CN110878117A - 肝癌的肿瘤标志物血清冷诱导型rna结合蛋白及其应用 - Google Patents
肝癌的肿瘤标志物血清冷诱导型rna结合蛋白及其应用 Download PDFInfo
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Abstract
本发明涉及肿瘤的早期诊断和生物医药领域,具体是指血清冷诱导型RNA结合蛋白(CIRP)作为肝癌肿瘤标志物的应用。本项发明描述了针对血清CIRP蛋白的浓度检测试剂在制备用于肝癌的早期肿瘤诊断或预后检测试剂中的应用。本发明通过标准ELISA法鉴定出血清中增高的CIRP蛋白分泌可作为早期肝癌的诊断标志物,同时可用于评估预后,具有显著的临床应用前景。
Description
技术领域
本发明属于生物医药技术领域,涉及肿瘤的早期诊断,特别涉及一种肝癌的肿瘤标志物血清冷诱导型RNA结合蛋白及其应用。
背景技术
原发性肝癌(hepatocellular carcinoma,HCC)是全球发病率和死亡率均较高的恶性肿瘤之一,严重危害人类健康,其全球发病率居第五位,病死率居第三位。近年来,随着慢性病毒性肝炎、非酒精性脂肪肝等人数的增加,其呈现出逐渐增高的发病率。据世界卫生组织国际癌症中心估计,每年全球肝癌新发病例约80万例,其中80%的新发病例发生于发展中国家,中国占55%,其死亡率在我国恶性肿瘤中位列第二位。尽管随着诊断技术及治疗手段的进步,近二十年来疗效已经有了一定的提高,但肿瘤的侵袭、转移、耐药等特性仍是影响肝癌治疗中长期疗效的重要障碍。同时,肝癌作为一种强侵袭性的恶性肿瘤,表现出广泛而复杂的生物学特性。截至目前,仍然没有一种较为可靠便捷的手段来预测肝癌患者的预后并指导临床进行个性化诊疗。因血清取样快捷的特点,其内所包含的多种生物标记物越来越多地被临床重视,并用于早期筛检诊断及临床评估肝癌预后。最广泛的应用是肝癌患者血清中甲胎蛋白(alpha-fetoprotein,AFP)的检测。虽然AFP水平升高是肝癌的重要危险因素,但其在肝癌早期筛检诊断中的灵敏性及特异性并不理想。因此积极探寻肝癌发生发展过程中的重要生物标志物,寻求可能阻断肝癌复发的敏感靶向分子,同时针对性地研究涉及肝癌早期诊断及预后评估的新型生物标志物,对于减低肝癌死亡率改善患者生存有着十分重要的意义。
血清冷诱导型RNA结合蛋白(Cold-inducible RNA-binding protein,CIRP)是一种在冷应激诱导后表达分泌,普遍分布于哺乳动物组织液及血清中的蛋白质,广泛参与多种疾病的炎症损伤,如失血性休克、缺血再灌注损伤、急性肺损伤等,可作为内源性炎症因子直接接到TNF-α、IL-6等炎性因子的表达过程。然而,血清CIRP作为肝功能受损后的重要炎性介质,其在肝癌中的应用却尚无研究发表。
发明内容
为了克服上述现有技术的缺点,本发明的目的在于提供一种肝癌的肿瘤标志物血清冷诱导型RNA结合蛋白及其应用,基于血清CIRP在肝癌患者和非肿瘤患者血清中的差异性表达分泌情况,为肝癌早期诊断及风险评估提供新的肿瘤标志物,并可用于制备早期肝癌诊断试剂和/或预后检测试剂,这对提高肝癌早期诊疗率及改善预后具有潜在的价值。
为了实现上述目的,本发明采用的技术方案是:
肝癌的肿瘤标志物,为血清冷诱导型RNA结合蛋白(CIRP)。
所述肝癌肿瘤标志物的定量检测试剂可用于制备早期肝癌诊断试剂或预后检测试剂。
