CN101760557B - 一种辅助诊断肝细胞癌的试剂盒 - Google Patents
一种辅助诊断肝细胞癌的试剂盒 Download PDFInfo
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- CN101760557B CN101760557B CN 201010118539 CN201010118539A CN101760557B CN 101760557 B CN101760557 B CN 101760557B CN 201010118539 CN201010118539 CN 201010118539 CN 201010118539 A CN201010118539 A CN 201010118539A CN 101760557 B CN101760557 B CN 101760557B
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Abstract
本发明公开了一种辅助诊断肝细胞癌的试剂盒。本发明提供的试剂盒包括检测Rab27B基因表达所用的化合物。上述的检测Rab27B基因表达所用的化合物在制备辅助诊断肝细胞癌的试剂盒中的应用也属于本发明的保护范围之内。上述检测Rab27B基因表达所用的化合物为以下1)或2)：1)检测Rab27B基因的cDNA的PCR引物；2)检测Rab27B蛋白的单克隆抗体或多克隆抗体。实验证明：LMS组(采血时两年内无复发转移组)Rab27B蛋白的浓度平均值为2.08ng/μl，HMS组(采血时一年内发生转移组)为7.03ng/μl，AMS组(采血时已经发生转移组)为17.80ng/μl，三组间的蛋白含量差异显著。
Description
技术领域
本发明涉及一种辅助诊断肝细胞癌的试剂盒。
背景技术
肝细胞癌(hepatocellular carcinoma，HCC)是一种常见的、致死率极高的恶性肿瘤，每年约650,000人死于HCC，且其发病率有增高的趋势。该病多发于东南亚和非洲，其中约有50％以上发生于中国，位居我国癌症死亡率的第二位。HCC早期诊断较难，病情进展快，预后水平低。手术切除一直被认为是HCC获得根治的最佳手段，但是仅有10-20％的病人适合通过手术来干预；除此之外，即使是根治性切除，5年内仍有60-70％的病人会出现转移复发而危及生命，而局部治疗的转移复发率则更高。HCC的转移是影响治疗效果和预后的最大障碍，是导致其高死亡率的主要原因。
近年来，针对HCC的转移进行了大量研究并找到了许多重要的分子，如：黏附分子、细胞骨架及调节蛋白、基质降解酶、癌基因及抑癌基因、血管生成的调控因子、肿瘤免疫相关分子等。也即HCC的转移过程与肿瘤细胞的黏附运动、细胞增殖、细胞外基质、机体免疫、肿瘤血管生成等多因素有关。尽管对HCC转移进行了大量研究，但目前HCC转移的分子机制仍不明确，尚未发现预测HCC复发转移的分子标志物和有效的治疗靶标。
甲胎蛋白(alpha fetoprotein，AFP)是目前最常用的HCC诊断标志物，但是AFP作为目前HCC筛查和病情监测一个最主要的生物标志物仍不理想，其诊断灵敏度、特异性和阳性预测率分别只有39％-65％、76％-94％和9％-50％。而且即使AFP阳性，也不能判断HCC有无转移。除AFP外，根据美国FDA规定进入II、III期研究的还有AFP-L3(AFP异质体3)、DCP(γ-脱羧基凝血酶原)、IGF-1(***-1)，此外有研究报道但未进一步研究验证的还有数十种标志物。但这些标志物在诊断HCC的灵敏度和特异性方面都很有限，特别是不能满足HCC早期、转移以及复发诊断的需要。
发明内容
本发明的目的在于提供一种辅助诊断肝细胞癌或辅助诊断肝细胞癌细胞转移的试剂盒。
本发明提供的试剂盒包括检测Rab27B基因表达所用的化合物。
上述检测Rab27B基因表达所用的化合物为以下1)或2)：
