摘要
目的探讨癌相关成纤维细胞(CAFs)来源的外泌体对骨肉瘤侵袭及转移的影响。方法利用人源骨肉瘤细胞株Saos-2采用差速贴壁法分离、培养CAFs,显微镜下观察CAFs的形态,免疫组织化学观察α-SMA蛋白表达对CAFs进行鉴定。培养CAFs,采用ExoQuick-TC外泌体提取试剂盒分离外泌体。通过透射电镜观察外泌体形态,Western blot检测外泌体特异性标记蛋白β-actin、CD63表达情况对外泌体进行鉴定。培养人源骨肉瘤细胞株Saos-2,分为外泌体组和对照组,采用细胞划痕及Transwell小室实验观察外泌体对Saos-2细胞迁移和侵袭能力的影响。选取一级5周龄BALB/c-nu雌性裸鼠12只,按编号随机分为外泌体组和对照组,每组6只。两组均采用皮下移植成瘤法在裸鼠右侧背部皮下接种人骨肉瘤Saos-2细胞与Matrigel基质胶混合悬液,建立裸鼠骨肉瘤肿瘤模型。造模结束后即刻进行尾静脉注射:外泌体组每只裸鼠通过尾静脉注射浓度为0.1μg/μL的外泌体悬液30μL,对照组裸鼠尾静脉注射等量生理盐水,两组均1次/周,共注射6次。第1次注射后每日观察裸鼠成瘤情况,从裸鼠右侧背部皮下可触及肿块开始,测量两组裸鼠肿块的最长径和最长径的最大垂直短径,计算肿瘤体积,此后每周测量1次,共测量6次。完成最后一次测量后腹腔麻醉断椎法处死裸鼠,完整取出肿块并称重。取裸鼠肺组织进行切片HE染色观察两组裸鼠肺转移情况。结果显微镜下观察CAFs呈长梭形,排列杂乱,无方向性;细胞免疫组织化学染色结果显示:CAFs的α-SMA蛋白表达阳性,提示CAFs提取成功。电镜观察外泌体呈70~100 nm圆形或椭圆形膜性囊泡状结构;Western blot结果显示外泌体中CD63蛋白呈高表达,β-actin蛋白表达较弱,提示成功分离CAFs来源外泌体。细胞划痕实验、侵袭实验结果显示:外泌体组细胞迁移距离、迁移率、侵袭细胞数目分别为(0.19±0.02)mm、0.81%±0.11%�
Objective To investigate the impact of cancer related fibroblasts-derived exosomes on promoting invasion and metastasis of osteosarcoma.Methods The osteosarcoma cell line Saos-2 was used to isolate cancer-associated fibroblasts(CAFs).Exosomes extract from the supernatants of CAFs and the morphology of exosomes was identified by electron microscopy.The expression of exosome-specific marker proteinβ-actin,CD63 were detected by Western blot.Cell scratches and Transwell chamber experiments were used to observe the effects of exosomes on the migration and invasion of Saos-2 cells.Twelve female BALB/c-nu nude mice of 5 weeks old were randomly divided into exosomes and control groups,with 6 in each group.The two groups were subcutaneously implanted by subcutaneous inoculation of human osteosarcoma Saos-2 cells and Matrigel mixed suspension to establish a nude mouse osteosarcoma model.Tail vein injection was performed after modeling.The exosome group was injected with a concentration of 0.1μg/μL of exosome suspension through the tail vein,30μL per nude mouse,once a week,for a total of 6 injections.Mice in the control group were injected with normal saline at the same dose.After the first injection,the tumor formation of the nude mice was observed daily.After one week of tumor implantation,the nude mice had a subcutaneous lesion on the right side.The longest diameter and the largest vertical orthogonal diameter of the two groups were measured,and the tumor volume was calculated,and then measured once a week for a total of 6 weeks.After the last measurement was completed,euthanasia was performed,and the lump was completely removed and weighed.The lung tissue of nude mice was taken for HE staining to observe the lung metastasis of the two groups.Results Under microscope,the CAFs showed long fusiform shape and disordered arrangement.Immunohistochemical staining showed that the expression ofα-SMA protein in CAFs was positive,suggesting that CAFs were successfully extracted.Exosomes were observed by Electron microscopy as
作者
陈成龙
孙雪娇
李志慧
孙晓娟
韩鹏飞
张龙
吕智
Chen Chenglong;Sun Xuejiao;Li Zhihu;Sun Xiaojuan;Han Pengfei;Zhang Long;Lyu Zhi(Department of Orthopaedics,the Second Clinical Medical College of Shanxi Medical University,Taiyuan 030001,China;Department of Orthopaedics,the Second Hospital of Shanxi Medical University,Shanxi Key Laboratory of Bone and Soft Tissue Injury Repair,Taiyuan 030001,China;Department of Biochemistry and Molecular Biology,School of Basic Medicine,Shanxi Medical University,Taiyuan 030001,China;Department of Clinical Medicine,the Second Clinical Medical College of Shanxi Medical University,Taiyuan 030001,China;Department of Orthopaedics,the Second Hospital of Shanxi Medical University,Taiyuan 030001,China)
出处
《中华解剖与临床杂志》
2019年第4期332-340,共9页
Chinese Journal of Anatomy and Clinics
基金
国家自然科学基金(81772867)
山西医科大学大学生创新项目(20160247).
关键词
骨肉瘤
外泌体
癌相关成纤维细胞
肺转移
肿瘤微环境
Osteosarcoma
Exosomes
Cancer-associated fibroblasts
Neoplasm metastasis
Tumor microenvironment
