型极化标志因子精氨酸酶-1（Arg1）以及炎症因子TNF-α和IL-1β的mRNA表达量。以上实验除mRNA表达量的检测样本数为3以外,其余各指标的检测样本数均为4。 结果 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组较正常对照组小鼠的肺组织水肿明显,炎症细胞浸润现象明显,凋亡、坏死细胞明显增多；CLP+ADSC外泌体组较单纯CLP组小鼠肺组织水肿症状明显减轻,炎症细胞浸润明显减少,细胞凋亡、坏死情况明显改善。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含量均明显增加（t值分别为50.82、30.81,P<0.05）；与单纯CLP组比较,CLP+ADSC外泌体组小鼠血清中的TNF-α和IL-1β含量均明显降低（t值分别为16.36、19.25,P<0.05）。术后24 h,与正常对照组比较,单纯CLP组小鼠肺组织中丙二醛含量明显升高（t=9.89,P<0.05）,SOD含量明显降低（t=5.01,P<0.05）；与单纯CLP组比较,CLP+ADSC外泌体组小鼠肺组织中丙二醛含量明显降低（t=4.38,P<0.05）,SOD含量明显升高（t=2.97,P<0.05）。术后24 h,与正常对照组相比,单纯CLP组小鼠的肺组织中CD86阳性细胞占比明显升高,CD206阳性细胞占比明显减少；与单纯CLP组相比,CLP+ADSC外泌体组小鼠肺组织中CD86阳性细胞占比明显减少,CD206阳性细胞占比明显升高。培养12 h后,与空白对照组比较,单纯LPS组RAW264.7细胞中ATP含量明显降低（t=6.28,P<0.05）；与单纯LPS组比较,LPS+ADSC外泌体组RAW264.7细胞中ATP含量明显升高（t=4.01,P<0.05）。培养12 h后,与空白对照组的（22±4）%比较,单纯LPS组RAW264.7细胞中线粒体活性氧的阳性细胞百分比（40±6）%明显增加（t=5.04,P<0.05）；与单纯LPS组比较,LPS+ADSC外泌体组RAW264.7细胞中线粒体活性氧的阳性细胞百分比（30±5）%明显降低（t=2.65,P<0.05）。培养12 h后,与空白对照组相比,单纯LPS组RAW264.7细胞的线粒体膜电位明显降低；LPS+ADSC外泌体组RAW264.7细胞的线粒体膜电位介于空白对照组和单纯LPS组之间。培养12 h后,与空白对照组相比,单纯LPS组RAW264.7细胞中TNF-α、IL-1β和iNOS的mRNA表达量均明显增加（t值分别为16.51、31.04、7.70,P<0.05）,Arg1的mRNA表达量降低但差异无统计学意义（P>0.05）；与单纯LPS组比较,LPS+ADSC外泌体组RAW264.7细胞中TNF-α、IL-1β和iNOS的mRNA表达量均明显降低（t值分别为11.38、22.58、5.28,P<0.05）,Arg1的mRNA表达量明显升高（t=7.66,P<0.05）。 结论 人ADSC外泌体可能通过改善LPS诱导的小鼠巨噬细胞线粒体功能障碍,抑制巨噬细胞向M1型极化,减轻炎症反应,从而发挥改善脓毒症小鼠肺损伤的作用。 Abstract:Objective To explore the effects and underlying mechanism of human adipose mesenchymal stem cells (ADSC)-derived exosomes on acute lung injury in septic mice. Methods The study was an experimental study. Human ADSC of passages 4-5 were selected, and exosomes in their supernatant were isolated and extracted by differential ultracentrifugation. Exosomes were then used after identification. Twenty-four adult male BALB/c mice were selected and divided into normal control group, simple cecal ligation and puncture (CLP) group, and CLP+ADSC-exosome group according to the random number table method (the grouping method was the same below), with 8 mice in each group. The mice in simple CLP group were injected with phosphate buffer after CLP surgery (to establish an animal model of acute lung injury in septic mice), the mice in CLP+ADSC-exosome group were treated according to the corresponding group name, and the mice in normal control group were only injected with phosphate buffer. At 24 hours after surgery, the morphology of lung tissue was observed by hematoxylin-eosin staining, the apoptosis of lung tissue cells was detected by in-situ end-labeling method, the content of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the serum of mice was detected by enzyme-linked immunosorbent assay, the content of malondialdehyde and superoxide dismutase (SOD) in lung tissue was detected by microplate reader, and the expressions of CD86 and CD206 in mouse lung tissue cells was detected by immunofluorescence method. Mouse macrophage RAW264.7 was taken and divided into blank control group, simple lipopolysaccharide (LPS) group, and LPS+ADSC-exosome group. The cells of LPS+ADSC-exosome group and simple LPS group were cultured by adding LPS+ADSC-exosome and LPS, respectively, and cells in blank control group were routinely cultured. Twelve hours after culture, the ATP content, the percentage of mitochondrial reactive oxygen species positive cells, as well as mitochondrial membrane potential in cells were detected by related detection kits. The mRNA expression levels of M1 polarization marker inducible nitric oxide synthase (iNOS), M2 polarization marker arginase-1 (Arg1), and inflammatory factors TNF-α and IL-1β in cells were detected by real-time fluorescence quantitative reverse-transcription polymerase chain reaction method. Three samples were used for mRNA expression detection, and four samples were used for the detection of the other indicators. Results At 24 hours after surgery, the structure of mouse lung tissues in normal control group was clear and intact without inflammatory cell infiltration. Compared with that in normal control group, the lung tissue edema as well as the infiltration of inflammatory cells of mice was much more obvious in simple CLP group. However, compared with that in simple CLP group, the lung tissue edema of mice in CLP+ADSC-exosome group was significantly alleviated, the infiltration