CN113025715A - Hop在预测胃癌预后中的应用 - Google Patents
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Abstract
本发明公开了HOP在预测胃癌预后中的应用，属于生物医学领域。具体地，本发明公开了HOP表达水平标记试剂在制备用于诊断胃癌或预测胃癌预后的试剂盒中的应用；HOP中和抗体在制备用于辅助胃癌化疗的药物中的应用以及HOP中和抗体在制备用于治疗胃癌的药物中的应用。本发明为胃部的诊断和预后提供了十分有效的标记物，并提供了新的用于治疗胃癌的药物，具有十分重要的临床价值和意义。
Description
技术领域
本发明属于生物医学领域，具体地，涉及HOP在预测胃癌预后中的应用。
背景技术
胃癌是常见的消化系统恶性肿瘤，高居我国各类癌症发病率和死亡率的第二位。肿瘤侵袭和迁移是其最主要的致死原因之一。
胃癌在临床上存在多个亟待解决的难点：（1）胃癌没有明显的早期症状和有效的诊断标志物，早期诊断困难；（2）大部分胃癌患者确诊时已有局部侵袭和转移，失去手术机会；（3）晚期胃癌对放疗、化疗均不敏感，现有的分子靶向治疗药物作用有限。
因此，阐明胃癌侵袭迁移的分子机制，寻找有效的预后诊断指标和干预靶点，对提高胃癌患者的生存期具有重大意义。而现有预测胃癌预后的生物标记物，限制了胃癌患者预后的提高。
发明内容
为了解决上述技术问题中的至少一个，发明人意外地发现，HOP在胃癌组织中表达较癌旁组织升高，而敲除HOP后可影响胃癌细胞株的侵袭迁移能力，同时发明人发现HOP中和抗体能增敏化疗药物5-FU的作用。最后，发明人发现HOP的高水平的表达与患者的预后较差成正相关，由此，完成本发明。
本发明的第一方面提供HOP表达水平标记试剂在制备用于诊断胃癌或预测胃癌预后的试剂盒中的应用。
HSP70/HSP90组织蛋白（HSP70/HSP90-Organing Protein，HOP），又称应激诱导蛋白，作为辅助伴侣分子或支架蛋白，调节热休克蛋白7（HSP70）和HSP90的连接和二聚化，并通过改变二聚体结构来调节HSP的功能，其氨基酸序列含有9个三角形四肽重复序列（Tetratrico peptide Repeat，TPR）。这些序列折叠成三个高度保守的TPR结构域：N端的TPR1，中段的TPR2A和C端的TPR2B，TPR结构域是HOP与其他蛋白结合执行功能的主要位点。除了作为接头蛋白外，HOP还通过调节几种蛋白复合物的功能，参与基因转录、信号转导和细胞分裂而具有独立的生物活性。HOP缺乏跨膜结构域或信号肽，HOP是一种细胞质蛋白。新的研究发现，HOP可以从细胞中分泌出来，并作为一种细胞因子，具有影响细胞增殖和凋亡的能力。
在本发明的一些实施方案中，所述HOP表达水平标记试剂为HOP抗体。在本发明的一些具体实施方案中，利用Western-Bloting方法检测HOP的表达水平；在本发明的另一些具体实施方案中，利用ELISA方法检测HOP的表达水平。
在本发明中，HOP抗体可以为任意商业化的或用于研究的能够有效结合的HOP抗体，也可以是通过抗体制备方法制备得到的HOP抗体。
在本发明的另一些实施方案中，所述HOP表达水平标记试剂为HOP转录本检测试剂。在本发明的一些具体实施方案中，所述HOP转录本为HOP基因mRNA。进一步地，所述HOP转录本检测试剂为引物。在本发明的一些优选实施方案中，利用qRT-PCR方法检测HOP转录本水平。
在本发明中，所述引物可以是任何商业化的或用于研究的能够有效扩增HOP转录本的引物，也可以是根据HOP基因或其转录本设计的任意引物。
本发明的第二方面提供HOP中和抗体在制备用于辅助胃癌化疗的药物中的应用。
在本发明的一些实施方案中，所述化疗是指5-FU化疗。
本发明的第三方面提供HOP中和抗体在制备用于治疗胃癌的药物中的应用。
本发明的第四方面提供一种用于辅助胃癌化疗或者治疗胃癌的药物，其特征在于，包括HOP中和抗体。
在本发明的一些实施方案中，所述化疗是指5-FU化疗。
本发明的有益效果
本发明相对于现有技术，具有以下有益效果：
本发明提供了一种新的诊断胃癌或预测胃癌预后的生物标记物——HOP。通过标记试剂检测HOP的表达水平，可以诊断胃癌并能预测胃部预后。
