CN115851942A - 诊断标志物gata4在胰腺“炎癌转化”中的应用 - Google Patents
诊断标志物gata4在胰腺“炎癌转化”中的应用 Download PDFInfo
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Abstract
本发明申请是202011531377.1的分案申请。本发明涉及诊断标志物GATA4在制备鉴别胰腺炎或胰腺癌炎症调节试剂或试剂盒中的应用，属于分子诊断技术领域。本发明通过在细胞中研究GATA4与“炎癌转化”的关系，分析GATA4的水平与CD68(巨噬细胞标记物)水平相关性，在胰腺癌组织中免疫荧光检测发现两者能共表达，从而提示GATA4可能参与胰腺癌发展过程中的炎症调节；用加入不同浓度LPS的诱导THP‑1分化的巨噬细胞培养基LMCM来诱导培养胰腺癌细胞，结果显示炎症能够促进胰腺癌细胞的增殖、迁移和侵袭。
Description
本申请是名为《诊断标志物GATA4在胰腺“炎癌转化”中的应用》的专利申请的分案申请，原申请的申请日为2020年12月22日，申请号为202011531377.1。
技术领域
本发明涉及分子诊断技术领域，具体地说，是诊断标志物GATA4在胰腺“炎癌转化”中的应用。
背景技术
胰腺癌(Pancreatic ductal adenocarcinoma,PDAC)恶性程度高，早期诊断难，现有治疗方法疗效差，因此其死亡率高，预后极差，其五年生存率仅有5％。虽然新的诊断和治疗方法的推出及应用使其它癌症的发病率和死亡率得到了控制，但是胰腺癌的发病率和死亡率却仍持续上升，2012年PDAC位列美国恶性肿瘤死亡率的第4位，而到2030年预测将会上升到第2位。深入研究胰腺癌发病的始动环节，从而早期诊断胰腺癌和/或采取措施预防胰腺癌的发生具有重要的科学意义和临床价值。
大量的研究表明，胰腺癌的发生与胰腺炎密切相关。流行病学调查显示急性或慢性胰腺炎患者相较正常人群其胰腺癌发病率增加5-6倍，而遗传性慢性胰腺炎患者则增加70倍。本课题组在临床实践中发现一个急性胰腺炎高发家族中有两人已确诊为胰腺癌。病理学研究显示胰腺炎和胰腺癌都起源于腺泡细胞，其中胰腺癌起源于腺泡细胞导管样化生(acinar-ductal metaplasiaADM)，这一病理变化在急性胰腺炎时短暂形成，慢性胰腺炎及胰腺癌时持续存在，胰腺炎症越重、持续时间越长，其病理结局越相似于胰腺癌，提示胰腺炎症可能促进胰腺癌的发生和发展。另外低级别胰腺上皮内瘤变(pancreaticintraepithelial neoplasiaPanIN)至高级别的PanIN进而最终变为胰腺癌均与慢性炎症信号密切关联。2016年TosteP.等人在Mol Cancer Research上发表文章指出在成年小鼠胰腺癌发病过程中需要KRAS基因突变及炎症过程同时参与。此外，尽管抗炎和抗氧化应激药物对胰腺癌的疗效结果不一，但仍有大量研究表明其应用对胰腺癌起着预防和/或治疗作用。如研究显示高剂量维生素C、E和硒的摄入能降低参与者患胰腺癌的风险。细胞及动物实验表明非甾体类抗炎药(non-steroidal anti-inflammatory drugs NSAIDs)如阿司匹林及抗炎药姜黄素在胰腺癌的预防和/或治疗中起作用。炎症除了与PDAC发生密切相关外，炎症信号通路也与PDAC的生长和转移密切相关。炎症因子如IL6和IL8能促进胰腺肿瘤细胞的迁移、侵袭和血管生成。