of inflammatory cells was significantly reduced, and the cell apoptosis and necrosis were significantly improved. Twenty-four hours after surgery, compared with that in normal control group, the levels of TNF-α and IL-1β in the serum of mice in simple CLP group were significantly increased (with t values of 50.82 and 30.81, respectively, P<0.05); compared with that in simple CLP group, the levels of TNF-α and IL-1β in the serum of mice in CLP+ADSC-exosome group were significantly decreased (with t values of 16.36 and 19.25, respectively, P<0.05). Compared with that in normal control group, the content of malondialdehyde in the lung tissue of mice in simple CLP group was significantly increased (t=9.89, P<0.05); and the content of SOD was significantly decreased (t=5.01, P<0.05); compared with that in simple CLP group, the content of malondialdehyde in the lung tissue of mice in CLP+ADSC-exosome group was significantly decreased (t=4.38, P<0.05), and the content of SOD was significantly increased (t=2.97, P<0.05). Twenty-four hours after surgery, compared with that in normal control group, the proportion of CD86 positive cells in the lung tissue of mice in simple CLP group was significantly increased, and the proportion of CD206 positive cells was significantly decreased; compared with that in simple CLP group, the proportion of CD86 positive cells in the lung tissue of mice in CLP+ADSC-exosome group was significantly decreased, and the proportion of CD206 positive cells was significantly increased. After 12 hours of culture, compared with that in blank control group, the ATP content of RAW264.7 cells in simple LPS group was significantly decreased (t=6.28, P<0.05); compared with that in simple LPS group, the ATP content of RAW264.7 cells in LPS+ADSC-exosome group was significantly increased (t=4.01, P<0.05). After 12 hours of culture, compared with (22±4)% in blank control group, (40±6)% of positive cells of mitochondrial reactive oxygen species in RAW264.7 cells in simple LPS group was significantly increased (t=5.04, P<0.05); compared with that in LPS group, (30±5)% of positive cells of mitochondrial reactive oxygen species in RAW264.7 cells in LPS+ADSC-exosome group was significantly decreased (t=2.65, P<0.05). After 12 hours of culture, compared with that in blank control group, the mitochondrial membrane potential of RAW264.7 cells in simple LPS group was significantly decreased; the mitochondrial membrane potential of RAW264.7 cells in LPS+ ADSC-exosome group was between those in blank control group and simple LPS group. After 12 hours of culture, compared with that in blank control group, the mRNA expressions of TNF-α, IL-1β, and iNOS in RAW264.7 cells in simple LPS group were significantly increased (with t values of 16.51, 31.04, and 7.70, respectively, P<0.05), and the decrease in the mRNA expression of Arg1 was not statistically significant (P>0.05); compared with that in simple LPS group, the mRNA expressions of TNF-α, IL-1β, and iNOS in RAW264.7 cells in LPS+ADSC-exosome group were significantly decreased (with t values of 11.38, 22.58, and 5.28, respectively, P<0.05), and the mRNA expression of Arg1 was significantly increased (t=7.66, P<0.05). Conclusions Human ADSC-exosomes may play a role in improving lung injury in septic mice by improving LPS-induced mitochondrial dysfunction in mice macrophages, inhibiting the polarization of macrophages toward M1, and reducing the inflammatory response. -
Key words:
- Sepsis /
- Acute lung injury /
- Mesenchymal stem cells /
- Exosomes /
- Macrophages /
- Mitochondrial dysfunction
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本文亮点：
(1)证实人脂肪间充质干细胞（ADSC）外泌体可改善内毒素/脂多糖诱导的小鼠巨噬细胞线粒体功能障碍,抑制巨噬细胞持续向M1型极化,减轻炎症反应。
(2)证实人ADSC外泌体可改善脓毒症小鼠急性肺损伤,减轻氧化应激、炎症反应,该作用可能通过调节巨噬细胞线粒体功能来实现。
Highlights:
(1)It was confirmed that human adipose mesenchymal stem cells (ADSC)-derived exosomes could improve lipopolysaccharide-induced mitochondrial dysfunction in mouse macrophages, inhibit the continuous polarization of macrophages toward M1, and reduce the inflammatory response.
(2)It was confirmed that human ADSC-exosomes could improve acute lung injury in septic mice, and alleviate oxidative stress and inflammatory response. This effect might be achieved by regulating the mitochondrial function of macrophages.
