【目的】  优化外泌体提取分离纯化的方法，并比较各种方法的外泌体提取效率。基于二代测序对AD患者外周血外泌体中miRNAs表达谱进行生物信息学分析，探究AD患者外周血中外泌体中miRNA-150-5p对KC的增殖、凋亡、活化、分化以及炎症的调控及作用机制。

【方法】 ①收集我院AD患者和健康人的外周血样本，分别采用超高速离心法和exoEasy Maxi Kit试剂盒法提取血清中的外泌体，采用电镜与纳米颗粒跟踪分析（Nanoparticle Tracking Analysis，NTA）方法比较两种方法下外泌体的分离效率。②应用二代测序技术检测AD患者和健康人（各5例）外周血外泌体中miRNAs及AD患者和健康人（各20例）外周血中mRNA，利用R 3.4.1筛选差异表达的miRNA，结合文献选取其中最具有研究价值的miRNA进行分析，利用TargetScanHuman7.1数据库预测该miRNA靶基因，与差异mRNA做交集分析，采用cytoscape 3.5.1及其插件ClueGO、CluePedia进行差异基因的GO功能、KEGG信号通路富集。③收集我院AD患者和健康人的外周血样本各30例分离血浆中外泌体。分别将0 μg/μl、20 μg/μl、40 μg/μl、80 μg/μl蛋白浓度的健康人外周血外泌体与KC共培养，采用CCK-8法检测各组细胞的增殖情况，采用Annexin V-FITC/PI双染法检测各组细胞的凋亡情况，找出最佳实验浓度。根据加入外泌体的外周血来源情况分为三组：AD组、健康人组和对照组，利用荧光定量-PCR（q-PCR）检测各组细胞的活化（K16）、炎症指标（TSLP、IL-25、IL-33、CXCL1、CXCL2）。④利用q-PCR检测AD患者和健康人外周血外泌体中miRNA-150-5p的水平差异。采用受试者工作特征（ROC）曲线评价组织中miRNA-150-5p在AD早期诊断中的效能。基于二代测序结果并结合文献分析选取其中与细胞增殖、凋亡、炎症相关的HMGA2基因进行下一步研究。利用免疫组化法与激光共聚焦定性对比AD患者和健康人皮肤组织中HMGA2的阳性表达。根据miRNA-150-5p转染情况将KC分成mimic组、inhibitor组和空转组。采用CCK-8法和流式检测Ki67法测定各组细胞的增殖情况，采用Annexin V-FITC/PI流式检测各组KC的凋亡水平，利用Western Blot（WB）检测各组HMGA2的表达水平，利用q-PCR检测各组细胞的活化（K16、SKALP）、分化（K10、IVL、FLG、LOR）、炎症指标（TSLP、IL-1、IL-20、IL-24、IL-25、IL-33、TNF-α、CXCL1、CXCL2）。采用双荧光素酶报告实验检测miRNA-150-5p与HMGA2基因的相互作用关系。⑤根据miRNA-150-5p和HMGA2的转染情况将KC分成空白组（未加任何刺激）、空转组（仅加入lipo）、siHMGA2组（小干扰RNA）、mimic组、inhibitor组和inhibitor+siHMGA2组（加入inhibitor+小干扰RNA）。利用q-PCR检测各组细胞中miRNA-150-5p和HMGA2的水平差异，以验证细胞的转染效率。采用q-PCR和WB分别从mRNA和蛋白水平检测各组Bcl-2、Bax、Caspase3、STAT3、NF-κB、TGF-β、Smad2的表达差异。

