CN116466073A - 一种肝癌病理标记物及其在制备肝癌检测药物中的应用和该药物 - Google Patents
一种肝癌病理标记物及其在制备肝癌检测药物中的应用和该药物 Download PDFInfo
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Abstract
本发明公开了一种肝癌病理标记物及其在制备肝癌检测药物中的应用和该药物，涉及生物医药领域。由于目前肝癌诊断病理标志物具有难以准确判定癌灶范围的局限性，本申请通过寻找在癌灶中及癌灶边缘皆明显具有表达差异的蛋白，利用RBBP8蛋白作为组织病理学诊断肝癌的新生物标志物，并可通过鲜有检测手段进行检测，以确证癌灶范围。RBBP8蛋白作为组织病理学诊断肝癌的新生物标志物，可以更全面判定肝癌癌灶范围，为肝癌诊断、患者治疗方式及预后提供更可靠的参考。
Description
技术领域
本发明涉及生物医药领域，尤其涉及一种肝癌病理标记物及其在制备肝癌检测药物中的应用和该药物。
背景技术
癌症的早期筛查、精确诊断分型对癌症患者治疗及预后评估至关重要。目前，基于特殊生物标志物的组织病理学证据是绝大多数癌症的诊断“金标准”。通过对患者疑似癌灶、淋巴结、癌旁组织、血液或分泌物等进行取材，测定生物学标志物表达量及分布，可判定癌灶范围及来源、癌细胞分化程度、侵袭范围等，对患者诊疗方案制定及生存预后评估具有决定性作用。除了应用于明确诊断，生物标志物对癌症与影像学证据结合，对包括肝癌、乳腺癌、膀胱癌、结肠癌等多种癌症的早诊筛查、监测治疗敏感性也有较大帮助。
近几十年来，由于生物技术的不断发展，涌现出大量用于诊断的生物标志物，其可为大分子蛋白、核苷酸(如microRNA或non-coding RNA、突变基因)、抗体、多肽等。目前可以通过ki67、CK7、CK19、Arginase-1、CD34、AFP等诊断肝癌。CD34作为内皮细胞标志物可于新生血管及毛细血管化肝窦中呈强阳性；细胞角蛋白CK7/19染色可示胆管反应，染色阳性为肺恶性肝细胞癌特征，反之示高分化恶性肝细胞癌浸润汇管区。Glypican-3、HSP70、GS在肝癌早期即于癌细胞中呈特异性高表达，因此当其中任意两种呈阳性时并结合临床可诊断早期肝癌。此外，Hep Par1、pCEA、CD10、CKs 7、8/18、19、20等可有助于鉴别原发性肝癌或转移性癌灶。其中，AFP是目前被认为肝癌早诊筛查的最佳指标，对于具有肝癌高风险人群而言，建议规律进行AFP、AFP-L3及DCP监测。
目前对于肝癌的病理分子诊断已较为成熟，但目前临床应用的原位生物标志物仍具有一定局限性。例如Ki67作为细胞增殖标记物，仅标记增生活跃的肝癌细胞，对于准确判定癌灶范围具有一定局限性，需要寻找一种新的肝癌病理标记物用于全面判定癌灶侵袭范围。
发明内容
本发明提供了一种肝癌病理标记物及其在制备肝癌检测药物中的应用和该药物，可以更全面为判定肝癌癌灶范围提供参考，同时为肝癌提供新的治疗靶点及方向。
为了解决上述技术问题，本发明目的之一提供了一种肝癌病理标记物，所述肝癌病理标记物包括RBBP8蛋白。
作为优选方案，所述肝癌病理标记物还包括Ki67蛋白。
为了解决上述技术问题，本发明目的之二提供了一种肝癌病理标记物在制备肝癌检测药物中的应用。
为了解决上述技术问题，本发明目的之三提供了一种肝癌检测药物，包括有用于检测RBBP8蛋白表达的试剂。
作为优选方案，还包括有用于检测Ki67蛋白表达的试剂。
作为优选方案，所述试剂为免疫组织化学染色试剂。
相比于现有技术，本发明实施例具有如下有益效果：
本申请通过寻找在癌灶中及癌灶边缘皆明显具有表达差异的蛋白，利用RBBP8蛋白作为组织病理学诊断肝癌的新生物标志物，并可通过鲜有检测手段进行检测，以确证癌灶范围，为肝癌诊断、患者治疗方式及预后提供更可靠的参考。
附图说明
图1：为本发明健康肝脏组织和患者肝脏肿瘤及癌旁组织关于RBBP8蛋白表达检测的免疫荧光组织化学染色结果；
图2：为本发明健康肝脏组织和患者肝脏肿瘤及癌旁组织关于RBBP8蛋白表达检测的免疫荧光组织化学染色统计结果。
具体实施方式