急性肺损伤（acute lung injury,ALI）或其严重形式的ARDS在大面积烧伤、危重多发伤、脓毒症等危重患者中较为常见,其特征是存在持续的肺部炎症和弥漫性肺泡损伤[1, 2, 3, 4]。巨噬细胞广泛分布于肺组织。在ALI早期阶段,巨噬细胞响应外源性微生物刺激,被迅速激活,随后通过产生多种细胞因子和向肺部招募更多的其他巨噬细胞及中性粒细胞等炎症细胞的形式促进炎症的持续发展,最终导致肺损伤[5, 6, 7, 8]。研究显示,线粒体功能障碍在巨噬细胞持续向M1型极化的过程中处于关键地位。在ALI、ARDS等多种疾病的发生发展中,线粒体功能障碍参与了过度炎症反应和活性氧过度产生[9, 10, 11, 12]。因此,在炎症早期围绕通过调控巨噬细胞线粒体功能障碍来控制巨噬细胞持续向M1型极化、过度炎症反应的研究,可能成为治疗ALI及ARDS的一种有效策略。
脂肪间充质干细胞（adipose mesenchymal stem cell,ADSC）是干细胞家族的重要成员,具有强大的免疫调节和抗炎能力,且易于获得[13, 14, 15]。近年来,大量研究表明,间充质干细胞分泌的可溶性因子和外泌体可以替代细胞作为抑制炎症和促进组织修复的选择[16, 17, 18, 19]。本研究通过对小鼠进行盲肠结扎穿孔（cecal ligation and puncture,CLP）来诱发ALI从而构建脓毒症动物模型,并采用人ADSC外泌体进行干预,旨在探究人ADSC外泌体对脓毒症小鼠ALI的潜在作用及分子机制。
1. 材料与方法
本实验研究遵循空军军医大学动物实验伦理委员会和国家有关实验动物管理和使用的规定。
1.1 动物和细胞及主要试剂与仪器来源
24只健康无特殊病原体级成年雄性BALB/c小鼠（体重21~26 g）由空军军医大学动物中心提供,许可证号：SYXK（军）2012-0022。人ADSC为本实验室冻存细胞,RAW264.7小鼠单核巨噬细胞白血病细胞系购自美国典型培养物保藏中心。
DMEM/F12、RPMI-1640培养基、无外泌体胎牛血清购自美国Gibco公司,ADSC专用培养基购自广州赛业生物科技有限公司,总mRNA提取试剂盒、ATP含量检测试剂盒、辣根过氧化物酶标记的山羊抗兔IgG多克隆抗体、线粒体超氧化物（mitochondrial superoxide,MitoSOX）检测试剂盒、线粒体膜电位检测试剂盒（荧光基团为JC-1）购自上海碧云天生物技术有限公司,反转录试剂盒及PCR检测试剂盒购自日本Takara公司,40 g/L多聚甲醛、HE染色试剂盒购自武汉赛维尔生物科技有限公司,丙二醛（脂质过氧化产物）和SOD（自由基代谢产物）检测试剂盒购自南京建成生物科技有限公司,一步法原位末端标记（TdT-mediated-dUTP nick-end labeling,TUNEL）细胞凋亡检测试剂盒（荧光基团为异硫氰酸荧光素）购自武汉伊莱瑞特生物科技股份有限公司,多聚-D-赖氨酸、4′,6-二脒基-2-苯基吲哚（4',6-diamidino-2-phenylindole,DAPI）、外泌体示踪染料PKH26红色荧光细胞连接试剂盒购自美国Sigma公司,兔抗小鼠CD86（M1型巨噬细胞标志物）单克隆抗体、兔抗小鼠CD206（M2型巨噬细胞标志物）单克隆抗体和异硫氰酸荧光素标记的兔抗人CD9单克隆抗体、兔抗人CD63单克隆抗体、兔抗人CD81单克隆抗体购自美国Cell Signaling Technology公司,小鼠TNF-α、IL-1β的ELISA试剂盒购自美国Becton Dickinson公司。
FACS AriaⅢ型流式细胞仪购自美国Becton Dickinson公司,二氧化碳培养箱购自美国Thermo Fisher Scientific公司,Optima™XPN型超高速离心机购自美国Beckman公司,Infinite M200 Pro型全波长多功能酶标仪购自瑞士TECAN公司,HT-7700型透射电子显微镜购自日本Hitachi公司,EVOS FL Auto 2型激光扫描共聚焦显微镜购自美国Invitrogen公司,FSX100型全自动生物图像导航仪购自日本Olympus公司,IQ5TM型实时荧光定量PCR仪购自美国Bio-Rad公司。
1.2 人ADSC外泌体的分离提取及表征
取人ADSC,采用ADSC专用培养基进行常规培养,选用第4或第5代细胞进行后续实验。参照文献[20]采用差速超高速离心法制备人ADSC外泌体,调整其质量浓度为2 μg/mL备用。采用透射电子显微镜在40 000倍放大倍数下观察外泌体形态,采用纳米颗粒跟踪分析仪检测外泌体粒径。常规采用流式细胞仪检测外泌体CD9、CD63、CD81阳性率,其中抗体为异硫氰酸荧光素标记的兔抗人CD9、CD63、CD81单克隆抗体（稀释比均为1∶200）。
1.3 动物实验
1.3.1 脓毒症小鼠模型的建立及分组与样本收集