【结果】  ①两种方法均成功提取出外泌体样物质——在透射电镜下观察到均一的微小囊泡，NTA镜下观察到提取物粒径在3nm～200nm之间。与超高速离心法相比，exoEasy Maxi Kit试剂盒法提取出的外泌体纯度更高。②在AD患者和健康人外周血外泌体中共筛选出差异表达miRNAs的共19个，其中上调miRNAs 14个，下调miRNAs 5个。结合文献选取其中最具有研究价值的差异表达的miRNA-150-5p进行下一步分析。数据库预测其靶基因共351个，与外周血中差异表达的mRNA取交集，共有PAX5、MUC21、SOGA3、HMGA2四个基因。通过GO注释共得到1339个生物学过程注释信息、204个细胞组分注释、130个分子功能注释信息，基因主要富集在基因表达的转录后调控、核运输、翻译调控、细胞-基质连接、mRNA结合等中。KEGG分析显示基因显著富集于STAT3信号通路、NF-κB信号通路、TGF-β/Smad2信号通路、凋亡信号通路等82个通路中。③加入40μg/μl蛋白浓度外泌体的KC增殖率较其他组别显著降低，而凋亡率明显升高（P＜0.001，P＜0.001）。较于对照组和健康人组，AD组中K16、TSLP、IL-25、IL-33、CXCL1、CXCL2的表达水平均呈不同程度上调趋势（P＜0.05）。④与健康人对比，AD患者外周血外泌体中miRNA-150-5p的表达水平显著降低（P=0.0018）。ROC曲线示miRNA-150-5p诊断AD的曲线下面积为0.723（95%CI 0.584～0.863）。免疫组化法与激光共聚焦结果均表明AD患者皮肤组织中HMGA2阳性表达比健康人呈升高趋势。较于inhibitor组和空转组，mimic组中HMGA2含量显著降低（P＜0.001）；KC细胞增殖率降低而凋亡率升高（P＜0.001，P＜0.001）；K16、SKALP、K10、IVL、TSLP、IL-1、IL-20、IL-24、IL-25、IL-33、TNF-α、CXCL2表达下调（P＜0.05），而LOR、FLG表达上调（P＜0.05）。双萤光素酶检验结果显示HMGA2为miRNA-150-5p的下游靶基因（P＜0.001）。⑤较于空白组和空转组，mimic组中miRNA-150-5p的表达水平明显升高（P＜0.05），inhibitor组和inhibitor+siHMGA2组中miRNA-150-5p的表达水平降低（P＜0.05）；mimic组、siHMGA组和inhibitor+siHMGA2组中HMGA2的表达水平不同程度的降低（P＜0.05），inhibitor组中HMGA2的表达水平呈升高趋势（P＜0.05）；mimic组、siHMGA2组和inhibitor+siHMGA2组中Bcl-2的表达水平不同程度的降低（P＜0.05）、Bax和Caspase3的表达水平不同程度升高（P＜0.05），inhibitor组中Bcl-2的表达水平升高（P＜0.05）、Bax和Caspase3的表达水平降低（P＜0.05）；mimic组、siHMGA2组和inhibitor+siHMGA2组中STAT3的表达水平降低（P＜0.05），而inhibitor组结果相反。

【结论】采用超高速离心法和试剂盒法均可应用于提取外周血中外泌体，但后者分离效率更高。AD患者外周血外泌体中miRNA-150-5p表达下调，后者可以靶向HMGA2基因通过STAT3和凋亡信号通路来促进KC的增殖、活化及炎症，从而参与AD的发生发展，有可能成为AD诊断和新型生物治疗的靶点。

【Objective】To optimize the method of exosomes extraction, and to compare the separating efficiency of each method.Based on the second-generation sequencing, profiles of miRNAs in exosomes derived from peripheral blood of AD patients were analyzed by bioinformatics. We aim to explore the mechanism of miRNA-150-5p in exosomes regulating the proliferation, apoptosis, activation and inflammation of KCs by targeting HMGA2.

【Methods】①Peripheral blood samples of patients with AD and healthy volunteers were collected and exosomes derived from serum were extracted by ultrahigh speed centrifugation and exoEasy Maxi kit, respectively. The separating efficiency of exosomes was verified by electron microscopy and NTA.②The miRNAs in exosomes and mRNA both derived from peripheral blood of AD patients and healthy volunteers were detected by Second generation sequencing. R 3.4.1 was used to screen miRNAs differentially expressed. The most valuable miRNA was selected for analysis in combination with literature review. Targetscan human 7.1 database was applied to predict the miRNA target genes, and the sequencing differential mRNA was involved for intersection. Cytoscape3.5.1 and its plugins ClueGO and CluePedia were used to enrich the GO function and KEGG signal pathway of genes.③Peripheral blood samples of 30 AD patients and 30 healthy volunteers were collected. The protein in exosomes was extracted and the protein concentration was determined. Exosomes of protein concentration 0μg/μl, 20μg/μl, 40μg/μl and 80μg/μl derived from healthy volunteers were respectively co-cultured with KC to find the optimum experimental concentration by detecting the proliferation and apoptosis of the cells. KCs were divided into three groups according to the source of exosomes: AD group, normal human group and control group. Markers of activation (K16) and inflammation (TSLP,IL-25,IL-33,CXCL1,CXCL2) were tested using q-PCR.④The level of miRNA-150-5p in exosomes derived from peripheral blood of AD patients and healthy volunteers was screened by means of q-PCR and the efficacy of miRNA-150-5p in diagnosis of AD was evaluated by ROC curve. HMGA2, related to cell proliferation, apoptosis and inflammation, was selected for further study based on the sequencing results and literature review. The expression of HMGA2 in the skin tissues of AD patients and healthy people was compared by immunohistochemistry and confocal laser. According to miRNA-150-5p transfected KC was divided into three groups: mimic group, inhibitor group and control group. The proliferation rate was measured by CCK-8 and the method of flow cytometry detecting Ki67. Annexin V-FITC/PI double staining was applied to test the apoptosis of cells in each group. WB was atopted to examine the expression of HMGA2. Q-PCR was undertaken to determine the activation (K16, SKALP), differentiation (K10, IVL, FLG, LOR), inflammation (TSLP, IL-1, IL-20, IL-24, IL-25, IL-33, TNF-α, CXCL1, CXCL2) of KCs in each group. The interaction between miRNA-150-5p and HMGA2 was detected by double luciferase report.⑤According to the transfection of miRNA-150-5p and HMGA2, KCs was divided into 6 groups: blank group (without any stimulation), idling group (with lipo only), siHMGA2 group (small interfering RNA), mimic group, inhibitor group and inhibitor + siHMGA2 group (inhibitor + small interfering RNA). Q-PCR was used to detect the difference of miRNA-150-5p and HMGA2 levels in each group to verify the transfection efficiency. The expression of Bcl-2, Bax, Caspase-3, STAT3, NF-κB, TGF-β and Smad2 were detected by q-PCR and WB.