下面将结合本发明实施例中的附图，对本发明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发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，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，都属于本发明保护的范围。
由于目前肝癌诊断病理标志物具有难以准确判定癌灶范围的局限性，本申请通过寻找在癌灶中及癌灶边缘皆明显具有表达差异的蛋白，并可通过鲜有检测手段进行检测，以确证癌灶范围，为肝癌诊断、患者治疗方式及预后提供更可靠的参考。RBBP8是参与细胞DNA修复的重要核蛋白，参与调控细胞周期、DNA复制等多方面，其被发现与多种肿瘤发生发展相关，包括乳腺癌、卵巢癌、膀胱癌等，但其尚未被作为肿瘤病理标志物。本发明拟利用RBBP8蛋白作为组织病理学诊断肝癌的新生物标志物，为更全面判定肝癌癌灶范围提供参考，为肝癌提供新的治疗靶点及方向。
实施例一
利用RBBP8蛋白表达鉴定人肝细胞癌的癌灶范围，具体包括以下步骤：
(1)患者肝脏肿瘤及癌旁组织的取样：术中取患者肝脏同时包含肿瘤及癌旁的样本，进行快速冰冻或浸于4％PFA中保存；
(2)取样标本固定、制片：对于快速冰冻样本，可置于OCT中，-80℃保存；对于浸于4％PFA的样本，可石蜡包埋保存，利用冰冻切片机或石蜡切片机进行切片，切片厚度为5μm，本申请的样品均采用石蜡包埋保存处理；
(3)样本染色处理：
A、对于冰冻切片，通过磷酸盐缓冲液(PBS)清洗三遍后，利用含0.5％ Triton-X100及3％驴血清的PBS封闭液进行封闭处理30分钟；利用含RBBP8抗体的封闭液进行过夜孵育；
B、对于石蜡切片，按照二甲苯、无水乙醇、95％乙醇、90％乙醇、80％乙醇、75乙醇顺序进行脱蜡水化，将切片浸于Tris-EDTA抗原复性液(Tris 1.4g,EDTA 0.37g,0.05％Tween20溶于1000ml ddH2O,)中，煮沸30分钟，待抗原复性液及切片自然冷却，磷酸盐缓冲液(PBS)清洗3次，每次10分钟，利用含0.5％Triton-X100及3％驴血清的PBS封闭液进行封闭处理30分钟，利用含RBBP8抗体和Ki67的封闭液进行过夜孵育；
C、上述A或B步骤的样品在第二日进行二抗孵育，弃去一抗，用含有0.2％ Tween20的PBS清洗3遍，每次10分钟，利用封闭液配制荧光二抗，避光孵育2小时，弃去二抗，用含有0.2％ Tween20的PBS清洗3遍，每次10分钟，进行DAPI染色，封片；
(4)镜检：在激光共聚焦显微镜或荧光显微镜或荧光病理扫描仪下，根据二抗荧光属性，选择适合的激发光进行检测，免疫荧光组织化学染色结果如图1所示，寻找RBBP8表达水平高的区域，即可定位为癌灶及其范围。
如图1所示，相较于ki67，RBBP8更倾向于高表达在癌灶边界细胞内，因此可明确癌灶范围。且在癌灶边界可见，一些ki67低表达的细胞中，RBBP8呈大面积高表达，因此二者结合可更全面地判定癌灶范围。如图2所示，Ki67可以标记增殖的肝癌细胞(即ki67+),从该图中可得出，在肝癌细胞中RBBP8蛋白表达量明显升高。
以上所述的具体实施例，对本发明的目的、技术方案和有益效果进行了进一步的详细说明，应当理解，以上所述仅为本发明的具体实施例而已，并不用于限定本发明的保护范围。特别指出，对于本领域技术人员来说，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (6)
1.一种肝癌病理标记物，其特征在于，所述肝癌病理标记物包括RBBP8蛋白。
2.如权利要求1所述的一种肝癌病理标记物，其特征在于，所述肝癌病理标记物还包括Ki67蛋白。
3.一种如权利要求1或2所述的肝癌病理标记物在制备肝癌检测药物中的应用。
4.一种肝癌检测药物，其特征在于，包括有用于检测RBBP8蛋白表达的试剂。
5.如权利要求4所述的一种肝癌检测药物，其特征在于，还包括有用于检测Ki67蛋白表达的试剂。
6.如权利要求4或5所述的一种肝癌检测药物，其特征在于，所述试剂为免疫组织化学染色试剂。
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