其中,制备血清冷诱导型RNA结合蛋白的定量检测试剂,是以血清中分泌或表达增高的血清冷诱导型RNA结合蛋白作为早期肝癌的诊断指标或肝癌预后欠佳的预测指标。
基于定量法,以所述血清冷诱导型RNA结合蛋白抗体制备肝癌检测试剂。
制备肝癌检测试剂时,定量法包含使用CIRP抗体的用于肝癌血清检测的化学发光酶免疫、电化学发光免疫测定、免疫酶联吸附法。
其中能特异性结合血清冷诱导型RNA结合蛋白或多肽的检测试剂为CIRP抗体或抗体片段。包含该CIRP抗体或抗体片段,可得到用于检测血清冷诱导型RNA结合蛋白分泌或表达水平的检测盒。
本发明中,针对肝癌患者与非肿瘤患者血清中CIRP表达分泌量的检测,发现相对于非肿瘤患者,肝癌患者血清中CIRP的分泌量显著升高,并且该蛋白的分泌高低与患者临床病理学指标有显著的相关性,具有重要的临床应用前景。
本发明中,同时利用H22细胞构建的原位肝癌小鼠肿瘤模型,发现相对于正常对照组,肝癌小鼠的血清CIRP水平明显增高,这进一步提示血清CIRP水平在肝癌发生发展中的重要作用。
与现有技术相比,本发明描述了含血清CIRP蛋白的浓度检测在制备用于肝癌的早期肿瘤诊断或预后检测试剂中的应用。本发明通过标准ELISA法鉴定出血清中增高的CIRP蛋白分泌可作为早期肝癌的诊断标志物,同时可用于评估预后,具有显著的临床应用前景。
附图说明
图1:血清CIRP在正常成人志愿者及肝癌患者血清中表达分泌情况。
图2:血清CIRP在非肿瘤患者组(慢性肝炎患者和单纯肝硬化的患者)和肝癌患者组中表达分泌情况:A.肝癌组为TNM分期早期的患者;B.肝癌组为肿瘤结节小于等于5cm的患者;C.肝癌组为血清AFP检测为阴性的患者。
图3:基于本组临床标本血清CIRP的检测结果,对血清CIRP水平在早期肝癌中的诊断潜能进行评估:A.肝癌患者中血清CIRP水平与血清AFP水平的相关性分析;B.肝硬化组和肝癌组患者(血清AFP水平大于或者小于等于20ng/ml)血清CIRP分泌表达水平;C.使用ROC模型预测血清CIRP、血清AFP以及两者合并后对肝癌的预测情况。(其中**P<0.01,*P<0.05)
图4:血清CIRP在原位移植瘤小鼠血清表达分泌情况:A.使用小动物体外荧光成像系统检测小鼠肝癌细胞H22原位种植瘤的生长情况;B.小鼠肝癌细胞H22原位种植成瘤组与对照组小鼠血清CIRP表达分泌水平。
具体实施方式
下面结合说明书附图和具体实施例,对本发明的技术方案进行详细的表述,但实施例本身并不对本发明做任何形式的限定。基于本发明中的实施例,本领域普通技术人员在没有付出创造性劳动前提下所获得的所有其他实施例,都属于本发明的保护范围。
本发明所述血清CIRP分泌增高包括细胞内CIRP向细胞外转移以及血清CIRP蛋白功能发挥的整个生物过程。
本发明涉及血清CIRP蛋白定量检测的试剂在制备用于早期肝癌的诊断和/或预后评估的试剂中的应用,血清中表达分泌增高的CIRP蛋白量是肝癌的指针和/或肝癌预后欠佳的表现。
本发明中,血清CIRP蛋白为动物来源,主要是指人类和小鼠来源。
本发明中,血清“CIRP蛋白”可等同于“血清生物标记物”、“生物标记物”、“肝癌肿瘤标志物”等,在具体实施例中如无特定备注,可予以替换。
本发明中作为肝癌肿瘤标志物的CIRP蛋白可包含CIRP蛋白、CIRP蛋白的天然变体以及CIRP蛋白或变体的片段,尤其是这些片段可被免疫学定量检测时。