取24只BALB/c小鼠,适应性饲养1周后按照随机数字表法（分组方法下同）分成正常对照组、单纯CLP组和CLP+ADSC外泌体组,每组8只。单纯CLP组和CLP+ADSC外泌体组小鼠均行CLP手术建立脓毒症模型[21, 22]。CLP术后2 h,对CLP+ADSC外泌体组小鼠行尾静脉注射100 μL由1.2制备的外泌体,单纯CLP组小鼠尾静脉注射等量无菌PBS。同时,正常对照组小鼠仅尾静脉注射等量无菌PBS。术后24 h（正常对照组小鼠取注射PBS后相同时间点）,参照文献[23, 24, 25],摘除小鼠眼球,取血备用；然后用颈椎脱臼法处死小鼠,将左肺置于冰上备用,将右肺采用40 g/L多聚甲醛固定,常规石蜡包埋,制成厚度为5 μm切片备用。
1.3.2 肺组织的形态学观察及细胞凋亡情况检测
取1.3.1中切片,常规行HE染色并采用全自动生物图像导航仪于激光扫描共聚焦显微镜100倍镜下观察肺组织形态。另取切片,按TUNEL细胞凋亡检测试剂盒说明书进行染色（阳性染色为绿色）,并用DAPI复染细胞核（阳性染色为蓝色）,然后于激光扫描共聚焦显微镜100倍放大倍数下观察肺组织细胞凋亡情况。
1.3.3 血清中炎症因子水平的检测
取1.3.1中的血标本,冰上静置30 min后于4 ℃条件下以1 600×g 离心15 min,小心吸取上层血清,按照ELISA试剂盒说明书步骤检测血清中的TNF-α和IL-1β含量。样本数为4。
1.3.4 肺组织的氧化应激水平检测
取1.3.1中小鼠肺组织,剪碎并裂解肺组织收集肺组织匀浆液。参照丙二醛和SOD检测试剂盒说明书,采用酶标仪分别于534 nm波长和452 nm波长处测定匀浆液的吸光度值,反映丙二醛和SOD含量。样本数为4。
1.3.5 肺组织中巨噬细胞表型的检测
取1.3.1中切片,进行抗原修复后常规进行免疫组织化学染色,一抗为兔抗小鼠CD86、CD206单克隆抗体（稀释比均为1∶100）,二抗为辣根过氧化物酶标记的山羊抗兔IgG多克隆抗体（稀释比为1∶200）,用苏木素染细胞核。在激光扫描共聚焦显微镜100倍放大倍数下观察阳性细胞占比情况。目标蛋白阳性染色为棕色,细胞核阳性染色为蓝色。样本数为4。
1.4 细胞实验
1.4.1 RAW264.7细胞对外泌体的内化情况
取100 μL由1.2制备的外泌体,加入4 μL的PKH26标记的细胞连接试剂,室温避光孵育5 min后,4 ℃、100 000×g离心1 h,获取沉淀,然后采用100 μL PBS重新悬浮备用。
调整RAW264.7细胞浓度为5×104个/mL,按照每孔100 μL接种于96孔板,培养至细胞融合达70%。取前述20 μL PKH26标记的人ADSC外泌体悬液,加到RAW264.7细胞培养液中,避光共培养12 h后用PBS清洗3次。用40 g/L多聚甲醛固定15 min,用DAPI染细胞核。在激光扫描共聚焦显微镜200倍放大倍数下观察细胞对PKH26标记的人ADSC外泌体的吞噬情况。外泌体阳性染色为红色,细胞核阳性染色为蓝色。
1.4.2 LPS诱导的RAW264.7细胞炎症模型及分组处理
取RAW264.7细胞,采用含体积分数10%无外泌体胎牛血清的RPMI-1640培养基常规进行细胞培养,待细胞生长至80%融合时,将细胞分为空白对照组、单纯LPS组和LPS+ADSC外泌体组。在LPS+ADSC外泌体组细胞培养基中先加入终质量浓度50 μg/mL人ADSC外泌体预孵育1 h,然后参照文献[26]进行LPS刺激；对单纯LPS组细胞仅进行同前的LPS刺激；对空白对照组细胞进行常规培养。
1.4.3 RAW264.7细胞中ATP含量检测
取1.4.2分组培养12 h后的细胞,根据ATP含量检测试剂盒说明书测定ATP量。样本数为4。
1.4.4 RAW264.7细胞中线粒体活性氧的检测
取1.4.2分组培养12 h后的细胞,加入5 μmol/L MitoSOX试剂孵育30 min,PBS洗涤3次后用流式细胞仪收集细胞。采用flowJo软件统计MitoSOX荧光强度并计算其阳性细胞百分比,反映细胞内线粒体活性氧的阳性细胞百分比。样本数为4。
1.4.5 RAW264.7细胞线粒体膜电位的检测
取1.4.2分组培养12 h后的细胞,根据线粒体膜电位检测试剂盒说明书加入含探针的试剂（稀释比为1∶1 000）,37 ℃避光孵育30 min。PBS洗涤3次,离心后弃上清液,加PBS重新悬浮细胞,采用全自动生物图像导航仪于激光扫描共聚焦显微镜200倍放大倍数下拍照,检测荧光情况。若呈现红色荧光则说明线粒体正常,即JC-1聚集在线粒体基质中形成聚合物；若呈现绿色荧光则说明线粒体膜电位下降或线粒体受损,即JC-1只能以单体的形式存在于细胞质中。样本数为4。
1.4.6 RAW264.7细胞M1/M2型极化标志因子及炎症因子的mRNA表达量检测
取1.4.2分组培养后12 h的细胞,提取总mRNA,采用实时荧光定量RT-PCR法检测巨噬细胞M1、M2型极化标志因子诱导型NOS（inducible nitric oxide synthase,iNOS）和精氨酸酶-1（arginase-1,Arg1）,炎症因子TNF-α和IL-1β的mRNA表达,引物序列见表1。按照荧光定量试剂盒说明书,以GAPDH为内参照,通过2-ΔΔCt法对TNF-α、IL-1β、iNOS和Arg1的mRNA表达进行定量分析。样本数为3。
