CN110018316B - Ddx20在制备肾透明细胞癌术后预后评估试剂盒中的应用 - Google Patents

Ddx20在制备肾透明细胞癌术后预后评估试剂盒中的应用 Download PDF

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CN110018316B
CN110018316B CN201910304731.8A CN201910304731A CN110018316B CN 110018316 B CN110018316 B CN 110018316B CN 201910304731 A CN201910304731 A CN 201910304731A CN 110018316 B CN110018316 B CN 110018316B
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林尧
王清水
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Abstract

本发明提供了DDX20在制备肾透明细胞癌术后预后评估试剂盒中的新应用。本发明人经过广泛而深入的研究，首次发现，采用免疫组化方法检测DDX20在肾透明细胞癌组织中的相对表达量，能够判断肾透明细胞癌患者出现肾透明细胞癌复发转移的风险。本发明的有益效果主要体现在：本发明提供了DDX20在制备肾透明细胞癌术后预后评估试剂盒中的应用，提示该蛋白能用于制备判断肾透明细胞癌患者预后的蛋白质分子标记，对于肾透明细胞癌病人术后监控和序贯治疗也具有重要的指导意义。

Description

DDX20在制备肾透明细胞癌术后预后评估试剂盒中的应用
技术领域
本发明涉及DDX20在制备肾透明细胞癌术后预后评估试剂盒中的应用。
背景技术
肾实质癌是来源于肾小管上皮细胞的腺癌，85％为透明细胞癌，还有一部分为颗粒细胞癌及混合细胞癌。癌中常有出血、坏死、囊变和钙化。生于肾实质内，长大后浸润、压迫、破坏肾盂肾盏，向肾包膜外发展，形成血管瘤栓或转移到淋巴结及其他脏器。
肾透明细胞癌的发生发展及预后与癌基因的激活和抑癌基因功能失活密切关联，是多因素诱导，多基因参与的复杂病理过程。近年来，肾透明细胞癌早期复发转移的分子机制是该领域的热门课题，深入阐明该分子机制，并在其特异性环节中导入靶向性治疗措施，有望很大幅度提高肾透明细胞癌术后总体治疗效果。因此，探寻切实有效的肾透明细胞癌肾透明细胞切除术后早期复发相关预警分子标志，阐明其与肾透明细胞癌早期复发转移的关系，这对提高肾透明细胞癌术后治疗效果、评估肾透明细胞癌早期复发风险、判断预后和个体化治疗有着至关重要的意义。
DDX20是ATP依赖的RNA解旋酶家族成员，参与RNA的各种代谢过程，如RNA二级结构变换、转录起始、线粒体RNA剪接、核糖体和剪接体装配、mRNA降解，以及维持mRNA的稳定性等。根据分布不同可参与胚胎发育、精子发生及细胞的生长和分裂等过程，但是该基因在肿瘤发生和转移方面的研究报道甚少。
本发明研究发现在肾透明细胞癌中的DDX20表达与病人术后预后存在显著的关系，暗示DDX20可作为肾透明细胞癌的术后预后的有效预警蛋白。
肾透明细胞癌做为对人类健康威胁最大的肿瘤之一，至今其发生的分子机制仍不清楚，对其的治疗也缺少特异性的分子靶点，而DDX20做为十分重要的肿瘤癌基因，目前尚无文献报道DDX20与判断肾透明细胞癌预后或肾透明细胞癌转移相关。
发明内容
本发明目的是提供DDX20在制备肾透明细胞癌术后预后评估试剂盒中的新应用。
本发明采用的技术方案是：
DDX20在制备肾透明细胞癌术后预后评估试剂盒中的应用。
本发明人经过广泛而深入的研究，首次发现，采用免疫组化方法检测DDX20在肾透明细胞癌组织中的相对表达量，能够判断肾透明细胞癌患者出现肾透明细胞癌复发转移的风险。基于DDX20表达量与肾透明细胞癌复发转移的相关性，以该蛋白作为预后标记物对其表达量进行检测可以用于指导肾透明细胞癌的预后判断，因此可将DDX20作为分子标记，利用DDX20单克隆抗体或多克隆抗体，结合免疫组化实验试剂，检测DDX20在肾透明细胞癌组织中的相对表达量。
所述试剂盒主要包括：人源DDX20单克隆抗体或多克隆抗体、免疫组化实验试剂。所述免疫组化实验试剂为本领域免疫组化实验中的常用试剂。
发明人在发现，DDX20在复发转移肾透明细胞癌组织中表达高于未复发转移肾透明细胞癌组织，可以推测DDX20在肾透明细胞癌术后复发转移发挥重要作用。查阅国内外文献，DDX20与肾透明细胞癌的发生以及复发转移的相关研究少，在本实验中，DDX20在肾透明细胞癌组织中的表达上调，而在癌旁和正常肾透明细胞组织中则表达下调，且与未复发转移组相比，DDX20在复发转移组中表达也上调，表明DDX20可能作为促进因子参与肾透明细胞癌发生发展过程。通过Kaplan-Meier生存曲线分析，DDX20表达程度与肾透明细胞癌患者的预后有关，DDX20高表达的患者预后不良(P<0.05)。
