CN112011610A - 一种用于癌症预后检测试剂盒 - Google Patents
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Abstract
本发明公开了一种用于癌症预后检测试剂盒,涉及医药卫生技术领域,具体为一种用于癌症预后检测试剂盒,试剂盒包括可检测ZNF467、SH3RF2、PPFIA2、MYT1、TROAP、GOLGA7B表达水平的检测试剂。该用于癌症预后检测试剂盒,相比于普通的试剂盒,本发明的试剂盒和方法能够准确预测癌症预后后果,尤其是对于前列腺癌的高风险诊断具有较高的预测价值和重要的基础及临床价值和广阔的应用前景,有效满足了人们的使用需求。
Description
技术领域
本发明涉及医药卫生技术领域,具体为一种用于癌症预后检测试剂盒。
背景技术
前列腺癌是严重威胁男性健康的恶性肿瘤之一,其发病率在西方男性中高居首位,相关死亡率位居第二,近年来因我国人口老龄化及生活方式西化,前列腺癌的发病率逐年攀升,呈现出“基数小、发展快”的特点,据中国肿瘤登记年报2017版报道,目前我国小城市前列腺癌发病率约5/10万,而大城市发病率是小城市的4倍,位居全国登记地区男性第六位高发恶性肿瘤,此外前列腺癌是发病率最高的男性泌尿生殖系统恶性肿瘤,其发病率随城市化发展程度逐渐上升,因为我国尚未全面普及PSA筛查,申请者对近期前列腺癌进行统计,初诊晚期前列腺癌患者比例远高于西方人群,但是前列腺癌也是一种异质性疾病,疾病的结局因人而异,差异非常大,许多临床和病理因素如血清前列腺特异性抗原水平、病理分期、肿瘤分级、是否切缘阳性、是否存在淋巴结转移等都与预后密切相关,美国泌尿外科学会指南中指出:Gleason分级、PSA水平和肿瘤分期是决定治疗方案的最重要的指标,但是由于术前Gleason评分的获取依赖病理医生和前列腺穿刺医生,医生的主观差异,术前穿刺的抽样误差等混杂因素,这一难题仍然未得到解决,同时在前列腺癌进展过程中,高危和低危患者为何预后相差甚远,其中进展机制有何不同,如何区分中危患者的进展方向都是前列腺癌临床诊疗工作中亟待解决的重要问题,同时也可以为阐明前列腺癌的发展机制及治疗提供新的思路和方法。
在国内,大多数国人对主动监测的接受程度低,造成了大量的过度治疗,浪费了宝贵的医疗资源,另一方面,因前列腺癌患者诊断时往往高龄,部分患者为得到积极干预,错过了最佳治疗窗,因此目前迫切需要研究前列腺癌危险度的基因预测模型,该模型不仅仅可以预测患者预后,还能判别该使用何种治疗手段,因此本项目将之称为决策模型,
功能基因组学又往往被称为后基因组学,它利用结构基因组所提供的信息和产物,发展和应用新的实验手段,通过在基因组或系统水平上全面分析基因的功能,使得生物学研究从对单一基因或蛋白质的研究转向多个基因或蛋白质同时进行系统的研究,这是在基因组静态的碱基序列弄清楚之后转入对基因组动态的生物学功能学研究,研究内容包括基因功能发现、基因表达分析及突变检测,基因的功能包括:生物学功能,如作为蛋白质激酶对特异蛋白质进行磷酸化修饰;细胞学功能,如参与细胞间和细胞内信号传递途径;发育上功能,如参与形态建成等,采用的手段包括经典的减法杂交、差示筛选,cDNA代表差异分析以及mRNA差异显示等,但这些技术不能对基因进行全面系统的分析,新的技术应运而生,包括基因表达的系统分析,cDNA微阵列,DNA芯片和序列标志片段显示技术、微流控芯片实验室等,
现有研究已经发现了部分与前列腺癌预后相关基因,但是大多数现有研究包含的数据集少,如2011年的一篇论文,mRNA expression signature of Gleason gradepredicts lethal prostate cancer,其基因模型包含了157个基因,而另一篇发表在2017年的clinical cancer research杂志上的改进的基因预后预测模型,也包含了30个基因,而2018年发表于癌症研究和临床肿瘤学杂志的一篇论文,Validation of a 10-genemolecular signature for predicting biochemical recurrence and clinicalmetastasis in localized prostate cancer,则是包含了10个基因的预测前列腺癌生化复发的模型,该论文还公开了采用该模型预测前列腺癌早期生化复发的结果:AUC值为0.65,由此可见,目前的前列腺癌基因预后模型,要么包含了太多的基因,不利于临床应用,要么包含了不算多的基因,但是预测效能较低,仍有待增强,需要从大量的前列腺癌基因中建立能够更加精准预测前列腺癌复发风险的模型。