Table 1. 实时荧光定量反转录PCR法检测RAW264.7细胞的各引物序列及产物大小基因名称 引物序列（5'→3'） 产物大小（bp） IL-1β 上游：TCCAGGATGAGGACATGAGCAC 105 下游：GAACGTCACACACCAGCAGGTTA iNOS 上游：ACTACTGCTGGTGGTGACAA 106 下游：GAAGGTGTGGTTGAGTTCTCTAAG TNF-α 上游：ACTCCAGGCGGTGCCTATGT 160 下游：GTGAGGGTCTGGGCCATAGAA Arg1 上游：ACATTGGCTTGCGAGACGTA 109 下游：ATCACCTTGCCAATCCCCAG GAPDH 上游：TGTGTCCGTCGTGGATCTGA 150 下游：TTGCTGTTGAAGTCGCAGGAG 注：IL为白细胞介素,iNOS为诱导型一氧化氮合酶,TNF为肿瘤坏死因子,Arg1为精氨酸酶-1,GAPDH为3-磷酸甘油醛脱氢酶 1.5 统计学处理
采用SPSS 20.0统计软件进行数据分析。计量资料数据均符合正态分布,以
2. 结果
2.1 人ADSC外泌体的表征
人ADSC外泌体呈茶托状结构,粒径分布在60~150 nm之间,见图1。人ADSC外泌体的CD9、CD63、CD81阳性率分别为（17.2±2.7）%、（15.4±1.4）%、（22.8±1.1）%。
图 1 人脂肪间充质干细胞外泌体的表征。1A.可见囊泡呈清晰的茶托状结构 透射电子显微镜×40 000；1B.纳米颗粒跟踪分析仪检测显示粒径为60~150 nm注：图1B为横坐标经过lg处理的数据形成的描记图2.2 人ADSC外泌体对CLP小鼠ALI的影响
2.2.1 对肺组织损伤的影响
术后24 h,正常对照组小鼠肺组织结构清晰完整,肺泡连接紧密、无炎症细胞浸润,肺间质较薄、无出血；单纯CLP组较正常对照组小鼠的肺组织水肿明显,炎症细胞浸润现象明显,凋亡、坏死细胞明显增多；CLP+ADSC外泌体组较单纯CLP组小鼠肺组织水肿症状明显减轻,炎症细胞浸润明显减少,细胞凋亡、坏死情况明显改善。见图2。
图 2 3组小鼠术后24 h肺组织损伤情况和肺组织细胞的凋亡情况。2A、2B、2C.分别为正常对照组、单纯CLP组、CLP+ADSC外泌体组小鼠肺组织损伤情况,图2B较图2A中的肺组织水肿情况明显,炎症细胞数明显增多,图2C较图2B肺组织水肿明显改善,炎症细胞数明显减少 苏木精-伊红×100；2D、2E、2F.分别为正常对照组、单纯CLP组、CLP+ADSC外泌体组小鼠肺组织细胞的凋亡情况,图2E较图2D中的凋亡、坏死细胞数明显增加,图2F较图2E凋亡、坏死细胞数明显减少 异硫氰酸荧光素-4',6-二脒基-2-苯基吲哚×100注：对单纯盲肠结扎穿孔（CLP）组小鼠行CLP后注射磷酸盐缓冲液,对CLP+脂肪间充质干细胞（ADSC）外泌体组小鼠进行组名相应的处理,对正常对照组小鼠仅注射磷酸盐缓冲液；凋亡细胞阳性染色为绿色,细胞核阳性染色为蓝色2.2.2 对炎症反应的影响
术后24 h,正常对照组、单纯CLP组和CLP+ADSC外泌体组小鼠血清中TNF-α含量分别为（76±4）、（362±11）、（221±14）pg/mL,IL-1β含量分别为（43±4）、（163±6）、（74±7）pg/mL,组间总体比较,差异有统计学意义（F值分别为793.40、437.00,P值均<0.001）。与正常对照组比较,单纯CLP组小鼠血清中TNF-α和IL-1β含量均明显增加（t值分别为50.82、30.81,P<0.001）；与单纯CLP组比较,CLP+ADSC外泌体组小鼠血清中的TNF-α和IL-1β含量均明显降低（t值分别为16.36、19.25,P<0.001）。
2.2.3 对肺组织氧化应激水平的影响
术后24 h,正常对照组、单纯CLP组和CLP+ADSC外泌体组小鼠肺组织匀浆液中丙二醛含量分别为（1.57±0.10）、（4.46±0.58）、（2.65±0.59）μmol/g,SOD含量分别为（64±7）、（37±8）、（53±7）U/g,组间总体比较,差异有统计学意义（F值分别为36.99、13.17,P值均<0.001）。与正常对照组比较,单纯CLP组小鼠肺组织中丙二醛含量明显升高（t=9.89,P<0.001）,SOD含量明显降低（t=5.01,P=0.002）；与单纯CLP组比较,CLP+ADSC外泌体组小鼠肺组织中丙二醛含量明显降低（t=4.38,P=0.005）,SOD含量明显升高（t=2.97,P=0.025）。
2.2.4 对肺组织中巨噬细胞表型的影响
术后24 h,与正常对照组相比,单纯CLP组小鼠的肺组织中CD86阳性细胞占比明显升高,CD206阳性细胞占比明显减少；与单纯CLP组相比,CLP+ADSC外泌体组小鼠肺组织中CD86阳性细胞占比明显减少,CD206阳性细胞占比明显升高。见图3。
图 3 3组小鼠术后24 h肺组织中巨噬细胞浸润及分化情况 辣根过氧化物酶-苏木素×100。3A、3B、3C.分别为正常对照组、单纯CLP组、CLP+ADSC外泌体组CD86（M1型巨噬细胞标志物）阳性细胞分布情况,图3B较图3A中的M1型巨噬细胞占比明显升高,图3C较图3B中的M1型巨噬细胞占比明显减少；3D、3E、3F.分别为正常对照组、单纯CLP组、CLP+ADSC外泌体组CD206（M2型巨噬细胞标志物）阳性细胞分布情况,图3E较图3D中的M2型巨噬细胞占比明显减少,图3F较图3E中的M2型巨噬细胞占比明显升高注：对单纯盲肠结扎穿孔（CLP）组小鼠行CLP后注射磷酸盐缓冲液,对CLP+脂肪间充质干细胞（ADSC）外泌体组小鼠进行组名相应的处理,对正常对照组小鼠仅注射磷酸盐缓冲液；细胞阳性染色均为棕色,细胞核阳性染色为蓝色2.3 人ADSC外泌体对RAW264.7细胞的影响