【Results】①Both methods successfully extracted exosomes—Uniform micro vesicles were observed under the transmission electron microscope, and the particle size of the extract was between 3nm and 200nm under the NTA microscope.However, the results showed that the purity of exosomes extracted by the kit method was higher than that by the ultrahigh speed centrifugation method.②A total of 19 miRNAs were differentially expressed in exosomes derived from peripheral blood of AD patients and healthy volunteers, of which 14 miRNAs were upregulated and 5 miRNAs were downregulated. The most valuable miRNA-150-5p was selected for further analysis. Database predicted that there were 351 target genes, and there were four genes, PAX5, MUC21, SOGA3 and HMGA2, in the intersection of the sequencingdifferential mRNA. GO annotation obtained 1339 biological process annotation information, 204 cell component annotation and 130 molecular functional annotation information. The genes are mainly enriched in posttranscriptional regulation of gene expression, nuclear transport, regulation of translation, cell-substrate junction, mRNA binding and so on. KEGG analysis showed that the genes were significantly enriched in 82 pathways, including STAT3, NF-κB, TGF-β/Smad2 and apoptotic signaling pathways.③Compare with other groups, the proliferation rate of KC with 40 μg/ul exosome was significantly lower and the apoptotic rate was higher(P<0.001, P<0.001). Compared with the control group and normal human group, the expression of K16，TSLP，IL-25，IL-33，CXCL1 and CXCL2 in the AD group increased in different degrees (P<0.05).④Compared with healthy volunteers, the level of miRNA-150-5p in exosomes derived from peripheral blood of AD patients decreased significantly (P=0.0018). ROC curve showed that the area under the curve of miRNA-150-5p was 0.723(95%CI 0.584～0.863). It is showed that the positive expression of HMGA2 in the skin tissues of AD patients was higher than that of healthy people. Compared with inhibitor group and control group, there is a significantly lower expression of HMGA2 in mimic group (P<0.001); the proliferation rate decreased and the apoptosis rate increased (P<0.001, P<0.001); K16, SKALP, K10, IVL, TSLP, IL-1, IL-20, IL-24, IL-25, IL-33, TNF-α, CXCL2 reduced (P<0.05), while the expression of LOR and FLG enhanced (P<0.05). Double luciferase report indicated that HMGA2 was the downstream target gene of miRNA-150-5p (P<0.001). ⑤Compared with the blank group and the idling group, the level of miRNA-150-5p in the mimic group was significantly over-expressed(P<0.05) and there is a lower expression of miRNA-150-5p in the inhibitor group and inhibitor+siHMGA2 group(P<0.05). The level of HMGA2 in the mimic group, siHMGA2 group and inhibitor+siHMGA2 group was downward regulated(P<0.05), and that in the inhibitor group was elevated(P<0.05). A decreased expression of Bcl-2 and an increased expression of Bax and Caspase-3 were found in mimic group, siHMGA2 group and inhibitor+siHMGA2 group(P<0.05). The level of Bcl-2 in inhibitor group is up-regulated, and the level of Bax and Caspase-3 downregulated(P<0.05). The expression of STAT3 in the mimic group, siHMGA2 group and the inhibitor + siHMGA2 group was lower than that in the inhibitor group(P<0.05).

【Conclusion】Ultrahigh speed centrifugation and kit method can be used to extract exosomes from peripheral blood, but the latter has higher separating efficiency.miRNA-150-5p up-regulated in exosomes from peripheral blood of AD patients and promote the proliferation, activation and inflammation of KC by targeting HMGA2 through STAT3 and apoptotic signaling pathway, thus participating in the occurrence and development of AD, which may become a target of AD diagnosis and new biotherapy.