本发明还提供了一种新的治疗胃癌或辅助胃癌化疗的药物或用途，为胃癌治疗提供了技术支持，具有重要的临床价值和意义。
附图说明
图1示出了HOP在胃癌组织及血清中的表达情况。A.分别采用Western blot（上图和左下图）和qRT-PCR方法（右下图）检测HOP在胃癌组织和配对癌旁正常组织中的表达；B.分析美国癌症和肿瘤基因图谱（The Cancer Genome Altas, TCGA）数据库中非配对（左图）和配对（右图）胃癌和癌旁组织中HOP的表达；C.采用ELISA方法检测10对年龄匹配健康人和胃癌患者外周血中HOP蛋白水平；D.采用ELISA法检测5位胃癌患者术前和术后第7天外周血中HOP蛋白的表达。
图2示出了敲低HOP显著抑制胃癌细胞AGS的侵袭、迁移能力的结果。
图3示出了外源性HOP信号能够促进胃癌细胞系增殖。a和c：不同浓度rhHOP对AGS细胞增殖的影响；b和d：不同浓度rhHOP对SCC-7901细胞增殖的影响；e：rhHOP对AGS和SCC-7901细胞增殖的影响。
图4示出了rhHOP蛋白以及HOP-N对胃癌细胞5-FU化疗敏感性影响的结果。
具体实施方式
为了使本发明所解决的技术问题、技术方案及有益效果更加清楚明白，以下结合实施例，对本发明进行进一步详细说明。
实施例
以下例子在此用于示范本发明的优选实施方案。本领域内的技术人员会明白，下述例子中披露的技术代表发明人发现的可以用于实施本发明的技术，因此可以视为实施本发明的优选方案。但是本领域内的技术人员根据本说明书应该明白，这里所公开的特定实施例可以做很多修改，仍然能得到相同的或者类似的结果，而非背离本发明的精神或范围。
除非另有定义，所有在此使用的技术和科学的术语，和本发明所属领域内的技术人员所通常理解的意思相同，在此公开引用及他们引用的材料都将以引用的方式被并入。
那些本领域内的技术人员将意识到或者通过常规试验就能了解许多这里所描述的发明的特定实施方案的许多等同技术。这些等同将被包含在权利要求书中。
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的仪器设备，如无特殊说明，均为实验室常规仪器设备；下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。
实施例 1 检测HOP在胃癌组织的表达情况
分别在癌旁组织及胃癌组织中应用Western-Bloting方法检测HOP的表达水平。具体过程如下：
①收集患者样本
受试者的伦理批准来自中山大学附属第一医院机构审查委员会(FAHSYSU；中国广州)，并获得每个患者的知情同意书。标本的新鲜配对样本来自于2014年在法合州立大学手术切除前未接受放疗或化疗而接受胃切除手术的原发性胃癌患者(n=39)。所有切除的组织立即冷冻在液氮中，并保存在-80℃下。血清样本采集自2015年FAHSYSU手术切除前未接受过放疗或化疗的胃癌患者(n=10)。这些血清样本是在手术前采集的，手术病理检查证实了胃癌的诊断。从10例胃癌患者中采集5例术前或术后血清标本。胃癌石蜡包埋组织取自病理科。简而言之，收集了2004年至2005年间在上海协和大学接受手术治疗的117名胃癌患者的样本，并对其进行了验证。
②Western-Bloting
用细胞裂解缓冲液(KeyGene，中国南京)提取总蛋白，用增强型BCA蛋白分析试剂盒(KeyGene)测定蛋白浓度。蛋白质用8-10%十二烷基硫酸钠聚丙烯酰胺凝胶电泳分离，电转移到聚偏氟乙烯膜(美国马萨诸塞州，比勒里卡，米利孔)。用5%牛血清白蛋白(TBS-T)封闭膜1h，在4°C用相应的一抗探查过夜，再与兔、小鼠过氧化物偶联二抗孵育1h，用增强型化学发光试剂(微孔)在放射自显影胶片上检测特异性条带。
结果如图1所示：与癌旁组织相比，HOP在胃癌组织中的表达升高；分别检测胃癌患者与健康对照者血清HOP水平，与健康对照者相比，胃癌患者血清中HOP的水平明显升高，而胃癌切除术后，患者血清的HOP水平明显下降。
实施例2 HOP对胃癌细胞AGS的侵袭、迁移能力的影响
发明人进一步研究HOP对胃癌细胞迁移、侵袭、细胞形态和EMT的影响。
①迁移（Migration）和侵袭（Invasion）
