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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继发性瘢痕性脱发（以下简称瘢痕性脱发）是烧伤整形外科中常见的疾病，指永久性毛发脱落伴毛囊不可逆的损害，毛囊区大量胶原纤维增生形成瘢痕的一类疾病［1, 2］，给患者的生活及心理带来困扰，尤其是对外貌有较高要求的个体，可能因此产生自卑、焦虑、抑郁等情绪［3］。目前对瘢痕性脱发的治疗方法有限，主要通过药物治疗或外科手术治疗［4］。自体毛发移植作为外科治疗手段之一，在1952年首次被用于脱发的治疗［5］，因具有操作简便、术后效果好、并发症发生率低等优点，近年来被用于瘢痕性脱发的临床治疗［6］。随着自体毛发移植在瘢痕性脱发治疗中应用的增加，其良好的治疗效果也使其应用范围进一步扩展，本文就自体毛发移植在瘢痕性脱发治疗中的应用研究进展进行简要综述。
1. 瘢痕性脱发与自体毛发移植
近年来临床对瘢痕性脱发的治疗不断改进，但治疗的效果仍不理想，在一定程度上仍受到瘢痕部分特性的影响，比如瘢痕区域组织硬化、血管缺乏，瘢痕区缺血、缺氧影响毛囊生长，特别是部分烧创伤康复后存在瘢痕面积大、分布不均、边界不规则等问题，加大了治疗难度［7］。
目前对于瘢痕性脱发的治疗观点不一致，未形成统一的临床标准。瘢痕性脱发的治疗以外科手术为主，传统的手术方式包括头皮缩减术、皮瓣修复术等，现常用手术方式有皮肤软组织扩张术、毛发移植等［4，8, 9, 10］。自体毛发移植术后瘢痕小而浅，同时自体毛发移植比皮瓣转移及皮肤软组织扩张术操作简便、治疗周期短，在门诊即可操作，可根据受区瘢痕周围毛发的特点进行毛发移植，克服了皮瓣修复术后毛发与周围毛发角度、方向、密度不一致的不足，可达到更好的美学效果。自体毛发移植全过程中不置入异物（如皮肤软组织扩张器）且并发症少，是一种较安全的外科手术［7，11, 12］。
自体毛发移植术是指将供区的部分毛发通过外科手术的方式种植于毛发缺失的部位，并使其在受区保持原有的特性生长。自体毛发移植以Orentreich［13］提出的安全供区为理论基础治疗各种类型的脱发，该理论基础也是指导自体毛发移植治疗瘢痕性脱发的关键原理。安全供区为人耳后和枕外隆突上下形成的“U”形区域，以双耳上方约2 cm的弧形连接线为上界，下界根据体格检查情况而定，供区内的毛发一生都处于优势生长周期。自体毛发移植因提取过程中能保留更多毛囊根部的干细胞、创伤小、并发症少等优点在瘢痕性脱发中的应用逐渐增加［9，14］，同时有研究表明自体毛发移植术可让患者看起来更年轻、更有吸引力和亲和力，在工作和社会生活中更易取得成功［15］，让患者的生活质量提高［16］。
临床中常用的自体毛发移植技术为毛囊单位移植（follicular unit transplantation，FUT）术与毛囊单位提取（follicular unit extraction，FUE）移植术［6，8，17］。FUT和FUE相似，都需从供区获得毛囊单位并将其移植于受区，区别在于FUT是在安全供区内切取头皮条再提取移植体，而FUE通过毛囊单位提取器直接在供区提取移植体。FUT术对于大面积瘢痕性脱发适用，但术后供区可出现线性瘢痕；而FUE移植术后瘢痕非常隐蔽，对供区的影响小，伤口愈合迅速，可一定程度上弥补FUT的不足，但是FUE移植术仍存在因器械和操作导致的移植物损害、耗时、二次提取困难等局限［6，18, 19］。目前较多患者适用FUE移植术，临床中选择FUT术还是FUE移植术没有明确的标准，有学者提出可根据FOX测试来选择［6］。