发明内容
针对现有技术的不足,本发明提供了一种用于癌症预后检测试剂盒,解决了上述背景技术中提出的问题。
为实现以上目的,本发明通过以下技术方案予以实现:一种用于癌症预后检测试剂盒,所述试剂盒包括可检测ZNF467、SH3RF2、PPFIA2、MYT1、TROAP、GOLGA7B表达水平的检测试剂。
可选的,所述检测试剂包括多核苷酸引物或探针。
可选的,所述多核苷酸引物的序列如SEQIDNO:1-10所示。
可选的,所述癌症选自前列腺癌。
可选的,所述癌症的进展及预后的确定包括以下步骤:
(a)检测样本的ZNF467、SH3RF2、PPFIA2、MYT1、TROAP、GOLGA7B的mRNA表达水平;
(b)根据步骤(a)获得的表达水平数据评估患者的癌症复发风险。
可选的,所述步骤(b)中通过以下方法评估患者的癌症生化复发风险及总体预后:
风险值=(0.0122×ZNF467表达水平)-(0.0075×SH3RF2表达水平)+(0.0080×PPFIA2表达水平)+(0.0167×MYT1表达水平)+(0.0233×TROAP表达水平)+(0.0374×GOLGA7B表达水平)。
本发明提供了一种用于癌症预后检测试剂盒,具备以下有益效果:
本发明的试剂盒和方法能够准确预测癌症预后后果,尤其是对于前列腺癌的高风险诊断具有较高的预测价值和重要的基础及临床价值和广阔的应用前景。
附图说明
图1为本发明实施例的6个基因为基础的基因决策模型示意图;
图2为本发明实施例的455例患者ROC曲线示意图;
图3为本发明实施例的455例患者生存曲线示意图;
图4为本发明实施例的141例患者ROC曲线示意图;
图5为本发明实施例的141例患者生存曲线示意图。
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。
术语“癌症”和“癌”是指或者描述哺乳动物中通常以异常或失控的细胞生长为特征的生理状态。癌症和癌症病理可以伴随着例如转移、干扰邻近细胞的正常机能、以异常水平释放细胞因子或其他分泌产物、抑制或加重炎性或免疫应答、瘤形成、癌前病变、恶性肿瘤、周围或远距离组织或器官如淋巴结的浸润等。尤其包括的是前列腺癌。
术语“预后”指对医学结果(medical outcome),例如不良或良好结果的预测(例如长期存活的可能性);消极的预后或不良结果包括复发、疾病进展(例如肿瘤生长或转移或耐药性)或死亡的预测。积极的预后或良好结果包括疾病好转(例如无疾状态)、改善(例如肿瘤消退)或稳定的预测。
请参阅图1至图5,本发明提供一种技术方案:一种用于癌症预后检测试剂盒,试剂盒包括可检测ZNF467、SH3RF2、PPFIA2、MYT1、TROAP、GOLGA7B表达水平的检测试剂;
检测试剂包括多核苷酸引物或探针;
癌症选自前列腺癌;
癌症的进展及预后的确定包括以下步骤:
(a)检测样本的ZNF467、SH3RF2、PPFIA2、MYT1、TROAP、GOLGA7B的mRNA表达水平;
(b)根据步骤(a)获得的表达水平数据评估患者的癌症复发风险;
步骤(b)中通过以下方法评估患者的癌症生化复发风险及总体预后:
风险值=(0.0122×ZNF467表达水平)-(0.0075×SH3RF2表达水平)+(0.0080×PPFIA2表达水平)+(0.0167×MYT1表达水平)+(0.0233×TROAP表达水平)+(0.0374×GOLGA7B表达水平)。
综上所述,该用于癌症预后检测试剂盒,
实施例1前列腺癌复发和/或存活后果或预后的评估模型的建立;
通过455例前列腺癌患者作为发现集,通过RNA-seq即转录组测序技术从其中提取了相应的基因表达数据,应用Lasso Cox回归模型分析预测这些患者生化复发密切相关的基因;最终确定和构建了以6个基因为基础的基因决策模型(见图1);并根据决策模型的风险,采用以下评分公式,得出风险值,将患者分为前列腺癌高危组和低危组;
风险值=(0.0122×ZNF467表达水平)-(0.0075×SH3RF2表达水平)+(0.0080×PPFIA2表达水平)+(0.0167×MYT1表达水平)+(0.0233×TROAP表达水平)+(0.0374×GOLGA7B表达水平);
