肿瘤来源外泌体调控癌细胞转移机制的研究进展

外泌体在细胞生理、病理过程中发挥重要调控作用,为细胞之间的信号传递搭建了桥梁,而且与癌细胞的远处转移调控密切相关。本文综述了外泌体特征、合成、分泌、成分、功能、生物发生以及肿瘤来源外泌体对肿瘤微环境、肿瘤细胞信号传递以及肿瘤血管生成等各个环节的调控功能及机制,并分析了外泌体在恶性肿瘤转移诊断和治疗方面的潜在价值和应用前景。     

外泌体携载阿霉素递送体系构建及体外抗肿瘤活性评价

目的 制备肝癌细胞Hep1-6外泌体并对化疗药物阿霉素（DOX）进行包载,以期实现对肿瘤细胞更高的靶向活性与杀伤作用。方法 采用梯度离心法对肿瘤细胞Hep1-6来源的外泌体进行制备分离;采用透射电镜技术、表面标记蛋白表征以及纳米颗粒追踪分析技术对外泌体的形态、特征蛋白、粒径分布和浓度进行表征;采用电穿孔方法实现外泌体对DOX的有效包载,制备包载阿霉素外泌体（EXO_DOX）。采用CCK-8法检测EXO_DOX与DOX（0.5、1、2、3、5、10 μg·mL^-1）体外对 Hep1-6细胞增殖的影响,采用激光共聚焦显微镜观察体外 Hep1-6细胞对 EXO_DOX与 DOX（1 μg·mL^-1）的靶向摄取作用。结果 所制备的外泌体具有形态良好 、 粒度均一的特性且具备外泌体特征膜蛋白 CD63、 CD81、 肿瘤易感基因101（TSG101）的表达;在电穿孔条件为150 V和75 μF下外泌体对DOX具备良好的包载特性;相比于单独给药DOX,在同等质量浓度下 EXO_DOX对 Hep1-6细胞增殖抑制作用显著增强（P＜0.05、0.01）,同时肿瘤细胞对 EXO_DOX的摄取更具靶向性。结论 制备的EXO_DOX较DOX具有更强的体外细胞毒活性,EXO_DOX表现出对肿瘤细胞更高的靶向特性。     

Extracellular vesicles: Future diagnostic and therapeutic tools for liver disease and regeneration

Extracellular vesicles are membrane fragments that can be produced by all cell types. Interactions between extracellular vesicles and various liver cells constitute an emerging field in hepatology and recent evidences have established a role for extracellular vesicles in various liver diseases and physiological processes. Extracellular vesicles originating from liver cells are implicated in intercellular communication and fluctuations of specific circulating extracellular vesicles could constitute new diagnostic tools. In contrast, extracellular vesicles derived from progenitor cells interact with hepatocytes or non‐parenchymal cells, thereby protecting the liver from various injuries and promoting liver regeneration. Our review focuses on recent developments investigating the role of various types of extracellular vesicles in acute and chronic liver diseases as well as their potential use as biomarkers and therapeutic tools.     

妊娠期糖尿病中胎盘外泌体对滋养细胞增殖、细胞周期及凋亡的影响

目的：探讨妊娠期糖尿病（GDM）中胎盘外泌体对滋养细胞增殖、细胞周期及细胞凋亡的影响。方法： 选取2018 年3 月至2019 年4 月间重庆市第七人民医院50 例诊断为GDM患者（GDM组）,另选取50 例正常孕产 妇作为对照组。分离纯化GDM组和对照组孕产妇外周血血浆中的胎盘外泌体,通过透射电镜观察外泌体的形 态,纳米粒径分析检测外泌体的直径,免疫印迹检测外泌体标志性蛋白CD63、TSG101 及胎盘标志性分子胎盘 碱性磷酸酶（PLAP）的表达。PKH67染色后荧光共聚焦显微镜观察体外培养滋养细胞系对外泌体的摄取情况; 应用MTT实验检测外泌体对滋养细胞增殖能力的影响,流式细胞术检测外泌体对滋养细胞周期的影响,Annexin V-FITC/PI 双染色结合流式细胞术检测外泌体对滋养细胞凋亡的影响。结果：成功从外周血中分离获得了胎盘外 泌体,形态呈典型的杯状或双凹状,直径为40 ～ 120 nm,CD63、TSG101、PLAP 表达呈阳性;与对照组相比, GDM组胎盘外泌体以浓度依赖性促进滋养细胞的增殖、细胞周期进展,并抑制滋养细胞的凋亡。结论：GDM中 胎盘外泌体可能通过促进滋养细胞的增殖、细胞周期进展,抑制滋养细胞凋亡等参与GDM的发生和发展。     