2.3.1 RAW264.7细胞内化人ADSC外泌体的情况
共培养12 h后,人ADSC外泌体成功被RAW264.7细胞吞入细胞质,见图4。
图 4 共培养12 h后RAW264.7细胞吞噬人脂肪间充质干细胞（ADSC）外泌体的情况 PKH26-4',6-二脒基-2-苯基吲哚×200。4A、4B、4C.分别为细胞中的外泌体染色、细胞核染色及复合染色情况,可见人ADSC外泌体成功被RAW264.7细胞吞噬注：人ADSC外泌体阳性染色为红色,细胞核阳性为蓝色2.3.2 人ADSC外泌体对RAW264.7细胞中ATP含量的影响
培养12 h后,空白对照组、单纯LPS组和LPS+ADSC外泌体组细胞中ATP含量分别为（13.8±1.5）、（7.9±1.2）、（12.5±2.0）nmol/mg,组间总体比较,差异有统计学意义（F=15.48,P=0.001）。与空白对照组比较,单纯LPS组细胞中ATP含量明显降低（t=6.28,P<0.001）；与单纯LPS组比较,LPS+ADSC外泌体组细胞中ATP含量明显升高（t=4.01,P=0.007）。
2.3.3 人ADSC外泌体对RAW264.7细胞线粒体活性氧的影响
培养12 h后,空白对照组、单纯LPS组和LPS+ADSC外泌体组细胞中线粒体活性氧的阳性细胞百分比分别为（22±4）%、（40±6）%、（30±5）%,组间总体比较,差异有统计学意义（F=12.61,P=0.002）。与空白对照组比较,单纯LPS组细胞中线粒体活性氧的阳性细胞百分比明显增加（t=5.04,P=0.002）；与单纯LPS组比较,LPS+ADSC外泌体组细胞中线粒体活性氧的阳性细胞百分比明显降低（t=2.65,P=0.038）。
2.3.4 人ADSC外泌体对RAW264.7细胞线粒体膜电位的影响
培养12 h后,与空白对照组相比,单纯LPS组细胞的线粒体膜电位明显降低；LPS+ADSC外泌体组细胞的线粒体膜电位介于空白对照组和单纯LPS组之间。见图5。
图 5 培养12 h后3组RAW264.7细胞中线粒体膜电位情况 JC-1×200。5A、5B、5C、5D.分别为空白对照组细胞呈蓝色荧光、绿色荧光、红色荧光及复合显色情况；5E、5F、5G、5H.分别为单纯LPS组细胞呈蓝色荧光、绿色荧光、红色荧光及复合显色情况；5I、5J、5K、5L.分别为LPS+ADSC外泌体组细胞呈蓝色荧光、绿色荧光、红色荧光及复合显色情况,图5F较图5B中的线粒体膜电位下降明显,图5J中的线粒体膜电位介于图5B与图5F之间,图5K中的正常线粒体荧光强度介于图5C与图5G之间注：在内毒素/脂多糖（LPS）+脂肪间质干细胞（ADSC）外泌体组和单纯LPS组细胞中分别加入LPS+ADSC外泌体、LPS进行培养,对空白对照组细胞进行常规培养；细胞核阳性染色为蓝色,受损或膜电位下降的线粒体阳性染色为绿色,正常线粒体阳性染色为红色2.3.5 人ADSC外泌体对RAW264.7细胞M1/M2型标志因子及炎症因子的影响
培养12 h后,空白对照组、单纯LPS组和LPS+ADSC外泌体组细胞中TNF-α、IL-1β、iNOS、Arg1的mRNA表达量组间总体比较,差异均有统计学意义（P<0.05）。与空白对照组比较,单纯LPS组细胞中TNF-α、IL-1β、iNOS的mRNA表达量均明显升高（P<0.05）,Arg1的mRNA表达量降低但差异无统计学意义（P>0.05）；与单纯LPS组比较,LPS+ADSC外泌体组细胞中TNF-α、IL-1β、iNOS的mRNA表达量均明显降低（P<0.05）,Arg1的mRNA表达量明显升高（P<0.05）。见表2。
Table 2. 培养12 h后3组RAW264.7细胞M1/M2型极化标志因子及炎症因子的mRNA表达量比较（组别 样本数 TNF-α IL-1β Arg1 iNOS 空白对照组 3 1.01±0.17 1.00± 0.09 1.00±0.05 1.00±0.04 单纯LPS组 3 85.60±8.87 1 648.67± 91.95 0.84±0.10 2.46±0.33 LPS+ADSC外泌体组 3 23.03±3.47 382.33± 31.26 1.35±0.07 1.44±0.07 F值 190.96 709.92 36.50 44.67 P值 <0.001 <0.001 <0.001 <0.001 t1值 16.51 31.04 2.40 7.70 P1值 <0.001 <0.001 0.077 <0.001 t2值 11.38 22.58 7.66 5.28 P2值 <0.001 <0.001 0.002 0.006 注：在内毒素/脂多糖（LPS）+脂肪间质干细胞（ADSC）外泌体组和单纯LPS组细胞中分别加入LPS+ADSC外泌体、LPS进行培养,对空白对照组细胞进行常规培养；IL为白细胞介素,iNOS为诱导型一氧化氮合酶,TNF为肿瘤坏死因子,Arg1为精氨酸酶-1；F值、P值为组间总体比较所得,t1值、P1值为空白对照组与单纯LPS组各指标比较所得,t2值、P2值为单纯LPS组与LPS+ADSC外泌体组各指标比较所得 3. 讨论
