目的 分离、鉴定小细胞肺癌(SCLC)患者血清中外泌体,探讨外泌体内信使RNA(mRNA)和长链非编码RNA(lncRNA)表达谱.方法 利用ExoRNeasy试剂盒分离5例SCLC患者血清外泌体,采用电镜、粒径分析、CD63和CD68两个表面标志物检测鉴定外泌体,提取RNA后,应用二代测序技术进行1例样本的转录组测序,分析mRNA和lncRNA表达谱,并在4例SCLC中采用实时荧光定量PCR验证测序结果.结果 电镜下可见大小均一,形态呈杯口状颗粒,粒径在40～130 nm间,CD63和CD81表达均呈阳性,与外泌体特征吻合.转录组测序共检测出1 822个mRNA和890个lncRNA分子,其中表达丰度最高的mRNA依次为血红蛋白β 链、血红蛋白A2和多角体蛋白促进因子结合蛋白,lncRNA依次为ARHGEF26-AS1、WARS2-IT1和DARS-AS1.进一步在其余样本中检测上述lncRNAs,发现其阳性表达率分别为75％、100％ 和50％,表明RNA-seq结果基本可靠.结论 经过形态、粒径大小及特征蛋白分析证实SCLC血清外泌体的存在,其内可检测到大量的mRNA和lncRNA分子,为深入探讨SCLC外泌体内RNA分子的临床应用提供基础数据.%Objective To isolate and identify the exosomes in serum from small cell lung cancer ( SCLC) patients and to reveal the expression profiles of message RNA ( mRNA) and long non-coding RNA ( lncRNA) in them. Methods Exosomes were extracted from serum sample of one case of SCLC patient using the exoRNeasy kit. The morphology and particle size of exosomes were characterized by use of electron microscope and Zetasizer particle size analyzer, respectively. Expression of two landmarks namely CD63 and CD68 was detected by the fluorescently-labeled flow cytometry. Moreover, expression profile was analyzed with the RNA-seq. Five SCLC serum samples were also chosen to validate expressions of four randomly selected lncRNAs. Results Cupping particle could be seen under electron microscopy with uniform particle size ranging from 40 to 130 nm, and the CD63 and CD81 landmarks were observed with positive expression, which were in accordance with the characteristics of exosomes. The transcriptome sequencing detected a total of 1 822 mRNA and 890 lncRNAs. The randomly selected 4 lncRNAs with high expression were further confirmed in another 4 SCLC serum exosomes by qPCR. Conclusion The serum exosomes from SCLC are identified by the morphology, particle size and landmarks. It comprised an abundant of mRNA and lncRNA molecules, which are possibly valuable biomarkers for SCLC prevention and clinical application.
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