FOX测试即从供区提取一些移植物并划分为5个等级，1级指容易提取毛囊单位且所有毛囊单位都是完整的；2级指第1次提取可能相对容易但会在供区产生瘢痕，使随后提取的过程变得困难，以及毛囊下部周围脂肪明显减少或横断率达20%；3级指术中提取的角度困难，所需的外科技能、经验和方向感要更好；4级指提取的毛囊根部大量周围脂肪破坏和较高的横断率；5级指提取的大部分毛囊遭到严重破坏，能移植的毛囊单位稀少。FUE移植术适用于FOX测试1~3级者，而FUT术适用于FOX测试4~5级者［6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20］，针对患者选择合适的手术方式，术后才能达到最佳效果。
2. 自体毛发移植在瘢痕性脱发中的应用及进展
自体毛发移植在瘢痕性脱发中的应用与非瘢痕处的应用有所差别，需考虑瘢痕特性。在受区方面，由于瘢痕的供氧及供血差，种植密度应比非瘢痕处的植发密度低，避免灌注不足引起坏死或感染。目前瘢痕处的植发密度尚存在争议，临床常采用的密度为20~25株/cm2［7， 21］。另外瘢痕处的硬度较大，需采用合适的力度进行打孔，从而避免因深度不够导致毛囊脱出。同时还应注意种植角度，因瘢痕厚度不一、表面凹凸不平，需时刻调整种植的角度和方向，才可使种植后的毛发看起来更自然，尤其是发际线、眉毛等处［22, 23］。在供区方面，自体毛发移植通常在安全供区内提取毛囊单位，对于头面部烧创伤患者可能出现安全供区内移植物不足的情况。有学者提出在安全供区内移植物不足时也可将其他体毛作为移植体，如安全供区外的毛发、胡须、胸毛等［24, 25, 26］也可取得较好的效果。自体毛发移植术中一般将提取的移植物体外储存于生理盐水中［17］，近年来提倡使用脂质体包裹的ATP作为储存介质的附加物，并作为术后愈合喷雾剂的辅助剂，以更利于毛囊单位的储存和成活［27］。自体毛发移植常见受区为头皮脱发区，现烧创伤后眉毛、胡须、会阴等部位也可行自体毛发移植，且能取得良好的效果［23，28］；移植前一般需剃发准备，对男性影响小，但对大部分女性存在较大影响，特别是在供区毛发长出来之前，所以发展了术前不用剃发的毛发移植术［29］。另外，与人工自体毛发移植相比，毛发移植器的出现大大提高了毛发移植效率，还可减少因术者疲劳导致的移植物横断［30, 31］。外科医师良好的专业素质、医院完善的设备、对瘢痕处于稳定期的患者进行毛发移植等都能极大地提高毛发移植成活率［11，31, 32］。
研究表明自体富血小板血浆（PRP）可改善瘢痕血液供应，术前在瘢痕处注射PRP可为后期在瘢痕处移植毛发提供有利条件［33, 34］，其释放大量的生长因子还可促进干细胞分化成毛囊增加毛发密度，同时又促进毛囊周围的血管丛生成［35, 36］，从而提高移植物的成活率。术中使用PRP可减少移植体的破坏，加快伤口愈合及激活休眠的毛囊［37］。目前还出现了一种源于猪膀胱基质的生物材料，其主要由胶原蛋白、弹性蛋白、纤维连接蛋白等组成，它可使瘢痕组织变软，促进创面的愈合以及刺激毛发的再生，但其发挥作用的具体机制目前尚不清楚，该生物材料可与PRP联合使用，提高PRP的作用效能［27］。研究证实低水平激光、微针治疗以及再生干细胞治疗都能通过Wnt/β连环蛋白信号通路促进毛发生长［10，38, 39］，其中脂肪源性干细胞可分泌多种细胞因子，促进血管生成和创面愈合，同时能刺激毛囊和诱导头发生长［40］。毛囊干细胞（HFSC）由于其强大的增殖力和多能性，被认为是皮肤再生医学供体细胞之一。Zhang等［41］研究显示可从大鼠尾部的皮肤样本中获得HFSC亚群，通过皮脂腺来捕获相邻的HFSC，未来这些技术应用于临床将增加供体来源，给移植物匮乏的患者带来希望。
