CN116179550A - 一种用于肝细胞癌药物敏感性检测的外泌体标志物获取方法 - Google Patents
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Abstract
本发明公开了一种用于肝细胞癌药物敏感性检测的外泌体标志物获取方法，该方法为对若干例患者在治疗前和服用待测药物进行治疗结束后24小时的血清外泌体代谢物进行分析，分析治疗前和治疗结束后的差异代谢物分子，从差异代谢物分子中筛选出表现最好的一种或多种为肝细胞癌患者药物敏感性代谢分子标志物。本发明可以发现药物敏感性代谢分子标志物。
Description
技术领域
本发明涉及生物医学领域，具体涉及一种用于肝细胞癌药物敏感性检测的外泌体标志物获取方法。
背景技术
肿瘤细胞之间以及肿瘤细胞与微环境中基质细胞之间的通讯促进了肿瘤细胞的转移，导致患者的生活质量降低，救治难度加大，甚至死亡。来自肿瘤细胞和基质细胞外泌体通过在细胞之间转运其内容物(mRNA、miRNA、lncRNA、蛋白质和脂质代谢分子)在肿瘤的发生发展和转移过程中发挥了举足轻重的作用。
外泌体(exosomes)是细胞经过“内吞-融合-外排”等一系列调控过程而形成的膜性囊泡，直径约为30-150nm，密度在1.13-1.21g ml-1，天然存在于血液、唾液、尿液及母乳等体液中。人体中大约有1014个外泌体，大约平均每个细胞产生1000-10000个。外泌体中含有核酸(DNA、miRNA、lncRNA、mRNA、tRF等)、蛋白质和氨基酸、糖、维生素、磷脂酰丝氨酸、胆固醇、神经酰胺等代谢分子，在细胞间物质和信息转导中发挥重要作用，研究表明其在免疫调控、肿瘤转移、血管生成以及生物标志物等领域都发挥着不可替代的作用。从外泌体这一微环境角度可以更好地解释细胞间通讯的传导，揭示疾病作用机制，同时寻找疾病诊断和预后的分子标志物，用于疾病的早诊和分型等。
肝细胞癌(HCC)是临床最常见的原发性肝癌和一种常见的致命性的恶性肿瘤，是全世界癌症死亡的主要原因之一，全世界每年大约有81万肝细胞癌患者死亡。其中中国的死亡人数占50％以上，平均每5min就有3人死于肝细胞癌。HCC的常见症状可分为肿瘤相关症状和肝功能失代偿导致的症状。HCC高危人群主要包括：具有乙型肝炎病毒(HepatitisBvirus，HBV)或丙型肝炎病毒(HepatitisC virus，HCV)感染、过度饮酒、非酒精性脂肪性肝炎、长期食用被黄曲霉毒素污染的食物、以及有肝癌家族史等人群，尤其是年龄>40岁的男性风险更大。
HCC治疗手段的选择取决于肿瘤位置、病变范围、是否转移及肝功能等因素。手术是唯一有可能治愈HCC的方法，包括肝脏部分切除和原位肝移植等。根治性手术切除目前仍是符合条件患者的标准治疗选择，但许多患者的预后仍然很差，术后五年内的复发风险通常超过50％。一项关于肝细胞癌根治性肝切除术后复发的动态风险预测研究共纳入了486位HCC患者，发现在术后5年内至少发生一次肝内复发的几率为31％，远处转移的几率为16.3，死亡的几率为19.8％。为了降低治愈性手术后的复发风险，可采用辅助治疗和新辅助治疗，包括新辅助局部治疗(经动脉化疗栓塞术、肝动脉灌注化疗和外照射放射治疗)和新辅助全身治疗(阻断剂、单抗和免疫检查点抑制剂)。
