脂肪间充质干细胞条件培养基及其外泌体促成骨作用的体外研究

目的研究大鼠脂肪间充质干细胞（ASC）条件培养基及其外泌体的体外促成骨作用。 方法分离培养原代大鼠ASC和骨髓间充质干细胞（BMSC）并进行鉴定;超速离心法收集/去除条件培养基中的外泌体,采用透射电镜法及粒径分析法鉴定所获取的外泌体;细胞计数试剂盒（CCK-8）法检测其对细胞增殖的影响;Transwell检测其对细胞迁移的影响;碱性磷酸酶（ALP）活性实验和实时荧光定量聚合酶链反应（PCR）检测其对成骨的影响。SPSS 20.0软件、Bonferroni检验进行统计学分析。 结果实验所获取的原代BMSC和ASC符合间充质干细胞鉴定标准。透射电镜及粒径分析结果表明所获取的外泌体具有典型的外泌体形态特征。CCK-8实验表明培养7 d后,各处理组中A₄50值存在差异（F= 157.74,P= 0.001）,BMSC增殖条件培养基组A₄₅₀值（2.71 ± 0.15）高于对照组（1.95 ± 0.11）;去外泌体组的A₄₅₀值（2.28 ± 0.34）低于条件培养基组。Transwell实验结果表明,培养24 h后各处理组中的迁移细胞数存在差异（F= 46.79,P= 0.001）,条件培养基组迁移细胞数（51.6 ± 1.3）高于对照组（28.6 ± 1.4）,去外泌体组迁移的细胞数（37.2 ± 2.2）低于条件培养基组。ALP活性实验结果表明,培养7 d后各处理组中的ALP活性存在差异（F= 78.43,P= 0.001）,条件培养基组ALP活性为（310 ± 11）,高于对照组（200 ± 24）,去外泌体组ALP活性为（240 ± 19）低于条件培养基组。实时荧光定量PCR检测成骨相关基因的表达,各处理组中的基因表达存在差异（F_ALP= 32.30,P_ALP= 0.001;F_RUNX2= 26.78,P_RUNX2= 0.001）。 结论成骨诱导后大鼠ASC条件培养基及其中的外泌体能够有效的促进BMSC增殖、迁移和成骨向分化,具有体外促成骨作用。     

癌相关成纤维细胞来源的外泌体在促骨肉瘤侵袭转移中作用的实验研究

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0.1 μg/μL的外泌体悬液30 μL,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%、(53.67±5.69) 个,均高于对照组的(0.81±0.11)mm、0.32%±0.11%、(31.67±8.08) 个,差异均有统计学意义(t=6.209、6.209、3.856, P值均＜0.01)。外泌体组和对照组12只裸鼠采用皮下移植成瘤法接种人骨肉瘤Saos-2细胞与Matrigel基质胶混合悬液,均造模成功,实验过程中无一例动物死亡。两组裸鼠均于第1次注射肿瘤细胞后1周触及肿块,肿瘤体积均随着时间的延长明显增大,差异均有统计学意义(F=49.428、8.477, P值均＜0.05)。两组间比较,第1、2次测量肿瘤体积,差异均无统计学意义(P值均＞0.05);而第3～6次测量肿瘤体积,外泌体组均大于对照组,差异均有统计学意义(t=4.697、4.825、3.516、2.706, P值均＜0.05)。测量6次后处死裸鼠,剥除肿瘤,外泌体组肿瘤质量(1.62±0.29)g大于对照组(1.00±0.43)g,差异有统计学意义(t=2.929, P＜0.05)。肺组织切片HE染色结果显示,两组裸鼠均发生肺转移,外泌体组肺组织转移灶数量为6.63(3,17)个,多于对照组的1.17(0,3)个,差异有统计学意义(W=36.000, P＜0.01)。结论 CAFs来源的外泌体可增加骨肉瘤细胞株Saos-2的迁移及侵袭能力,同时促进骨肉瘤的生长和肺转移的形成。     

The emerging roles of exosomal miRNAs in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a unique subtype of head and neck cancer that is endemic to Southern China and Southeast Asia. Due to the concealed location and intrinsic invasiveness of this disease, majority of NPC patients are diagnosed with advanced stages (III and IV) and poor prognosis. Chemoradiotherapy resistance is a major problem for NPC patients, leading to incomplete local elimination, recurrence and metastasis. Therefore, it is of great significance to seek novel biomarkers and effective therapeutic regimen for clinical management of this deadly cancer. Exosomes are tiny membrane vesicles with a lipid bilayer secreted by most cells in the body, which are widely distributed in various body fluids. They are functionally active in different physiopathological process by carrying and transmitting important signal molecules such as miRNA, mRNA, protein, lipid, etc. Exosomal miRNAs play an important role in tumorigenesis and development of NPC. They are extensively involved in NPC cell proliferation, migration, invasion, neovascularization, radiotherapy resistance and the regulation of tumor immune microenvironment through intercellular communication and control of gene expression. Moreover, exosomal miRNAs can be used as valuable biomarkers for early diagnosis and therapeutic targets of NPC.     