以上流行病学研究、动物模型及病人资料均提示胰腺炎与胰腺癌的发生发展存在密切的关系。研究胰腺“炎癌转化”的分子机制并进而预防和治疗胰腺癌具有重要的理论意义和临床应用价值。
关于本发明诊断标志物GATA4在胰腺“炎癌转化”中的应用目前还未见报道。
发明内容
本发明的目的是针对现有技术的不足，提供诊断标志物GATA4在胰腺“炎癌转化”中的应用。
为实现上述目的，本发明采取的技术方案是：
本发明先提供了GATA4作为胰腺“炎癌转化”诊断标志物在制备鉴别胰腺炎与胰腺癌的试剂或试剂盒中的应用。
本发明还提供了GATA4作为胰腺“炎癌转化”诊断标志物在制备鉴别胰腺炎与胰腺炎向胰腺癌转化的试剂或试剂盒中的应用。
本发明还提供了GATA4的抑制剂在制备防治胰腺“炎癌转化”的药物中的应用。
优选地，所述“炎癌转化”均是指胰腺炎向胰腺癌单方向转化。
优选地，所述GATA4的抑制剂是降低GATA4表达量的物质。
优选地，所述GATA4的抑制剂选自小分子化合物或生物大分子。
优选地，所述生物大分子是以GATA4蛋白或其转录本为靶序列、且能够抑制GATA4蛋白表达或基因转录的小干扰RNA、dsRNA、shRNA、微小RNA、反义核酸；或能表达或形成所述小干扰RNA、dsRNA、微小RNA、反义核酸的构建物。
本发明还提供了GATA4的抑制剂在制备防治胰腺癌细胞增殖、迁移、侵袭的药物中的应用。
本发明还提供了GATA4作为胰腺“炎癌转化”诊断标志物在制备胰腺癌预后风险判断的试剂或试剂盒中的应用。
本发明优点在于：
1、本发明研究证实GATA4的表达水平在胰腺“炎癌转化”过程中的关键性作用，首次发现GATA4可作为胰腺“炎癌转化”的标志物，深入研究胰腺癌发病的始动环节，从而早期诊断胰腺癌和/或采取措施预防胰腺癌的发生，具有优异的准确度、特异度和灵敏度，具有重要的科学意义和临床价值。
2、独特的资源优势：拥有胰腺炎及胰腺癌完整随访资料，初步建立了胰腺炎症及胰腺癌患者的血样本和组织样本库，目前共计200余例；并已熟练构建急慢性胰腺炎和原位胰腺癌动物模型。
3、多学科交叉研究模式：充分发挥临床肿瘤学、生物信息学及分子生物学的研究特点，并进行有机结合，是转化医学多学科交叉研究模式的创新。
4、系统的深入研究：在既往研究基础上，以转录因子GATA4为切入点，解析胰腺“炎癌转化”的炎症动态基因调控网络，深度挖掘新的关键节点并进行功能研究，在已建立的模式动物和临床样本库中验证，体现了研究的系统性，连续性和深入性。
5、研究手段的多维性：有机整合临床肿瘤学、分子生物学以及生物信息学方法对胰腺炎恶性转化机制从分子水平到整体层面以及动物模型、临床样本进行多维研究，所得出的结论将更加可信。
附图说明
附图1是临床标本组织实验流程图。
附图2是实施例2结果：表明GATA4在胰腺癌组织及细胞中高表达，A：RT-PC显示GATA4在胰腺癌组织中表达增加；B：Western-blot结果显示GATA4在胰腺癌组织中表达上调；C：免疫组化的结果显示GATA4在胰腺癌组织中表达上调；D：免疫荧光检测发现GATA4的水平与CD68水平正相关。
附图3实施例3结果，该实验用加入不同浓度LPS(0、50、100ng/ml)的诱导THP-1分化的巨噬细胞培养基LMCM来诱导培养胰腺癌细胞，结果表明巨噬细胞培养基促进胰腺癌细胞的增殖、迁移和侵袭，A:CCK-8实验证实巨噬细胞培养基促进胰腺癌细胞增殖；B、C:克隆形成实验证实巨噬细胞培养基促进胰腺癌细胞克隆形成；以及Transwell细胞迁移及侵袭能力实验证实巨噬细胞培养基促进胰腺癌细胞迁移及侵袭能力增强。