ALI是脓毒症患者常见的并发症之一,属于临床常见的危重症,发病急、致死率高[27, 28, 29, 30, 31]。其特征是肺泡上皮细胞和内皮细胞损伤、肺部炎症细胞浸润及充血性水肿等,严重感染、创伤后的肺部或全身不可控的炎症是主要发病诱因。目前以原发病的对症治疗、呼吸支持为主,总体上缺乏特效的药物及治疗手段,治疗效果不太理想[1,32, 33]。因此,深入探究ALI发生的机制,寻找ALI预防措施及新型治疗药物具有重要意义。
巨噬细胞常通过吞噬作用释放活性氧以及产生炎症细胞因子来发挥免疫调节作用。ALI发生时,大量炎症细胞被募集,其中肺泡巨噬细胞首先被激活并产生大量的活性氧,最终造成细胞和组织损伤,甚至导致MODS。因此,抑制炎症反应及减轻细胞氧化应激损伤,是减轻脓毒症组织器官损伤的重要治疗策略[10,34, 35, 36, 37, 38]。本团队的前期研究及其他学者的研究均显示,ADSC外泌体可以调控巨噬细胞,减轻巨噬细胞引起的炎症反应进而发挥其保护作用[20,39, 40]。本研究显示,CLP+ADSC外泌体组较单纯CLP组小鼠肺水肿症状明显减轻,炎症细胞浸润明显减少,细胞凋亡、坏死情况明显改善,同时检测到炎症反应因子TNF-α、IL-1β的表达水平明显降低,氧化产物丙二醛含量明显降低,抗氧化物质SOD含量明显升高,这与前述研究结果类似。本研究还显示,CLP+ADSC外泌体组较单纯CLP组小鼠肺组织中CD86阳性细胞即M1型巨噬细胞占比减少、CD206阳性细胞即M2型巨噬细胞占比增多；ADSC外泌体可以下调LPS诱导的M1型巨噬细胞特异性标志物iNOS的mRNA表达,而上调M2型巨噬细胞特异性标志物Arg1的mRNA表达,这表明ADSC外泌体可以抑制LPS诱导的巨噬细胞炎症反应,促进巨噬细胞向M2型极化,通过抑制巨噬细胞炎症反应进而改善脓毒症诱导的ALI。
线粒体是细胞内的主要产能结构,线粒体发生氧化磷酸化的过程不仅可以为细胞生长提供生物能量和生物中间体,还可以通过代谢-表观遗传调节决定细胞状态和功能[41, 42, 43]。在有害物质的刺激下,线粒体易发生肿胀、碎裂,基质密度降低和嵴的数量减少,最终引起线粒体功能障碍,表现为线粒体活性氧增加、ATP产生减少、DNA损伤以及由此产生的氧化应激引起细胞损伤[44, 45, 46, 47, 48]。线粒体功能障碍引起的氧化应激增强可促进大量活性氧生成,进而激活核苷酸结合寡聚化结构域样受体蛋白3等多种途径,促进M1型巨噬细胞的极化,导致一系列瀑布样炎症反应[49, 50, 51, 52]。研究显示,在ALI、ARDS等多种疾病的发病过程中,线粒体功能障碍参与了过度炎症反应和过度活性氧产生[9, 10,53]。本研究观察到单纯LPS组相较于空白对照组的RAW264.7细胞ATP含量降低,线粒体膜电位下降、线粒体活性氧生成增加。活性氧是细胞有氧代谢过程中产生的中间产物,可与不饱和脂肪酸形成脂质活性氧,从而破坏细胞内膜结构,造成细胞损伤。线粒体作为氧化磷酸化的关键场所,参与了活性氧的产生和代谢,线粒体不仅是活性氧的主要来源,也是活性氧作用的主要靶点。LPS在诱导炎症反应的同时可进一步促进活性氧生成,继而导致线粒体功能障碍及膜电位下降,最终导致线粒体功能紊乱[54, 55]。因此,恢复线粒体功能稳态对于维持巨噬细胞的正常状态和防止其对LPS刺激的过度反应至关重要。
总之,本研究显示人ADSC外泌体可被RAW264.7细胞内吞或吞噬,进而改善LPS诱导的巨噬细胞线粒体功能障碍,使细胞内线粒体活性氧水平显著降低,线粒体膜电位得到恢复,从而重新编程巨噬细胞的代谢状态,维持ATP供应,抑制巨噬细胞持续向M1型极化,减轻炎症反应,并表现出对脓毒症小鼠肺损伤的抑制作用。后期本团队将探究在脓毒症诱发的ALI过程中,人ADSC外泌体改善巨噬细胞线粒体功能障碍,促进组织损伤修复的确切作用机制等。
白晓智：实验设计、实施研究、起草文章；陶克、官浩：研究指导、论文修改、经费支持；刘洋：分析、解释数据；郝彤、张浩：实施研究、采集数据所有作者均声明不存在利益冲突 -
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图 1 人脂肪间充质干细胞外泌体的表征。1A.可见囊泡呈清晰的茶托状结构 透射电子显微镜×40 000；1B.纳米颗粒跟踪分析仪检测显示粒径为60~150 nm
注：图1B为横坐标经过lg处理的数据形成的描记图
图 2 3组小鼠术后24 h肺组织损伤情况和肺组织细胞的凋亡情况。2A、2B、2C.分别为正常对照组、单纯CLP组、CLP+ADSC外泌体组小鼠肺组织损伤情况,图2B较图2A中的肺组织水肿情况明显,炎症细胞数明显增多,图2C较图2B肺组织水肿明显改善,炎症细胞数明显减少 苏木精-伊红×100；2D、2E、2F.分别为正常对照组、单纯CLP组、CLP+ADSC外泌体组小鼠肺组织细胞的凋亡情况,图2E较图2D中的凋亡、坏死细胞数明显增加,图2F较图2E凋亡、坏死细胞数明显减少 异硫氰酸荧光素-4',6-二脒基-2-苯基吲哚×100