综上所述，DDX20在肾透明细胞癌组织中高表达，DDX20的高表达与肾透明细胞癌患者术后复发转移有关。DDX20可以作为肾透明细胞癌预后的一个重要候选分子标记物。
优选的，所述人源DDX20多克隆抗体由序列为SEQ ID NO.1所示的DDX20免疫兔子获得，可自行制备，也可采用市购商品。
具体的，所述免疫组化实验试剂包括：二甲苯、乙醇、3％H2O2(水溶液)、3％BSA封闭液(以PBS配制)、DAB显色试剂、苏木素、辣根过氧化物酶(用于标记二抗)、PBS(pH7.4)、0.01M EDTA修复液。
本发明所述试剂盒的使用方法如下：
(a)病理标本来自于肾透明细胞癌患者活检组织或术中、术后的病理取材。
(b)免疫组化方法利用SP染色法，具体步骤如下：
(c)制备肾透明细胞癌组织石蜡切片，60℃烤箱过夜。
(d)切片脱腊。依次浸泡：二甲苯I：10min；二甲苯II：10min；二甲苯III：10min。
(e)切片水化。依次浸泡：无水乙醇：3min；90％(v/v)乙醇：3min；80％乙醇：3min；75％乙醇：3min。
(f)PBS清洗3次，每次5min。
(h)EDTA抗原高压修复：切片放入0.01M EDTA修复液浸泡，沸水浴5min，冷却至室温。PBS清洗3次，每次5min。
(I)加入300μL的3％(w/w)过氧化氢水溶液，37℃10min。PBS清洗3次，每次5min。
(J)加入300μL的3％(w/w)BSA封闭液(PBS配制)，37℃1h。PBS清洗3次，每次5min。
(K)加入一抗：DDX20抗体浓度：1：500，4℃冰箱放置16h后取出，室温复温15min，然后PBS洗4次，每次5min。
(L)滴加二抗，所述的二抗为辣根过氧化物酶标记羊抗兔IgG(购自福州迈新试剂公司，即用型，无需稀释)，37℃45min。PBS洗4次，每次5min。
(M)PBS洗3次，每次5min。DAB(DAB显色试剂盒，购自上海生工)显色2-10min，镜下观察；双蒸水洗止显色，苏木素复染10s，用自来水冲洗浸泡。
(N)脱水。依次浸泡：75％乙醇:2min；80％乙醇：2min；90％乙醇：2min；无水乙醇：2min。
(O)用电吹风吹干，加入中性树胶，盖玻片覆盖。
(P)利用显微镜和成像装置随机选取肾透明细胞癌组织和癌旁组织3个视野拍摄，利用Aperio Image Scope软件对组织样本的相片进行扫描，扫描后采用该软件的Algorithms(Positive Pixel Count V9)程序对每个样本进行阳性强度计算，计算数据如下：
Figure BDA0002029431090000021
Figure BDA0002029431090000031
(Q)每个组织样本的免疫组织化学评分计算为Positivity×Log10[255/Iavg]，其中Positivity＝NPositive/NTotal，即阳性率，计算方法为阳性象素数量/显色总数量；Iavg＝(Iwp+Ip+Isp)/(Nwp+Np+Nsp)，即阳性平均强度，计算方法为阳性平均强度＝(弱阳性象素总强度+阳性象素总强度+强阳性象素总强度)/(弱阳性象素数量+阳性象素数量+强阳性象素数量)，即为该组织的免疫组化评分，用于后续分析。
(L)采用SPSS18.0进行统计分析，检验指标与临床资料之间的计数资料采用Pearson卡方检验，计量资料采用t检验。检测指标与临床预后的分析采用KaPlan-Meier生存分析，对数秩和检验(log-ranktest)比较生存曲线的差别。本发明显示DDX20与肾透明细胞癌的预后具有显著的相关性，为预测肾透明细胞癌的复发转移及术后的生存率提供一条全新途径，对肾透明细胞癌患者的预后有重要作用。当癌组织DDX20免疫组化评分高于0.452时，肾透明细胞癌易出现复发转移，肾透明细胞癌患者术后易死亡。
本发明的有益效果主要体现在：本发明提供了DDX20在制备肾透明细胞癌术后预后评估试剂盒中的应用，提示该蛋白能用于制备判断肾透明细胞癌患者预后的蛋白质分子标记，对于肾透明细胞癌病人术后监控和序贯治疗也具有重要的指导意义。
附图说明
图1为DDX20在肾透明细胞癌患者术后无复发转移的组织样本中表达情况；
图2为DDX20在肾透明细胞癌患者术后复发转移的组织样本中表达情况；
图3为肾透明细胞癌组织中DDX20低表达组与高表达组生存曲线；
具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：
实施例1：
(a)病理标本来自于肾透明细胞癌患者活检组织或术中、术后的病理取材。
(b)免疫组化方法利用SP染色法，具体步骤如下：
(c)制备肾透明细胞癌组织石蜡切片，60℃烤箱过夜。
(d)切片脱腊。依次浸泡：二甲苯I：10min；二甲苯II：10min；二甲苯III：10min。