系统或局部姑息手段常用于因肝功能较差难以耐受手术或因肿瘤多发不能手术的患者，或在等待肝移植过程中控制肿瘤生长。目前临床治疗不可手术HCC的一线治疗药物主要有获得FDA批准的索拉菲尼(Sorafenib)和乐伐替尼(Lenvatinib)。索拉菲尼是一种阻断Raf激酶、血管内皮生长因子受体(VEGFR)与血小板衍生化生长因子受体(PDGFR)的多重酪氨酸激酶抑制剂。尽管索拉菲尼在治疗安全性与有效性存在不足，它仍是近15年内唯一用于晚期HCC全身治疗的药物。乐伐替尼作为新型的酪氨酸激酶抑制剂，其作用靶点包括VEGFR 1-3、成纤维细胞生长因子受体(FGFR 1-4)、PDGFRa与RET，其疗效与索拉菲尼相比没有显著性改善。当患者病情出现恶化，或对索拉菲尼/乐伐替尼出现不耐受性和抗药性时，二线治疗是改善HCC患者整体生存质量的有效治疗手段。《Perez M,et al.Dynamic riskprofiling of hepatocellular carcinoma recurrence after curative intent liverresection Hepatology》公开了目前二线治疗包括瑞戈非尼(Regorafenib)、卡博替尼(Cabozantinib)、雷莫芦单抗(Ramucirumab)、依维莫司(Everolimus)、阿西替尼(Axitinib)和布立尼布(Brivanib)的靶向治疗以及免疫关卡抑制剂(PD-L1)和嵌合抗原受体T细胞(CAR-T)的免疫治疗。文献《The Role of Extracellular Vesicles as Shuttlesof RNA and Their Clinical Significance as Biomarkers in HepatocellularCarcinoma》中，Neri详细报道了外泌体中非编码RNA(ncRNA)在HCC发生的肿瘤细胞形成、血管再生和肿瘤细胞转移过程中的作用，认为外泌体ncRNA作为HCC诊断与预后标志物的前景广阔。
虽然免疫治疗的兴起为肝细胞癌的治疗提供了新途径，其实用价值亦得到越来越多的肯定和关注，但是目前有效治疗HCC的包括阻断剂、单抗、免疫关卡抑制剂和CAR-T的药物仍然匮乏，HCC患者在临床治疗效果也存在较大差异。而药物治疗过程中产生的耐药性一方面制约了各类药物在HCC治疗的广泛应用，另一方面由于HCC患者对药物治疗敏感性不一，亟待发现针对药物治疗敏感性的生物标志物为制定HCC个性化治疗方案提供科学依据。
发明内容
为了克服上述现有技术的不足，本发明提供了一种用于肝细胞癌药物敏感性检测的外泌体标志物获取方法。
本发明采用的技术方案是：
本发明提供了一种用于肝细胞癌药物敏感性检测的外泌体标志物获取方法，该方法为对若干例患者在治疗前和服用待测药物进行治疗结束后24小时的血清外泌体代谢物进行分析，分析治疗前和治疗结束后的差异代谢物分子，从差异代谢物分子中筛选出表现最好的一种或多种为肝细胞癌患者药物敏感性代谢分子标志物。
进一步地，其包括以下步骤，1)患者治疗前，获取患者的血清，提取外泌体代谢物；2)获取患者服用待测药物进行治疗结束后24小时的血清，提取外泌体代谢物；3)对比步骤1)和2)外泌体代谢物，使用三重四级杆质谱确定差异代谢物分子；4)筛选得出肝细胞癌患者药物敏感性代谢分子标志物。
进一步地，所述差异代谢物分子包括氨基酸、糖类或脂质。
进一步地，步骤1)和2)中，使用高速离心机对患者血清进行外泌体分离，将分离得到的外泌体经超声破壁和甲醇提取后，得到外泌体代谢物。
进一步地，步骤3)中，用三重四级杆质谱确定差异代谢物分子确定差异代谢物分子，包括20种上调代谢物和20种小调差异代谢物分子。
进一步地，步骤4)中，筛选得出5种差异代谢物分子作为肝细胞癌患者药物敏感性代谢分子标志物。
