乳腺癌转移病人血清外泌体蛋白质分析

doi:10.16139/j.1007-9610.2019.02.012

摘要/Abstract

摘要： 目的研究乳腺癌转移与未转移病人血清外泌体内差异蛋白表达情况。方法收集乳腺癌淋巴结转移或远处转移（metastatsis,M+）病人血清25例,乳腺癌淋巴结未转移且未远处转移M-病人血清25例。应用EIQ3试剂盒分离血清外泌体。挑选4例M+及4例M-病人血清,通过NTA粒径分析、CD9和CD63表面标志物鉴定外泌体。通过蛋白质质谱分析及生物信息学分析,筛选乳腺癌病人血清外泌体差异表达的蛋白质,并进行蛋白质印迹验证。结果血清提取物粒径在20~200 nm,CD9和CD63表达均呈阳性,符合外泌体特征。两组外泌体经质谱共鉴定出301种蛋白质,筛选出16种差异蛋白,其中的玻连蛋白（vitronectin, VN）及人组织激肽释放酶结合蛋白（kallistatin,KAL）在M+组表达上调,遮蔽蛋白（obscurin, OBSCN）则在M-组下调。通过蛋白质印迹验证,3种差异蛋白质结果与质谱结果一致。结论乳腺癌病人血清中存在外泌体,其中外泌体来源蛋白VN、KAL及OBSCN有望作为预测乳腺癌转移的生物标志物。

关键词: 乳腺癌, 外泌体, 质谱, 生物标志物

Abstract: Objective To study differentially expressed protein in serum exosome of the patients with both metastatic and non-metastatic breast cancer. Methods Serum was collected from both 25 breast cancer patients with metastasis of lymph node or distant organ (M+ group) and 25 breast cancer patients without any metastasis (M- group). Serum exosomes were separated with EIQ3 analytic kit in 4 breast cancer patients in M+ group and 4 breast cancer patients in M- group.These serum exosomes were recognized by CD9 and CD63 as surface markers using western blotting, and the size of exosomes were meaured by nanosight tracking analysis（NTA）. Protein mass spectrometry and bioinformatics analysis were performed to identify the differentially expressed proteins in serum exosomes of breast cancer patients, which were also quantified by Western blot. Results The particle size by nanosight tracking analysis ranged from 20 to 200 nm and the expression of CD9 and CD63 were positive. Totally there were 301 proteins identified by mass spectrometry and among them 16 proteins differentially expressed from serum exosomes in M+ group and M- group. The expression of vitronectin(VN) and kallistatin (KAL) was up-regulated, while the expression of obscurin(OBSCN) was down-regulated in M+ group compared with those in M- group using Western blotting, which was consistent with the results of analysis by mass spectrometry. Conclusions The serum exosomes in breast cancer patients are present identified by particle size and markers. VN, KAL and OBSCN in exosomes could serve as potential candidate biomarkers for prediction of metastatic breast cancer.

Key words: Breast cancer, Exosome, Mass spectrum, Biomarker

中图分类号:

R737.9

曹丽辰, 田宝星, 瞿飞麟, 张庆华, 郭善禹. 乳腺癌转移病人血清外泌体蛋白质分析[J]. 外科理论与实践, 2019, 24(02): 141-148.

CAO Lichen, TIAN Baoxing, QU Feilin, ZHANG Qinghua, GUO Shanyu. Study on serum exosome proteins in metastatic breast cancer patients[J]. Journal of Surgery Concepts & Practice, 2019, 24(02): 141-148.

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