(e)切片水化。依次浸泡：无水乙醇：3min；90％(v/v)乙醇：3min；80％乙醇：3min；75％乙醇：3min。
(f)PBS清洗3次，每次5min。
(h)EDTA抗原高压修复：切片放入0.01M EDTA修复液浸泡，沸水浴5min，冷却至室温。PBS清洗3次，每次5min。
(I)加入300μL的3％(w/w)过氧化氢水溶液，37℃10min。PBS清洗3次，每次5min。
(J)加入300μL的3％(w/w)BSA封闭液(PBS配制)，37℃1h。PBS清洗3次，每次5min。
(K)加入一抗：DDX20抗体浓度：1：500，4℃冰箱放置16h后取出，室温复温15min。PBS洗4次，每次5min。
(L)滴加二抗，所述的二抗为辣根过氧化物酶标记羊抗兔IgG(购自福州迈新试剂公司，即用型，无需稀释)，37℃45min。PBS洗4次，每次5min。
(M)PBS洗3次，每次5min。DAB(DAB显色试剂盒，购自上海生工)显色2-10min，镜下观察；双蒸水洗止显色，苏木素复染10s，用自来水冲洗浸泡。
(N)脱水。依次浸泡：75％乙醇：2min；80％乙醇：2min；90％乙醇：2min；无水乙醇：2min。
(O)用电吹风吹干，加入中性树胶，盖玻片覆盖。
(P)利用显微镜和成像装置随机选取肾透明细胞癌组织和癌旁组织3个视野拍摄，利用Aperio Image Scope软件对组织样本的照片进行扫描，扫描后采用该软件的Algorithms(Positive Pixel Count V9)程序对每个样本进行阳性强度计算，计算数据如下：
Figure BDA0002029431090000041
Figure BDA0002029431090000051
(Q)每个组织样本的免疫组织化学评分计算为Positivity×Log10[255/Iavg]，其中Positivity＝NPositive/NTotal，即阳性率，计算方法为阳性象素数量/显色总数量；Iavg＝(Iwp+Ip+Isp)/(Nwp+Np+Nsp)，即阳性平均强度，计算方法为阳性平均强度＝(弱阳性象素总强度+阳性象素总强度+强阳性象素总强度)/(弱阳性象素数量+阳性象素数量+强阳性象素数量)，即为该组织的免疫组化评分，用于后续分析。DDX20高低表达标准以40例肾透明细胞癌组织中DDX20表达评分的中位数(0.452)为界。
(L)采用SPSS18.0进行统计分析，检验指标与临床资料之间的计数资料采用Pearson卡方检验，计量资料采用t检验。检测指标与临床预后的分析采用KaPlan-Meier生存分析，对数秩和检验(log-ranktest)比较生存曲线的差别。
按照上述方法，本发明在40例肾透明细胞癌病人的肿瘤组织中检测结果如图1-2所示：DDX20无复发转移组中的表达(图1)低于复发转移组(图2)。
DDX20与肾透明细胞癌患者预后的关系：
通过Kaplan-Meier生存曲线分析，DDX20的表达程度与肾透明细胞癌患者的预后相关(图3)。
实施例2：
取某肾透明细胞癌术后肿瘤样本进行石蜡包埋切片，并利用以上所述的免疫组织化学方法进行检测，经计算，其癌组织的DDX20免疫组化组织评分为0.632。经过术后随访发现，该患者在术后第40个月发生肾透明细胞癌复发转移，术后48个月死亡。
实施例3：
取某肾透明细胞癌术后肿瘤样本进行石蜡包埋切片，并利用以上所述的免疫组织化学方法进行检测，经计算，其癌组织的DDX20免疫组化组织评分为0.299。经过术后随访发现，该患者在术后5年均未发现转移复发，健在。
由以上试验结果可知，通过采用免疫组化的方法检测DDX20分子相对表达量能预测肾透明细胞癌远处转移风险以及患者术后的生存或死亡。当癌组织DDX20的免疫组化评分高于0.452时，肾透明细胞癌易出现复发转移，肾透明细胞癌患者术后易死亡。显然DDX20与肾透明细胞癌具有相关性，因此，以DDX20作为蛋白质分子标记对其表达量进行检测能预测肾透明细胞癌手术后复发转移等事件，并判断预后。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等同物界定。
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Claims (2)

1.DDX20在制备肾透明细胞癌术后预后评估试剂盒中的应用，其特征在于：以DDX20作为分子标记，利用人源DDX20单克隆抗体或人源DDX20多克隆抗体，结合免疫组化实验试剂，检测DDX20在肾透明细胞癌组织中的相对表达量。
2.根据权利要求1所述的DDX20在制备肾透明细胞癌术后预后评估试剂盒中的应用，其特征在于：所述人源DDX20多克隆抗体由序列为SEQ ID NO.1所示的DDX20免疫兔子获得。
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