进一步地，步骤4)中，通过绘制每种差异代谢物分子的ROC曲线并计算AUC值，确定药物敏感性代谢分子标志物。
本发明的有益效果是：
本发明创造性的提出通过血清外泌体代谢组学分析，寻找发现预测HCC药物治疗敏感性的生物标志物的方法，可以发现药物敏感性代谢分子标志物。
附图说明
图1为本发明差异化代谢物的获取过程原理图；
图2为根据本发明HCC药物敏感性分子机制获取原理图；
图3为本发明特征性物质选取的ROC曲线。
具体实施方式
为更进一步阐述本发明为实现预定发明目的所采取的技术手段及功效，以下结合附图及较佳实施例，对依据本发明的具体实施方式、结构、特征及其功效，详细说明如后。
下面将结合本申请实施例中的附图，对本申请实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本申请一部分实施例，而不是全部的实施例。以下对至少一个示例性实施例的描述实际上仅仅是说明性的，决不作为对本申请及其应用或使用的任何限制。基于本申请中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本申请的示例性实施方式。如在这里所使用的，除非上下文另外明确指出，否则单数形式也意图包括复数形式，此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
实施例1
本发明采用促凝管收集血液，经离心处理后取上清液，-80℃冻存待用。提取多组血清样本外泌体代谢物，通过高速离心手段分离血清外泌体，通过纳米粒子粒度分析和透射电镜分析，实现对所分离的血清外泌体的质控分析，避免样本之间的外泌体分离提取的批间差。采用高效液相色谱-高分辨质谱(QE HF-X)分析(LC-MS/MS)，得到的质谱原始数据经过CD(Compound Discoverer 3.0)工作站进行数据处理和分析，鉴定代谢物分子并测定样品之间的真实差异和阐明分子功能、生物学过程和信号通路等生物学信息。结合生物代谢理论，通过多变量统计分析方法分析比较肝细胞癌患者接受药物治疗前后的差异代谢组学图谱，筛选出与肝细胞癌患者药物治疗敏感性相关的特征代谢物分子，包括氨基酸、糖类、脂质类等物质。同时建立相关检测方法的技术标准，以便于临床检验使用的标准化、规范化、合法化。
本发明方法为通过高效液相色谱-高分辨质谱对两种药物在对60例HCC患者治疗前和治疗结束后24小时的血清外泌体代谢物进行分析，对获得的质谱原始数据进行处理和分析，分析治疗前和治疗结束后的差异代谢物分子，通过统计学分析整理出每种药物治疗方案中的患者的血清外泌体差异代谢分子进行相关的富集分析包括分子功能和生物学过程分析，如图1所示，最终确定HCC患者药物敏感性代谢分子标志物。
实施例2
本发明的基础为高通量血清外泌体代谢组学分析技术的建立
使用高速离心机对保存待用的血清样本进行外泌体分离，对提取出的外泌体进行纳米粒子粒度分析(ZetaView Nanoparticle-tracking analysis，Particle Metrix)和透射电子显微镜分析(JEM 1010，JEOL)以检查所提取的外泌体的物理特征。将分离得到的外泌体经超声破壁和甲醇提取后，得到外泌体代谢物。
