胰岛素样生长因子结合蛋白7转染的人脂肪间充质干细胞外泌体对恶性黑素瘤A375细胞凋亡的影响  

Impact of exosomes from IGFBP7-transfected human adipose-derived mesenchymal stem cells on apoptosis of malignant melanoma A375 cells

作　　者：罗亭 景海霞 王润超 王恒[1] 刘文韬[1] LUO Ting;JING Haixia;WANG Runchao;WANG Heng;LIU Wentao(Department of Dermatology,Shiyan Taihe Hospital,Hubei University of Medicine,Shiyan,Hubei 442000,China;Hubei University of Medicine(Department of Dermatology,Shiyan Taihe Hospital),Shiyan,Hubei 442000,China)

机构地区：[1]十堰市太和医院(附属湖北医药学院)皮肤病中心,湖北十堰442000 [2]湖北医药学院附属十堰市太和医院皮肤病中心,湖北十堰442000

出　　处：《中国热带医学》2024年第7期864-868,共5页China Tropical Medicine

基　　金：湖北省十堰市科学技术局项目(No.18Y24)。

摘　　要：目的研究A375细胞对胰岛素样生长因子结合蛋白7(insulin-like growth factor binding protein 7,IGFBP7)转染的人脂肪间充质干细胞(human adipose-derived mesenchymal stem cells,ADSCs)外泌体(ADSCs;IGFBP7;外泌体)的摄取能力以及摄取后凋亡情况的影响,为恶性黑素瘤的基因治疗提供新的思路。方法采用组织块贴壁培养法,将脂肪干细胞原代培养至第2代,采用细胞爬片免疫荧光法进行细胞表面标记物的鉴定。将购买的IGFBP7-GFP过表达载体转染至ADSCs细胞,构成ADSCs;IGFBP7;细胞,荧光定量聚合酶链反应(polymerase chain reaction,PCR)检测其IGFBP7 mRNA水平。提取外泌体,用电子显微镜分析其超微结构,并用免疫印迹(Western blot)法鉴定CD63、CD81蛋白的表达。将ADSCs;IGFBP7;外泌体作用于A375细胞,用荧光显微镜观察A375细胞摄取ADSCs;IGFBP7;外泌体的能力,用流式细胞仪检测A375细胞的凋亡情况。结果细胞爬片免疫荧光结果示,分离出的细胞CD44呈阳性表达,CD34呈阴性表达,提示分离培养的细胞符合脂肪间充质干细胞的特异性表型。与对照组相比,转染组的IGFBP7 mRNA表达增高(t=11.50,P<0.01),提示IGFBP7-GFP过表达载体转染至ADSCs细胞,构成ADSCs;IGFBP7;细胞。透射电镜下可见外泌体具有较明显的异质性,发现ADSCs;IGFBP7;外泌体形态呈大小不一的球膜结构,中间颜色较深,边缘处有低密度物质;Western blot结果示,外泌体标志蛋白CD63、CD81均阳性表达,提示外泌体提取成功。与A375细胞组相比,将ADSCs;IGFBP7;外泌体与A375细胞共同培养,用PKH26标记外泌体,6 h后A375细胞在摄取ADSCs;IGFBP7;外泌体。A375细胞组、A375细胞+ADSCs;IGFBP7;外泌体组的A375细胞凋亡率分别为(0.743±0.050)%、(4.990±0.196)%。A375细胞+ADSCs;IGFBP7;外泌体组凋亡率升高,与A375细胞组相比差异有统计学意义(t=20.93,P<0.001)。结论ADSCs;IGFBP7;外泌体可被A375细胞摄取并促进其凋亡。Objective To analyze the uptake capacity of the malignant melanoma cell line A375 towards exosomes derived from insulin-like growth factor binding protein 7(IGFBP7)transfected human adipose-derived mesenchymal stem cells(ADSCs)and the subsequent effects on apoptosis,providing new insights for gene therapy in malignant melanoma.Methods Primary adipose-derived stem cells were prepared by adherent culture of tissue blocks in vitro and passaged to obtain the second-generation.Cell surface markers were identified by cell climbing immunofluorescence.IGFBP7-GFP overexpression vectors were transfected into ADSCs to create ADSCsIGFBP7,followed by measuring the mRNA level of IGFBP7 by fluorescent quantitative PCR.Exosomes were isolated and extracted,and their ultrastructure was observed under electron microscopy.The protein expressions of CD63 and CD81 proteins were detected using Western blot.ADSCsIGFBP7 exosomes were applied to A375 cells,with the uptake capacity of A375 cells for ADSCsIGFBP7 exosomes being observed under a fluorescence microscope.Apoptosis of A375 cells was detected using flow cytometry.Results Immunofluorescence results showed positive expression of CD44 and negative expression of CD34 in the isolated cells,indicating that the isolated and cultured cells displayed a specific phenotype of ADSCs.IGFBP7 mRNA expression in the transfection group was higher compared to the control group(t=11.50,P<0.001),suggesting successful transfection of IGFBP7-GFP overexpression vector into ADSCs to construct ADSCsIGFBP7 cells.Transmission electron microscopy revealed that the exosomes had distinct heterogeneity,showing the morphology of exosomes of ADSCsIGFBP7 appeared as spherical structures of varying sizes with a darker color in the center and low-density substances at the edges.Western blot results indicated positive expression of exosome marker proteins CD63 and CD81,which confirmed the successful extraction of exosomes.After the exosomes of ADSCsIGFBP7 were co-cultured with A375 cells for 6 hours and the exosomes

关 键 词：恶性黑素瘤 外泌体 胰岛素样生长因子结合蛋白7 人脂肪间充质干细胞 

分 类 号：R756[医药卫生—皮肤病学与性病学] R392.9[医药卫生—临床医学]