3. 自体毛发移植治疗瘢痕性脱发的局限性
瘢痕厚薄不均、质地柔韧性不同、血运条件差等使得在瘢痕处行自体毛发移植本身就具有挑战性，虽然相对其他外科手术安全性高［32］，但仍有毛囊炎、移植物半脱位形成囊肿或者移植物“蹦出”等情况发生，部分患者存在术后疼痛、脉管损伤引起受区中央坏死、手术部位色素沉着等并发症［6， 42］。瘢痕处移植成活率一般较低，可能要多次手术才能达到满意的毛发密度且供区毛发密度会降低［7］。自体毛发移植更适用于面积小、散在的瘢痕性脱发，对于广泛不规则的瘢痕性脱发需分阶段重建治疗，前期需要行皮瓣转移修补瘢痕规则部分，后期行自体毛发移植修补散在部分，如此多种方式结合以使术后效果更好且移植后毛发更自然［7］。对于肥厚的瘢痕组织，自体毛发移植可能面临血供不良的问题，合适的移植体及打孔深度能提高移植物的成活率，同时应根据局部血供情况适当降低植发密度［21］。萎缩性瘢痕很可能因打孔深度不够导致移植体脱出，需要术者改变打孔角度，但临床常见的贴骨瘢痕即便改变种植的角度进行自体毛发移植效果仍较差，可先行皮肤软组织扩张术，后期行皮瓣转移或瘢痕下自体脂肪移植后再移植毛发。但皮肤软组织扩张术并发症较多且治疗时间长，而自体脂肪移植可改善瘢痕的凹陷度、柔韧度，从而改善受区条件利于毛发的种植及成活［43, 44, 45］。
此外，自体毛发移植治疗瘢痕性脱发的效果评价较少，瘢痕处移植的毛发成活率本就不高，所以一般不采用有创（包括微创）的方法评估术后效果。皮肤镜作为一种无创检查［46］，可观察毛囊单位的形态和特征、发根、毛细血管等，也可观察瘢痕的情况，为评估瘢痕处自体毛发移植术后效果提供了一种选择，可直观地观察术后毛发在瘢痕上的种植情况。Liu等［47］应用FACE-Q量表评估自体毛发移植患者的满意度，并通过统计分析得出其可能成为基于临床证据的毛发移植术后效果评价量表。还有学者认为将术后毛发密度测定与患者与观察者瘢痕评估量表（POSAS）结合是一种很有前景的评估方式［48, 49］，其中POSAS涉及观察者和患者的观点，适用于术后效果的综合评估，但还需进一步量化、优化评估标准。
4. 总结与展望
综上所述，自体毛发移植在瘢痕性脱发治疗中的应用客观有效，但只改变了毛发的空间分布，并没有使毛发的总数量增加，有限的供区毛发不利于反复或者大面积的毛发移植。在基础研究方面还需深入，解决毛发供体不足的问题，如毛囊干细胞分离克隆、自体脂肪源性干细胞重建新生毛囊等［27， 50］；另外应深入研究如何使瘢痕变成更合适的受区，提高毛发移植后的成活率。现临床对自体毛发移植治疗瘢痕性脱发的研究规模较小，手术方法及术后评估缺乏统一标准，只有进一步扩大研究的规模，不断积累手术经验，完善手术及术后评估标准，自体毛发移植才能更好地应用于瘢痕性脱发的治疗。未来随着人们对毛发移植和瘢痕认识的加深、科学的进步，自体毛发移植技术在瘢痕性脱发治疗中的应用也会越来越成熟。
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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1. 孙倩，张贺. SD大鼠“环形固定法”皮肤移植模型建立及评估. 临床军医杂志. 2023(01): 53-56 . 
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