1)检测Rab27B基因的cDNA的PCR引物；
2)检测Rab27B蛋白的单克隆抗体或多克隆抗体。
上述检测Rab27B蛋白的单克隆抗体或多克隆抗体均可从商业途径获得。
上述Rab27B蛋白的氨基酸序列如序列表中序列2所示；所述Rab27B基因是所述Rab27B蛋白的编码基因，优选是序列表中序列1所示的基因。
上述试剂盒还包括酶联免疫吸附剂测定(ELISA)所用的显色液。
上述试剂盒还包括酶联免疫吸附剂测定(ELISA)所用的反应终止液。
上述的检测Rab27B基因表达所用的化合物在制备如下1)或2)的试剂盒中的应用也属于本发明的保护范围之内：
1)辅助诊断肝细胞癌的试剂盒；
2)辅助诊断肝细胞癌细胞转移的试剂盒。
上述检测Rab27B基因表达所用的化合物为以下1)或2)：
1)检测Rab27B基因的cDNA的PCR引物；
2)检测Rab27B蛋白的单克隆抗体或多克隆抗体。
上述检测Rab27B蛋白的单克隆抗体或多克隆抗体均可从商业途径获得。
上述Rab27B蛋白的氨基酸序列如序列表中序列2所示；所述Rab27B基因是所述Rab27B蛋白的编码基因，优选是序列表中序列1所示的基因。
上述试剂盒还包括酶联免疫吸附剂测定(ELISA)所用的显色液。
上述试剂盒还包括酶联免疫吸附剂测定(ELISA)所用的反应终止液。
实验证明：LMS组(采血时两年内无复发转移组)Rab27B蛋白的浓度平均值为2.08ng/μl，HMS组(采血时一年内发生转移组)为7.03ng/μl，AMS组(采血时已经发生转移组)为17.80ng/μl，三组间的蛋白含量差异显著。通过ROC曲线分析，将LMS组作为阴性病例，AMS组和HMS组作为阳性病例计算得出诊断的最佳工作点为诊断特异度是97.06％，灵敏度为63.64％；将LMS和HMS组作为阴性病例，AMS组作为阳性病例计算得出诊断的最佳工作点为诊断特异度是98.53％，灵敏度为34.38％，因此Rab27B可作为诊断肝细胞癌是否已经发生转移的潜在标志物。
附图说明
图1为不同HCC病人血清中Rab27B的蛋白含量。
图2为ROC曲线分析Rab27B的诊断价值，(A)ROC分析Rab27B诊断转移潜能：LMS组作为阴性病例，HMS和AMS组作为阳性病例；(B)ROC分析Rab27B诊断是否已经转移：HMS和LMS组作为阴性病例，AMS组作为阳性病例。
具体实施方式
下面结合具体实施例对本发明作进一步说明，但本发明并不限于以下实施例。
下述实施例中，如无特殊说明，均为常规方法。
实施例1、检测不同肝细胞癌病人血清中Rab27B的蛋白含量
本实施例使用的是酶联免疫吸附剂测定(enzyme linked immunosorbent assay，ELISA)。
1、标准曲线的制作
分别取Rab27B标准蛋白(购自Santa Cruz公司，货号sc-22993p)0ng、0.01ng、0.02ng、0.05ng、0.1ng、0.2ng、0.5ng、1ng、1.5ng和2ng包被酶标板，每个梯度做2个平行孔，4℃孵育过夜后，洗液洗涤3次；每孔加200μl 3％的牛血清白蛋白(Jackson公司)(BSA-IgG free)封闭，37℃孵育2h，洗涤3次；每孔加入Rab27B多抗(购自Santa Cruz公司，货号sc-22993)(1∶1000PBS-BT稀释)100μl，4℃孵育过夜后，洗涤3次；每孔加入HRP-驴抗羊二抗(Jackson公司)(1∶7500 PBS-BT稀释)100μl，37℃孵育1h，洗涤3次；加入A、B显色液(北京康为世纪)(1∶19)100μl/孔，37℃显色至蓝色；加入终止液80μl/孔，450nm波长测定OD值；绘制标准曲线。
2、肝细胞癌病人血清样本
肝细胞癌病人血清收集自北京大学临床肿瘤医院，年龄为40至70岁，其中LMS组(采血时两年内无复发转移组)30例；HMS组(采血时一年内发生转移组)30例；AMS组(采血时已经发生转移组)30例。所有病例均经手术及病理检查确诊。