使用高分辨(QExactive HF-X)对来自“平台构建队列”样本的血清外泌体代谢物进行分析。基于代谢物的极性，以不接色谱柱的形式考察在质谱中的响应，选择正离子或负离子扫描模式，优化离子源、离子传输以及碰撞能量等质谱条件。然后优化色谱分离条件，包括分离色谱柱类型、梯度洗脱曲线、流动相流速和色谱柱柱温箱的温度等。包括精确质量数的母离子、高分辨的子离子扫描图、保留时间、色谱条件等。最后对样本进行LC-MS/MS分析，对获得的原始数据通过CD(Compound Discoverer,ThermoFisher)软件进行代谢物的鉴定分析。通过比较HCC混合样与健康对照混合样中对应的各类代谢物的相对定量分析，评价从血清外泌体分离、外泌体代谢物提取到LC-MS/MS分析的重现性，五次重复试验的CV<15％。所建立的血清外泌体代谢组学质谱分析技术用于HCC患者药物治疗敏感性研究。
本发明包括以下步骤，
1)患者选用60人，由60例HCC患者组成，根据药物治疗敏感性分成两组(“敏感性好”和“敏感性差”)并在性别、年龄、既往史、癌症家族史上进行匹配。最后入组“敏感性好”30例，入组“敏感性差”30例。
患者治疗前，获取患者的血清，提取外泌体代谢物。
具体为：采用促凝管收集血液，经离心处理后取上清液，-80℃冻存待用。提取多组血清样本外泌体代谢物，通过高速离心手段分离血清外泌体，具体为使用高速离心机对患者血清进行外泌体分离，将分离得到的外泌体经超声破壁和甲醇提取后，得到外泌体代谢物。
2)获取患者服用待测药物进行治疗结束后24小时的血清，提取外泌体代谢物；具体为：采用促凝管收集血液，经离心处理后取上清液，-80℃冻存待用。提取多组血清样本外泌体代谢物，通过高速离心手段分离血清外泌体，具体为使用高速离心机对患者血清进行外泌体分离，将分离得到的外泌体经超声破壁和甲醇提取后，得到外泌体代谢物。
3)对比步骤1)和2)外泌体代谢物，使用三重四级杆质谱(LC-MRM MS)确定差异代谢物分子。
色谱-质谱方法(LC-MS/MS和LC-MRM MS)是开展代谢组学最成熟和可靠的方法，高分辨质谱是质谱领域的高端技术，灵敏度更优越、定性结果更为准确、检测速度更为迅速，可以筛查出更多的代谢物分子。Zhou等在对小细胞肺癌患者的药物治疗监控中发现使用LC-MS/MS可以快速检测血浆中12种代谢物。
4)筛选得出肝细胞癌患者药物敏感性代谢分子标志物。
依据HCC患者接受药物治疗(每种药物治疗入组60例HCC患者)在不同用药阶段的血清外泌体代谢组学分析，以治疗前为对照，计算出每位HCC患者在治疗前后鉴定出代谢物的相对定量信息(即：比值＝“治疗结束/治疗前”)。以比值2.0为cutoff，整理出上调代谢分子和小调代谢分子，具体整理出最优的20种上调代谢物和最优的20种小调差异代谢物分子。
上调代谢物为cutoff>0.2，小调代谢物为cutoff＜0.2。
通过绘制每种差异代谢物分子的ROC曲线并计算AUC值，确定药物敏感性代谢分子标志物。
最终筛选得出表现最好的5种差异代谢物分子作为肝细胞癌患者药物敏感性代谢分子标志物。
实施例3
在实施例2的基础上，本发明的进一步研究为，对HCC患者药物敏感性标志物的验证和评估研究如下：
对整理出最优的20种上调代谢物和最优的20种小调差异代谢物分子用“验证队列”(150例敏感性好、150例敏感性差)进行靶向分析，根据每种标志物的ROC曲线分析，计算AUC值以获得最佳界定点(灵敏度和特异性)并从中筛选出10种表现最好的代谢分子进行“评估队列”分析。
所述“验证队列”由300例HCC患者组成，根据药物治疗敏感性分成两组(“敏感性好”和“敏感性差”)并在性别、年龄、既往史、癌症家族史上进行匹配。最后入组“敏感性好”150例，入组“敏感性差”150例。
所述“评估队列”由300例HCC患者组成，根据药物治疗敏感性分成两组(“敏感性好”和“敏感性差”)并在性别、年龄、既往史、癌症家族史上进行匹配。最后入组“敏感性好”150例，入组“敏感性差”150例。