Exosomes: Promising biomarkers and targets for cancer

The review article entitled "Exosomes as potential diagnosis and treatment for liver cancer " recently published in World Journal of Gastrointestinal Oncology 2022; 14: 334-347 concluded that exosomes can be used as effective biomarkers or therapeutic biotargets in liver cancer. Exosomes are a hot spot in the field of tumor diagnosis and treatment research. We had also previously published a review on exosomes and tumors. In this letter to the editor, we summarize the clinical application prospects and current challenges of exosomes.     

外泌体在乳腺癌中的研究进展

外泌体是一类由自体细胞分泌的纳米级囊泡样小体,参与细胞及内环境之间的物质运输和信号传递。外泌体可以通过在肿瘤细胞和细胞外基质间运输细胞内的核酸、蛋白和脂类等物质,影响肿瘤的发生和发展,甚至可以影响肿瘤的治疗。不同类型的肿瘤细胞分泌的外泌体,在肿瘤的发生过程中发挥着不同的作用。近来,与乳腺癌相关的外泌体逐渐成为一项新的研究热点,并发现乳腺癌细胞可以刺激分泌某些特定类型的外泌体,并有望成为早期乳腺癌筛查的新型生物学标志。外泌体传递的核酸、蛋白质等物质在乳腺癌的发生、转移和治疗耐药中起到了重要作用。同时外泌体也可以将抗肿瘤药物转运出乳腺癌细胞导致耐药。但外泌体作为肿瘤药物载体时,表现出极低的免疫原性和生物毒性,将为今后的肿瘤靶向治疗提供可行的技术基础。     

子痫前期孕妇血浆外泌体中LncRNA MALAT1和miR-206的表达水平及临床意义

目的 探究长链非编码RNA肺腺癌转移相关转录本1(LncRNA MALAT1)、微小RNA-206(miR-206)在子痫前期(PE)孕妇血浆外泌体中的表达水平及临床意义.方法 选取2017年1月至2019年12月在郑州市妇幼保健院住院分娩的PE孕妇42例为PE组,另选取同期在本院分娩的健康孕妇42例为对照组.根据入组...     

Evolving utility of exosomes in pancreatic cancer management

Despite the development of newer oncological treatment, the survival of patients with pancreatic cancer (PC) remains poor. Recent studies have identified exosomes as essential mediators of intercellular communications and play a vital role in tumor initiation, metastasis and chemoresistance. Thus, the utility of liquid biopsies using exosomes in PC management can be used for early detection, diagnosis, monitoring as well as drug delivery vehicles for cancer therapy. This review summarizes the function, and clinical applications of exosomes in cancers as minimally invasive liquid biomarker in diagnostic, prognostic and therapeutic roles.     

Molecular lipid species in urinary exosomes as potential prostate cancer biomarkers

BackgroundExosomes have recently appeared as a novel source of noninvasive cancer biomarkers, since these nanovesicles contain molecules from cancer cells and can be detected in biofluids. We have here investigated the potential use of lipids in urinary exosomes as prostate cancer biomarkers.MethodsA high-throughput mass spectrometry quantitative lipidomic analysis was performed to reveal the lipid composition of urinary exosomes in prostate cancer patients and healthy controls.ResultsControl samples were first analysed to characterise the lipidome of urinary exosomes and test the reproducibility of the method. In total, 107 lipid species were quantified in urinary exosomes. Several differences, for example, in cholesterol and phosphatidylcholine, were found between urinary exosomes and exosomes derived from cell lines, thus showing the importance of in vivo studies for biomarker analysis. The 36 most abundant lipid species in urinary exosomes were then quantified in 15 prostate cancer patients and 13 healthy controls. Interestingly, the levels of nine lipids species were found to be significantly different when the two groups were compared. The highest significance was shown for phosphatidylserine (PS) 18:1/18:1 and lactosylceramide (d18:1/16:0), the latter also showed the highest patient-to-control ratio. Furthermore, combinations of these lipid species and PS 18:0-18:2 distinguished the two groups with 93% sensitivity and 100% specificity. Finally, in agreement with the reported dysregulation of sphingolipid metabolism in cancer cells, alteration in specific sphingolipid lipid classes were observed.ConclusionThis study shows for the first time the potential use of exosomal lipid species in urine as prostate cancer biomarkers.