本发明中所述“CIRP蛋白”应包含CIRP的完整氨基酸序列,以及CIRP多肽。
所使用血清CIRP浓度检测的酶联免疫吸附试剂是商用试剂盒,可直接购买成品。除非特别说明,本发明采用的试剂、方法和设备均为本技术领域常规试剂、方法和设备。
实施例1:非肿瘤患者和肝癌患者血清中CIRP表达分泌检测
一.临床血清样本的采集
材料:非肿瘤患者血清(包括单纯肝硬化患者和正常成人志愿者)与肝癌患者血清均采集自西安交通大学第一附属医院。共收集了从2012年至2016年间诊断为肝癌的173例患者的血清。肝癌患者的诊断均通过增强CT扫描、血管造影及组织病理学检查获得。同时获得124例无肝病史的健康志愿者,71例肝硬化患者以及39例慢性肝炎的患者。整个采集及实验检测过程通过西安交通大学第一附属医院伦理委员会认证。所有血清样本在采集前均已签署知情同意书。
试验步骤:
1.临床取静脉血3ml,置于试管中静置30min;
2.待凝后3000r/min离心5min;
3.取上清液即为获取的血清;
4.所有血清在获取后30分内迅速储存于-80°冰箱中冷藏备用。
二.双抗体夹心法检测冷诱导型RNA结合蛋白
材料及试剂:CIRP单克隆抗体、CIRP-HRP抗体、0.1M碳酸盐包被缓冲液(pH=9.6)、多孔聚苯乙烯反应板、0.1%BSA溶液、0.1M PBS溶液(pH=7.0)、TMB底物显色液、2M硫酸溶液、Tween-20溶液。
试验步骤:
1.使用0.1M碳酸盐包被缓冲液(pH=9.6)稀释CIRP单克隆抗体至蛋白质浓度为1-10ug/ml;
2.在聚苯乙烯板的反应孔内加入100ul稀释液;
3.4℃静置过夜;
4.弃掉孔内液体,轻轻甩干;
5.使用含0.1%Tween-20的PBS洗涤缓冲液清洗3次,每次5分钟(洗涤液下同);
6.将稀释后的临床肝癌血清100ul加入反应孔内,同时设置标准孔、空白孔和待测样品孔;
7.轻轻晃动,盖上覆膜,避光置于37℃温箱孵育1小时;
8.倒尽孔内液体,用洗涤液清洗3次;
9.在各个反应孔内加入新鲜配制的酶标抗体100ul;
10.轻轻晃动,盖上覆膜,避光37℃温箱孵育1小时;
11.到尽孔内液体,清洗3次;
12.在各个反应孔内加入新鲜配制的TMB底物显色液;
13. 37℃避光显色20分钟;
14.在各个反应孔内加入50ul 2M硫酸溶液终止反应;
15.酶标仪主波长450nm下读取检测结果(O.D.值)。
三.结果:
图1所示,基于各组临床血清ELISA检测结果,相对于无肝病史的正常志愿者的血清CIRP平均水平0.19±0.01ng/ml,肝癌患者血清中CIRP平均浓度为7.63±0.72ng/ml,其表达分泌水平显著升高(P<0.001)。该结果表明血清CIRP分泌水平的异常增高与肝癌的发生密切相关。
图2所示,获取AFP阴性的肝癌患者与两组非肿瘤患者的血清CIRP水平进行对比。发现AFP阴性的肝癌患者血清CIRP水平显著高于非肿瘤患者的肝硬化组和慢性肝炎组(P<0.05),这可能提示即使在血清AFP阴性的肝癌患者中,血清CIRP也能作为重要的肿瘤标志物存在。
图3所示,基于本组临床标本血清CIRP的检测,对其在肝癌中的诊断潜能进行评估。(图A)通过肝癌患者血清CIRP水平与血清AFP之间的相关性进行评估,发现其与AFP的水平无相关性(P>0.05);(图B)正常血清AFP水平(即≤20ng/ml)的肝癌患者血清CIRP水平显著高于单纯的肝硬化患者(P<0.05),表明联合使用血清CIRP和AFP在肝癌早期诊断中具有重要意义。(图C)使用ROC分析,发现血清CIRP联合AFP对早期肝癌诊断的敏感性和特异性均显著提高,分别为65.2%和95.5%。