所有细胞培养试剂和细胞培养池放在37℃温育，待测细胞培养至对数生长期，消化细胞，用PBS和无血清培养基先后洗涤1次，用无血清培养基悬浮细胞，计数，调整浓度为5×105/mL；如进行侵袭（Invasion）实验，将Matrigel胶从-20℃取出于4℃冰箱过夜，在4℃条件下将Matrigel胶用冰冷的无血清的细胞培养基稀释至300μL/mL，取100μL均匀涂抹一层于细胞培养池的PET膜上表面，然后将培养池轻轻放入24孔板孔内，37℃放置3h左右，取出于超净工作台过夜干燥。如进行迁移（Migration）实验，则跳过这一步骤。在下室（即24孔板底部)加入600~800μL含20％血清的培养基，上室加入200μl细胞悬液，继续在孵箱培养24h；将其下表面浸泡在4%多聚甲醛通用组织固定液中，固定15~30min，用0.5%结晶紫染色，镜检，并计算PET膜下表面的细胞数，计算中间和四周5个视野，取平均值。镜下对染色后的细胞进行计数，计数值高，则该组细胞迁移/侵袭能力强。
结果显示（图2）：敲低HOP的表达显著抑制胃癌细胞AGS的侵袭、迁移能力；提示HOP具有促进胃癌细胞侵袭迁移的能力。
②细胞增殖实验
采用细胞计数试剂盒(CCK-8)检测胃癌细胞株（AGS和SGC-790）增殖情况。取生长状态良好的对数生长期细胞接种于96孔细胞培养板(KeyGene)上，密度为2×10~3个/孔，培养液为100μL，在有或无HOPNA和rhHOP的条件下经CM处理48h后，每孔培养板中加入10μLCCK-8试剂，37°C孵育2h。以IgG作为对照（Control）。用酶标仪测波长为450 nm的吸光度（OD）值。实验重复3次，取实验结果的平均值作为最终实验结果。按公式计算生长抑制率＝[(对照组OD－实验组OD)/对照组OD]×100%，以分组为横坐标，生长抑制率为纵坐标，绘制细胞生长抑制率柱状图。
结果显示（图3）：外源性HOP信号能够促进胃癌细胞系增殖。
实施例3 重组人HOP蛋白以及HOP-中和抗体对胃癌细胞5-FU化疗敏感性影响的体外实验
（1）构建胃癌PDX小鼠模型：
①签署患者知情同意书，收集刚离体胃癌组织标本。
②麻醉裸鼠，皮下种植胃癌组织。2-3月可触及皮下瘤（P1代）。成瘤后进行传代，在P3代时进行药物筛选。
③对组织切片进行染色：H&E染色观察肿瘤细胞，Ki67、TUNEL及CD31染色分别检测肿瘤增殖、凋亡和血管生成等情况。
④分别提取人体胃癌组织和皮下瘤组织的RNA，进行转录组测序，比对两者基因表达谱的情况。
（2）探索HOP中和抗体对化疗药5-FU的增敏作用：
HOP中和抗体(HOP-NA)购自Abnova(中国台湾)。
①选取P3代的PDX小鼠（皮下肿瘤体积达到350mm3），随机分到空白对照组、HOP中和抗体组、5-FU组以及联合处理组，分别腹腔注射生理盐水、HOP中和抗体以及5-FU。
②定期观察、测量皮下肿瘤的大小，观察不同处理对皮下瘤的生长影响。
③当任何一只小鼠皮下肿瘤体积达到700mm3时终止实验，完整分离肿瘤并称重；比较不同处理组成瘤的情况，检测增殖、凋亡和血管生成等指标的差异。
结果如图4所示，采用重组人HOP蛋白（rhHOP）刺激胃癌细胞AGS，显著增加细胞活力；HOP中和抗体（HOP-NA）显著增强传统化疗药5-氟尿嘧啶（5-FU）对胃癌细胞的杀伤作用。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
Claims (10)
1.HOP表达水平标记试剂在制备用于诊断胃癌或预测胃癌预后的试剂盒中的应用。
2.根据权利要求1所述的应用，其特征在于，所述HOP表达水平标记试剂为HOP抗体。
3.根据权利要求1所述的应用，其特征在于，所述HOP表达水平标记试剂为HOP转录本检测试剂。
4.根据权利要求3所述的应用，其特征在于，所述HOP转录本为HOP基因mRNA。
5.根据权利要求4所述的应用，其特征在于，所述HOP转录本检测试剂为引物。
6.HOP中和抗体在制备用于辅助胃癌化疗的药物中的应用。
7.根据权利要求6所述的应用，其特征在于，所述化疗是指5-FU化疗。
8.HOP中和抗体在制备用于治疗胃癌的药物中的应用。
9.一种用于辅助胃癌化疗或者治疗胃癌的药物，其特征在于，包括HOP中和抗体。
10.根据权利要求9所述的药物，其特征在于，所述化疗是指5-FU化疗。
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