决策基因mRNA表达值代入模型中计算出风险值,风险值大于1.6判定为前列腺癌高危组,风险值小于1.6判定为前列腺癌低危组;
将455例前列腺癌患者的风险值分别计算出,结合患者的临床资料,绘制ROC曲线,见图2,可见早期生化复发生存的AUC值为0.73;并根据risk score分为的前列腺癌高危组和低危组,绘制生存曲线,见图3,可见两组总体预后存在着显著差异,HR=0.28,95%CI:0.18–0.44;
实施例2用于前列腺癌预后的多基因试剂盒;
本实施例中用于前列腺癌预后的多基因试剂盒包括:总RNA提取试剂Trizol、氯仿(三氯甲烷)、异戊醇、无水乙醇、DEPC水(DD1005)、焦磷酸乙二酯(DEPC)、抗RNA酶液(RNaseZap)、反转录试剂盒、iQ SYBR Green Supermix、序列为SEQIDNO:1-10的多核苷酸引物;
利用试剂盒对各基因进行实时定量PCR(qRT-PCR)检测后,初始数据结果以Ct值表示,即每个反应体系内的荧光信号达到设定的阈值所需要的循环数;每一样品的Ct值与该样品起始拷贝数的对数存在线性关系,起始拷贝数越多,则Ct值越小;采用CT法计算各基因mRNA表达水平,并进行标准化处理;
根据上述步骤获得的各基因mRNA表达水平,代入以下前列腺癌生化复发预测模型计算前列腺癌生化复发的风险值,风险值大于1.6判定为前列腺癌生化复发高危组,风险值小于1.6判定为前列腺癌生化复发低危组;
风险值=(0.0122×ZNF467表达水平)-(0.0075×SH3RF2表达水平)+(0.0080×PPFIA2表达水平)+(0.0167×MYT1表达水平)+(0.0233×TROAP表达水平)+(0.0374×GOLGA7B表达水平);
实施例3样本RNA的制备和预处理;
(1)组织RNA提取实验用枪头、镊子在DEPC水中浸泡过夜后高温高压灭菌,预冷4℃离心机,实验台、移液枪、手套等用抗RNA酶液(RNase Zap)擦拭;
(2)组织匀浆取50~100mg组织样品,用无菌手术刀片稍切碎后置于1.5mLEP管中,加入500μLTrizol试剂,用电动组织研磨器充分匀浆,然后补充加入500μLTrizol试剂;
(3)每1mLTrizol试剂匀浆样品中加入0.2mL氯仿,盖紧EP管盖;剧烈振荡15秒,室温放置3min;4℃12000rpm离心15min(提前预冷离心机);离心后混合体系将分为上层的无色水相,中层的蛋白质和下层的红色酚氯仿相;DNA溶于氯仿分布于下层,RNA溶于水相分布于上层;水相的体积为匀浆时加入Trizol试剂体积的60%左右;
(4)RNA沉淀,将上层水相转移至新的EP管中;每1mLTrizol试剂匀浆样品中加入0.5mL异丙醇;混匀后室温放置10min,4℃下12,000rpm离心10min;离心后将在管壁底部和侧壁上可见白色沉淀物;
(5)RNA清洗,轻轻弃去上清,向每1mLTrizol试剂匀浆样品中加入1mL75%的乙醇,清洗RNA沉淀;振荡,4℃下10,000rpm离心5min;
(6)RNA溶解,轻弃乙醇溶液,空气中干燥RNA沉淀约5~10min;注意切勿完全干燥RNA沉淀,否则将大大降低RNA的可溶性;溶解RNA时,加入适量无RNase的DEPC水,用移液枪反复吹打,确保RNA充分溶解后,将RNA溶液保存于-80℃冰箱;
1)测量前先用溶解RNA用的DEPC水调零;
2)用移液枪吸取2μLRNA样品滴加至测量基座的表面;
3)轻轻合上基座后,液滴会自动在上下基座之间形成液柱,完成测定后电脑中即显示RNA溶液的各种参数包括RNA浓度和纯度等;A260/A280的比值是一种常用的评估RNA纯度的参数,一般认为其比值范围1.8~2.1表示RNA纯度较好;
4)一次检测完成后,用擦镜纸轻轻擦去上下基座表面液体,即可进行下一个样品的检测
ii)琼脂糖凝胶电泳;
1)制胶:称取1g琼脂糖加入100mL1×TAE buffer中,置于微波炉加热至沸腾,灌制凝胶板,待胶凝后取下梳子,将凝胶板放入电泳槽内,加入适量的1×TAE buffer至液面完全覆盖胶面;
2)准备RNA样品:取3μgRNA,加3倍体积的甲醛上样染液,再加EB于甲醛上样染液中至EB终浓度为10ug/mL,将体系加热至70℃孵育5min使样品变性;