实施例2:小鼠肝癌细胞株H22在C57小鼠原位成瘤后血清CIRP的表达
一.H22细胞腹腔接种
1.冻存小鼠肝癌H22细胞获取后,在37℃温水中持续摇晃,使细胞快速融化;
2.低速离心5min,弃掉上清液;
3.使用无菌生理盐水重悬细胞,调整浓度至1×107/ml;
4.小鼠腹部碘伏充分消毒,腹腔注射0.2ml细胞重悬液;
5.注射7天后抽取腹水进行细胞培养传代。
6.使用Luciferase对第三代传代细胞进行转染;
7.无菌获取转染后的H22细胞进行后续的肝脏注射。
二.小鼠肝癌原位成瘤模型构建
1.从西安交通大学医学院动物试验中心获取6-8周龄C57BL/6小鼠11只,体重为20±2g;
2.无菌获取上述荧光素标记的传代细胞,加入PBS液混匀;
3.低速离心5min,弃掉上清液;
4.继续使用PBS液清洗2次;
5.使用无菌生理盐水重悬细胞,显微镜下计数,调整细胞浓度为1×107/ml;
6. 4%水合氯醛麻醉小鼠,操作台仰卧位固定;
7.碘伏消毒腹部皮肤,腹中线纵行切口,充分暴露左肝叶;
8.微量注射器抽取50ul上述细胞悬液,低角度刺入肝脏实质约0.5cm缓慢悬液注射;
9.逐层关腹,术后体温恢复处理,并密切观察生命体征。
二.小动物体外荧光成像检测肿瘤情况
1.于术后14天将小鼠常规麻醉后放入成像暗箱平台;
2.软件控制,选择合适视野;
3.明场获取背景图,同时暗场获取荧光信号;
4.明场和暗场的背景图叠加后可以直观的显示动物体内特异光子的部位和强度,完成成像操作;
5.成瘤小鼠眼球取血后,按照上述操作,使用ELISA法检测成瘤小鼠血清中CIRP的分泌表达情况。
四.结果:
如图4所示,C57小鼠在肝脏原位注射肝癌细胞H22后14天明确成瘤,获取小鼠血清后使用ELISA法检测CIRP水平,与正常对照组小鼠相比明显升高,具有统计学差异(P<0.05)。该结果与人类患者中检测结果一致,表明血清CIRP分泌水平异常增高与肝癌发生密切相关。
Claims (7)
1.肝癌的肿瘤标志物,为血清冷诱导型RNA结合蛋白(CIRP)。
2.权利要求1所述肝癌肿瘤标志物的定量检测试剂用于制备早期肝癌诊断试剂或预后检测试剂的应用。
3.根据权利要求2所述应用,其特征在于,制备血清冷诱导型RNA结合蛋白的定量检测试剂,以血清中分泌或表达增高的血清冷诱导型RNA结合蛋白作为早期肝癌的诊断指标或肝癌预后欠佳的预测指标。
4.根据权利要求2所述应用,其特征在于,基于定量法,以所述血清冷诱导型RNA结合蛋白抗体制备肝癌检测试剂。
5.根据权利要求4所述应用,其特征在于,制备肝癌检测试剂时,定量法包含使用CIRP抗体的用于肝癌血清检测的化学发光酶免疫、电化学发光免疫测定、免疫酶联吸附法。
6.根据权利要求4所述应用,其特征在于,能特异性结合血清冷诱导型RNA结合蛋白或多肽的检测试剂为CIRP抗体或抗体片段。
7.用于检测血清冷诱导型RNA结合蛋白分泌或表达水平的检测盒,其包括权利要求6所述的CIRP抗体或抗体片段。
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