注：对单纯盲肠结扎穿孔（CLP）组小鼠行CLP后注射磷酸盐缓冲液,对CLP+脂肪间充质干细胞（ADSC）外泌体组小鼠进行组名相应的处理,对正常对照组小鼠仅注射磷酸盐缓冲液；凋亡细胞阳性染色为绿色,细胞核阳性染色为蓝色
图 3 3组小鼠术后24 h肺组织中巨噬细胞浸润及分化情况 辣根过氧化物酶-苏木素×100。3A、3B、3C.分别为正常对照组、单纯CLP组、CLP+ADSC外泌体组CD86（M1型巨噬细胞标志物）阳性细胞分布情况,图3B较图3A中的M1型巨噬细胞占比明显升高,图3C较图3B中的M1型巨噬细胞占比明显减少；3D、3E、3F.分别为正常对照组、单纯CLP组、CLP+ADSC外泌体组CD206（M2型巨噬细胞标志物）阳性细胞分布情况,图3E较图3D中的M2型巨噬细胞占比明显减少,图3F较图3E中的M2型巨噬细胞占比明显升高
注：对单纯盲肠结扎穿孔（CLP）组小鼠行CLP后注射磷酸盐缓冲液,对CLP+脂肪间充质干细胞（ADSC）外泌体组小鼠进行组名相应的处理,对正常对照组小鼠仅注射磷酸盐缓冲液；细胞阳性染色均为棕色,细胞核阳性染色为蓝色
图 4 共培养12 h后RAW264.7细胞吞噬人脂肪间充质干细胞（ADSC）外泌体的情况 PKH26-4',6-二脒基-2-苯基吲哚×200。4A、4B、4C.分别为细胞中的外泌体染色、细胞核染色及复合染色情况,可见人ADSC外泌体成功被RAW264.7细胞吞噬
注：人ADSC外泌体阳性染色为红色,细胞核阳性为蓝色
图 5 培养12 h后3组RAW264.7细胞中线粒体膜电位情况 JC-1×200。5A、5B、5C、5D.分别为空白对照组细胞呈蓝色荧光、绿色荧光、红色荧光及复合显色情况；5E、5F、5G、5H.分别为单纯LPS组细胞呈蓝色荧光、绿色荧光、红色荧光及复合显色情况；5I、5J、5K、5L.分别为LPS+ADSC外泌体组细胞呈蓝色荧光、绿色荧光、红色荧光及复合显色情况,图5F较图5B中的线粒体膜电位下降明显,图5J中的线粒体膜电位介于图5B与图5F之间,图5K中的正常线粒体荧光强度介于图5C与图5G之间
注：在内毒素/脂多糖（LPS）+脂肪间质干细胞（ADSC）外泌体组和单纯LPS组细胞中分别加入LPS+ADSC外泌体、LPS进行培养,对空白对照组细胞进行常规培养；细胞核阳性染色为蓝色,受损或膜电位下降的线粒体阳性染色为绿色,正常线粒体阳性染色为红色
Table 1. 实时荧光定量反转录PCR法检测RAW264.7细胞的各引物序列及产物大小
基因名称 引物序列（5'→3'） 产物大小（bp） IL-1β 上游：TCCAGGATGAGGACATGAGCAC 105 下游：GAACGTCACACACCAGCAGGTTA iNOS 上游：ACTACTGCTGGTGGTGACAA 106 下游：GAAGGTGTGGTTGAGTTCTCTAAG TNF-α 上游：ACTCCAGGCGGTGCCTATGT 160 下游：GTGAGGGTCTGGGCCATAGAA Arg1 上游：ACATTGGCTTGCGAGACGTA 109 下游：ATCACCTTGCCAATCCCCAG GAPDH 上游：TGTGTCCGTCGTGGATCTGA 150 下游：TTGCTGTTGAAGTCGCAGGAG 注：IL为白细胞介素,iNOS为诱导型一氧化氮合酶,TNF为肿瘤坏死因子,Arg1为精氨酸酶-1,GAPDH为3-磷酸甘油醛脱氢酶 
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Table 2. 培养12 h后3组RAW264.7细胞M1/M2型极化标志因子及炎症因子的mRNA表达量比较（
组别 样本数 TNF-α IL-1β Arg1 iNOS 空白对照组 3 1.01±0.17 1.00± 0.09 1.00±0.05 1.00±0.04 单纯LPS组 3 85.60±8.87 1 648.67± 91.95 0.84±0.10 2.46±0.33 LPS+ADSC外泌体组 3 23.03±3.47 382.33± 31.26 1.35±0.07 1.44±0.07 F值 190.96 709.92 36.50 44.67 P值 <0.001 <0.001 <0.001 <0.001 t1值 16.51 31.04 2.40 7.70 P1值 <0.001 <0.001 0.077 <0.001 t2值 11.38 22.58 7.66 5.28 P2值 <0.001 <0.001 0.002 0.006 注：在内毒素/脂多糖（LPS）+脂肪间质干细胞（ADSC）外泌体组和单纯LPS组细胞中分别加入LPS+ADSC外泌体、LPS进行培养,对空白对照组细胞进行常规培养；IL为白细胞介素,iNOS为诱导型一氧化氮合酶,TNF为肿瘤坏死因子,Arg1为精氨酸酶-1；F值、P值为组间总体比较所得,t1值、P1值为空白对照组与单纯LPS组各指标比较所得,t2值、P2值为单纯LPS组与LPS+ADSC外泌体组各指标比较所得
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