Circulating exosomal noncoding RNAs as prognostic biomarkers in human hepatocellular carcinoma

Exosomal noncoding RNAs (ncRNAs) have unique expression profiles reflecting the characteristics of a tumor, and their role in tumor progression and metastasis is emerging. However, the significance of circulating exosomal ncRNAs in the prognosis of hepatocellular carcinoma (HCC) remains to be elucidated. We therefore determined the prognostic significance of circulating exosomal ncRNAs (miRNA-21 and lncRNA-ATB) for human HCC. This prospective study enrolled 79 HCC patients between October 2014 and September 2015. Exosomes were extracted from serum samples using the ExoQuick Exosome Precipitation Solution. To validate the isolation of the exosomes from serum, immunoblotting for exosome markers and characterization of nanoparticle using NanoSight were performed. NcRNAs were isolated from exosomes using the miRNeasy serum/plasma micro kit. Both circulating exosomal miRNA-21 and lncRNA-ATB were related to TNM stage and other prognostic factors, including the T stage and portal vein thrombosis. Multivariate analysis using the Cox regression test identified that both higher miRNA-21 and higher lncRNA-ATB were independent predictors of mortality and disease progression, along with larger tumor size and higher C-reactive protein (all p < 0.05). The overall survival and progression-free survival were significantly lower in patients with higher circulating levels of exosomal miRNA-21 (≥0.09) and lncRNA-ATB (≥0.0016) (log-rank test: p < 0.05). In conclusion, our study has provided strong evidence that circulating exosomal ncRNAs (miRNA-21 and lncRNA-ATB) are novel prognostic markers and therapeutic targets for HCC.     

超速离心法与QIAGEN膜亲和柱法提取前列腺癌细胞培养上清外泌体的方法学比较

目的 对超速离心法与QIAGEN膜亲和柱法提取前列腺癌细胞上清外泌体优缺点进行比较,为外泌体相关基础实验研究及临床检测提供方法学参考。方法 收集前列腺癌细胞上清分别用两种方法提取外泌体,进行电镜鉴定、BCA定量测定蛋白浓度、Western blot检测标志蛋白以及Zetaview颗粒粒径、浓度及电势分析,比较两种方法提取外泌体的优缺点。结果 电镜下两种方法均可观察到具有膜结构的典型的类似于茶托状结构的外泌体,但膜亲和柱法提取外泌体背景中有类似蛋白聚合物等杂质的存在,超离背景纯净,每个视野下的外泌体数量要明显多于QIAGEN膜亲和柱法; Western blot测定标志蛋白,根据BCA定量同等上样量超速离心法可观察到明显的β-actin,ALIX,CD63以及EGFR条带,而QIAGEN膜亲和柱法仅可观察到β-actin,ALIX以及EGFR条带,但均较超速离心法条带弱; Zetaview粒径分析,超速离心法粒径分布峰值为116 nm,膜亲和柱法提取外泌体分布峰值为122 nm,均符合外泌体的粒径分布范围; 电势分析两者均为负值,符合外泌体的电势分布; 而颗粒数与蛋白浓度比值超速离心法提取外泌体要高于QIAGEN膜亲和柱法(t=13.47,P<0.01)。结论 超速离心法与膜亲和柱法均可以从细胞上清中提取外泌体,超速离心法步骤繁琐,时间较长,但外泌体纯度高,杂质少,BCA蛋白定量与实际所含外泌体蛋白量基本一致; QIAGEN膜亲和柱法方法简单,可快速从细胞上清中提取到外泌体,但纯度不及超速离心法,BCA蛋白定量比实际所含外泌体蛋白量要高,超速离心法适用于外泌体的各方面研究,而QIAGEN膜亲和柱法由于有杂蛋白的污染,因此更适用于外泌体核酸分析相关研究。     