3、测定病人血清样本中Rab27B的含量(图1)
血清样本稀释浓度(1∶3500)，3组病人血清每例样本每孔加入100μl包板，做一个平行复孔，4℃孵育过夜后，洗液洗涤3次；每孔加200μl3％的牛血清白蛋白(BSA-IgG free)封闭，37℃孵育2h，洗涤3次；每孔加入Rab27B多抗(1∶1000PBS-BT稀释)100μl，4℃孵育过夜后，洗涤3次；每孔加入HRP-驴抗羊二抗(1∶7500PBS-BT稀释)100μl，37℃孵育1h，洗涤3次；加入A、B显色液(1∶19)100μl/孔，37℃显色至蓝色；加入终止液80μl/孔，450nm波长测定OD值；根据各自的OD值，计算各血清样本中Rab27B的含量。结果如图1所示，根据所得到的Rab27B蛋白的标准曲线以及各组病人血清样本所测得的OD值，计算所得各组病人血清样本中Rab27B蛋白的含量，其中LMS组Rab27B蛋白的浓度平均值为2.08ng/μl，HMS组为7.03ng/μl，AMS组为17.80ng/μl，对各组样本中Rab27B蛋白含量进行统计学分析，发现各组之间的目的蛋白的含量差异具有显著的统计学意义，也即各组之间差异显著。
4、统计学分析(图2)
通过Excel软件对各组病人血清样本中Rab27B蛋白含量进行统计学描述分析，对各组之间通过单因素方差分析各组间的差异；通过GraphPad Prism软件绘制ROC曲线分析Rab27B对于肝细胞癌转移的鉴别诊断价值。结果如图2所示：A图将LMS组作为阴性病例，AMS组和HMS组作为阳性病例，分析结果可知：曲线下面积为0.9011，根据阳性似然比(灵敏度/1-特异度)计算得出诊断的最佳工作点为诊断特异度是97.06％，灵敏度为63.64％，因此Rab27B可作为诊断肝细胞癌转移的潜在标志物。B图将LMS和HMS组作为阴性病例，AMS组作为阳性病例，分析结果可知：曲线下面积为0.9344，根据阳性似然比(灵敏度/1-特异度)计算得出诊断的最佳工作点为诊断特异度是98.53％，灵敏度为34.38％，因此Rab27B可作为诊断肝细胞癌是否已经发生转移的潜在标志物。
序列表
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Claims (10)
1.检测Rab27B基因表达所用的化合物在制备辅助诊断肝细胞癌细胞转移的试剂盒中的应用。
2.如权利要求1所述的应用，其特征在于：所述检测Rab27B基因表达所用的化合物为以下1）或2）：
1）检测Rab27B基因的cDNA的PCR引物；
2）检测Rab27B蛋白的单克隆抗体或多克隆抗体。
3.如权利要求1或2所述的应用，其特征在于：所述Rab27B蛋白的氨基酸序列如序列表中序列2所示；所述Rab27B基因是序列表中序列1所示的基因。
4.如权利要求3所述的应用，其特征在于：所述试剂盒还包括酶联免疫吸附剂测定所用的显色液。
5.如权利要求4所述的应用，其特征在于：所述试剂盒还包括酶联免疫吸附剂测定所用的反应终止液。
6.一种辅助诊断肝细胞癌细胞转移的试剂盒，包括检测Rab27B基因表达所用的化合物。
7.如权利要求6所述的试剂盒，其特征在于：所述检测Rab27B基因表达所用的化合物为以下1）或2）：
1）检测Rab27B基因的cDNA的PCR引物；
2）检测Rab27B蛋白的单克隆抗体或多克隆抗体。
8.如权利要求6或7所述的试剂盒，其特征在于：所述Rab27B蛋白的氨基酸序列如序列表中序列2所示；所述Rab27B基因是序列表中序列1所示的基因。
9.如权利要求8所述的试剂盒，其特征在于：所述试剂盒还包括酶联免疫吸附剂测定所用的显色液。
10.如权利要求9所述的试剂盒，其特征在于：所述试剂盒还包括酶联免疫吸附剂测定所用的反应终止液。
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