3)电泳:上样后,5~6V/cm电压下电泳,至溴酚蓝指示剂进入胶内至少2~3cm;
4)紫外透射光下观察结果:经过变性RNA电泳后,在凝胶成像系统上可见28SrRNA、18SrRNA和5SrRNA三条带;观察到28SrRNA条带的强度约为18SrRNA的2倍,而且5SrRNA条带较弱,说明总RNA未发生明显降解;
实施例4决策基因mRNA检测;
(1)采用全式金公司的TransScript1stStrandcDNASynthesisSuperMix进行第一链cDNA的合成,反应体系的配制在冰上进行,反应体系如下:
(2)采用Bio-Rad公司的iQ SYBR Green Supermix进行实时定量PCR反应;一般引物终浓度为0.2μM可以得到较好的结果,反应性能不佳时,可在0.1~1.0μM范围内调整引物浓度,实时定量PCR试验引物序列如下:
(3)反应体系
试剂 | 使用量 |
模板(cDNA稀释20倍) | 2μL |
PCR前引物 | 0.4μL |
PCR后引物 | 0.4μL |
iQ SYBR Green Supermix | 10μL |
DdH2O | 7.2μL |
PCR引物分别为:
ZNF467的扩增引物为:SEQIDNO:1、SEQIDNO:2;
SH3RF2基因的扩增引物为:SEQIDNO:3、SEQIDNO:4;
PPFIA2基因的扩增引物为:SEQIDNO:5、SEQIDNO:6;
MYT1基因的扩增引物为:SEQIDNO:7、SEQIDNO:8;
TROAP基因的扩增引物为:SEQIDNO:9、SEQIDNO:10;
GOLGA7B基因的扩增引物为:SEQIDNO:11、SEQIDNO:12;
(4)反应程序
温度 | 时间 |
95℃ | 3min预变性 |
95℃ | 15s 45Cycles |
55℃ | 60s(收集荧光) |
55-90℃ | 每2~5秒上升0.5℃溶解曲线 |
(5)对各基因mRNA的表达进行定量;
实时定量PCR反应结束后对Real time PCR扩增曲线及熔解曲线进行数据分析处理,初始数据结果以Ct值表示,即每个反应体系内的荧光信号达到设定的阈值所需要的循环数;每一样品的Ct值与该样品起始拷贝数的对数存在线性关系,起始拷贝数越多,则Ct值越小;采用ΔΔCT法计算各基因mRNA表达水平;
实施例5前列腺癌预后预测模型与前列腺癌总体生存的相关性分析;
采用本发明的试剂盒进行预测,对141例验证集的前列腺癌患者进行了验证,通过上述实施例的方法,将验证人群风险值计算出并分为高危组和低危组,绘制ROC曲线及生化复发曲线,见图4和图5,结果显示在验证人群中本发明的方法对前列腺癌患者总体预后有很强的预测价值,早期生化复发的总体生存ACU值分别为0.72,HR=0.39,95%CI:0.25-0.61。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
Claims (6)
1.一种用于癌症预后检测试剂盒,其特征在于:所述试剂盒包括可检测ZNF467、SH3RF2、PPFIA2、MYT1、TROAP、GOLGA7B表达水平的检测试剂。
2.根据权利要求1所述的一种用于癌症预后检测试剂盒,其特征在于:所述检测试剂包括多核苷酸引物或探针。
3.根据权利要求2所述的一种用于癌症预后检测试剂盒,其特征在于:所述多核苷酸引物的序列如SEQIDNO:1-10所示。
4.根据权利要求1所述的一种用于癌症预后检测试剂盒,其特征在于:所述癌症选自前列腺癌。
5.根据权利要求1所述的一种用于癌症预后检测试剂盒,其特征在于:所述癌症的进展及预后的确定包括以下步骤:
(a)检测样本的ZNF467、SH3RF2、PPFIA2、MYT1、TROAP、GOLGA7B的mRNA表达水平;
(b)根据步骤(a)获得的表达水平数据评估患者的癌症复发风险。
6.根据权利要求5所述的一种用于癌症预后检测试剂盒,其特征在于:所述步骤(b)中通过以下方法评估患者的癌症生化复发风险及总体预后:
风险值=(0.0122×ZNF467表达水平)-(0.0075×SH3RF2表达水平)+(0.0080×PPFIA2表达水平)+(0.0167×MYT1表达水平)+(0.0233×TROAP表达水平)+(0.0374×GOLGA7B表达水平)。
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