MicroRNA‐204‐5p: A novel candidate urinary biomarker of Xp11.2 translocation renal cell carcinoma

Xp11.2 translocation renal cell carcinoma (Xp11 tRCC) is a rare sporadic pediatric kidney cancer caused by constitutively active TFE3 fusion proteins. Tumors in patients with Xp11 tRCC tend to recur and undergo frequent metastasis, in part due to lack of methods available to detect early‐stage disease. Here we generated transgenic (Tg) mice overexpressing the human PRCC‐TFE3 fusion gene in renal tubular epithelial cells, as an Xp11 tRCC mouse model. At 20 weeks of age, mice showed no histological abnormalities in kidney but by 40 weeks showed Xp11 tRCC development and related morphological and histological changes. MicroRNA (miR)‐204‐5p levels in urinary exosomes of 40‐week‐old Tg mice showing tRCC were significantly elevated compared with levels in control mice. MicroRNA‐204‐5p expression also significantly increased in primary renal cell carcinoma cell lines established both from Tg mouse tumors and from tumor tissue from 2 Xp11 tRCC patients. All of these lines secreted miR‐204‐5p‐containing exosomes. Notably, we also observed increased miR‐204‐5p levels in urinary exosomes in 20‐week‐old renal PRCC‐TFE3 Tg mice prior to tRCC development, and those levels were equivalent to those in 40‐week‐old Tg mice, suggesting that miR‐204‐5p increases follow expression of constitutively active TFE3 fusion proteins in renal tubular epithelial cells prior to overt tRCC development. Finally, we confirmed that miR‐204‐5p expression significantly increases in noncancerous human kidney cells after overexpression of a PRCC‐TFE3 fusion gene. These findings suggest that miR‐204‐5p in urinary exosomes could be a useful biomarker for early diagnosis of patients with Xp11 tRCC.     

Exosomes containing ErbB2/CRK induce vascular growth in premetastatic niches and promote metastasis of bladder cancer

Locally advanced and metastatic invasive bladder cancer (BC) has a poor prognosis, and no advanced therapies beyond cisplatin‐based combination chemotherapy have been developed. Therefore, it is an urgent issue to elucidate the underlying mechanisms of tumor progression and metastasis of invasive BC for the development of new therapeutic strategies. Here, we clarified a novel role of exosomes containing ErbB2 and CRK in a formation of premetastatic niches and subsequent metastases. CRK adaptors were overexpressed in invasive UM‐UC‐3 BC cells. In an orthotopic xenograft model, metastases to lung, liver, and bone of UM‐UC‐3 cells were completely abolished by CRK elimination. Mass spectrometry analysis identified that ErbB2 was contained in UM‐UC‐3‐derived exosomes in a CRK‐dependent manner; the exosomes significantly increased proliferation and invasion properties of low‐grade 5637 BC cells and HUVECs through FAK and PI3K/AKT signaling pathways. In athymic mice educated with UM‐UC‐3‐derived exosomes, i.v. implanted UM‐UC‐3 cells were trapped with surrounding PKH67‐labeled exosomes in lung and led to development of lung metastasis with disordered vascular proliferation. In contrast, exosomes derived from CRK‐depleted BC cells failed to induce these malignant features. Taken together, we showed that CRK adaptors elevated the expression of ErbB2/3 in BC cells, and these tyrosine kinase/adaptor units were transferred from host BC cells to metastatic recipient cells by exosomes, leading to vascular leakiness and proliferation and contributing to the formation of distant metastasis. Thus, CRK intervention with ErbB2/3 blockade might be a potent therapeutic strategy for patients with ErbB2 overexpressing advanced and metastatic BC.