对“验证队列”筛选出的10种代谢物在200例“评估队列”(100例敏感性好、100例敏感性差)进行靶向分析，根据每种标志物的ROC曲线分析，计算AUC值以获得最佳界定点(灵敏度和特异性)并从中筛选出5种表现最好的代谢物作为预测HCC药物敏感性的生物标志物。
进一步地，本发明还可以后续用于更为深入研究，HCC患者药物敏感性代谢分子标志物的验证与评估研究。
分别将上调代谢分子和下调代谢分子带入KEGG数据库进行信号通路分析以定位上下游催化基因/酶，结合HCC患者确诊原因、临床发病机理和药物治疗敏感性情况，探讨药物治疗敏感性的分子机制，见图2。
使用三重四级杆质谱(LC-MRM MS)对HCC患者药物敏感性代谢分子标志物(20种上调代谢物和20种小调代谢物)通过验证队列分析，从中筛选出10种表现最好的代谢分子，随后通过评估队列分析最终筛选出5种现最好的代谢分子作为潜在标志物用于预测HCC药物治疗敏感性。应用生化免疫方法和串联质谱试剂方法，研发快速检测体外诊断试剂产品，更有利于大批量临床样本的检测和分析。
本发明建立了肝细胞癌患者血清中广谱小分子代谢物的筛查方法，质控了方法的可靠性，发现了甘氨胆酸等12种特征性物质，最终确定了乙酰肉毒碱、溶血软磷脂、油酰胺等潜在标志物。图3示出了实施例2中，肝硬化-肝癌中，ROC曲线提示最大AUC：0.815，预测的敏感度和特异度分别为88.9％和70.6％。
以上所述实施方式为本发明的优选实施例，而非本发明可行实施的穷举。对于本领域的技术人员而言，在不脱离本发明的精神和实质的情况下，做出的各种改进，都应当被认为包含在本发明的保护范围内。
Claims (7)
1.一种用于肝细胞癌药物敏感性检测的外泌体标志物获取方法，其特征在于，对若干例患者在治疗前和服用待测药物进行治疗结束后24小时的血清外泌体代谢物进行分析，分析治疗前和治疗结束后的差异代谢物分子，从差异代谢物分子中筛选出表现最好的一种或多种为肝细胞癌患者药物敏感性代谢分子标志物。
2.根据权利要求1所述的一种用于肝细胞癌药物敏感性检测的外泌体标志物获取方法，其特征在于，其包括以下步骤，1)患者治疗前，获取患者的血清，提取外泌体代谢物；2)获取患者服用待测药物进行治疗结束后24小时的血清，提取外泌体代谢物；3)对比步骤1)和2)外泌体代谢物，使用三重四级杆质谱确定差异代谢物分子；4)筛选得出肝细胞癌患者药物敏感性代谢分子标志物。
3.根据权利要求1所述的一种用于肝细胞癌药物敏感性检测的外泌体标志物获取方法，其特征在于，所述差异代谢物分子包括氨基酸、糖类或脂质。
4.根据权利要求2所述的一种用于肝细胞癌药物敏感性检测的外泌体标志物获取方法，其特征在于，步骤1)和2)中，使用高速离心机对患者血清进行外泌体分离，将分离得到的外泌体经超声破壁和甲醇提取后，得到外泌体代谢物。
5.根据权利要求2所述的一种用于肝细胞癌药物敏感性检测的外泌体标志物获取方法，其特征在于，步骤3)中，用三重四级杆质谱确定差异代谢物分子确定差异代谢物分子，包括20种上调代谢物和20种小调差异代谢物分子。
6.根据权利要求5所述的一种用于肝细胞癌药物敏感性检测的外泌体标志物获取方法，其特征在于，步骤4)中，筛选得出5种差异代谢物分子作为肝细胞癌患者药物敏感性代谢分子标志物。
7.根据权利要求6所述的一种用于肝细胞癌药物敏感性检测的外泌体标志物获取方法，其特征在于，步骤4)中，通过绘制每种差异代谢物分子的ROC曲线并计算AUC值，确定药物敏感性代谢分子标志物。
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