附图4是实施例3结果：表明GATA4在胰腺癌细胞中高表达且受肿瘤相关巨噬细胞的影响。
附图5是实施例3结果：表明干扰GATA4能够明显降低LMCM对胰腺癌细胞增殖活性的促进作用，A、B：CCK-8实验证实干扰GATA4能够明显降低LMCM对胰腺癌细胞BxPC-3及PANC-1的增殖活性，呈浓度依赖性；C-D:克隆形成实验证实干扰GATA4能够明显降低LMCM对胰腺癌细胞BxPC-3及PANC-1的克隆形成能力，呈浓度依赖性。
附图6和附图7是实施例3结果：其中，图6以及图7均表明干扰GATA4能够明显降低LMCM对胰腺癌细胞迁移和侵袭的促进作用，图6是Transwell细胞迁移及侵袭能力实验证实干扰GATA4能够明显降低LMCM对胰腺癌细胞BxPC-3的迁移及侵袭能力的促进作用，呈浓度依赖性；图7是Transwell细胞迁移及侵袭能力实验证实干扰GATA4能够明显降低LMCM对胰腺癌细胞PANC-1的迁移及侵袭能力的促进作用，呈浓度依赖性。
附图8是实施例3结果：表明过表达GATA4能够促进LMCM对胰腺癌细胞的增殖、迁移和侵袭能力，A：CCK-8实验证实过表达GATA4能够明显促进胰腺癌细胞ASPC-1的增殖活性，呈时间依赖性；B、C:克隆形成实验证实过表达GATA4能够促进LMCM对胰腺癌细胞ASPC-1的克隆形成能力促进作用，呈浓度依赖性；D-G:Transwell细胞迁移及侵袭能力实验证实过表达GATA4能够明显促进LMCM对胰腺癌细胞ASPC-1的迁移及侵袭能力的促进作用，呈浓度依赖性。
具体实施方式
下面结合具体实施方式，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明记载的内容之后，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。
实施例1
预实验
iTRAQ质谱分析筛选胰腺炎和胰腺癌组织样本的差异表达基因，提示GATA4在胰腺癌组织中高表达。
同位素标记相对和绝对定量iTRAQ技术是由美国应用生物系统公司(AppliedBiosystems Incorporation，ABI)开发的一种多肽体外标记技术。该技术采用了4种或8种同位素标签，通过标记肽段(Peptide)特异氨基酸位点，然后进行串联质谱分析，可灵活比较多种不同样本中蛋白质的相对含量和绝对含量。iTRAQ实验中，经过蛋白质提取获得组织细胞中的全蛋白，全蛋白在胰酶作用下被酶解(Trypsin Digestion)成肽段，iTRAQ试剂对所获得的全部肽段进行标记。iTRAQ试剂主要由3部分组成：报告基团(Reportgroup)、平衡基团(Balancegroup)和肽反应基团(Amine-specificreactive group)。以4标试剂为例：报告基团相对分子质量分别为114、115、116和117；平衡基团相对分子质量分别为31、30、29和28，肽反应基团将iTRAQ试剂与肽段上赖氨酸(Lysine，K)及N端氨基酸残基相连，在其上4种报告基团通过平衡基团与反应基团相连，这样就形成了4种相对分子质量均为145的等量异位标签。iTRAQ试剂在标记肽段样本后，报告基团和平衡基团的平衡分子量都为145，因此改变任一iTRAQ试剂，不同同位素标记的同一多肽在第一级质谱检测中分子量都完全相同。而在串联质谱(MSMS)中，同重元素标记肽段的报告基团、平衡基团和肽反应基团之间的连接键断裂，平衡基团中性丢失，同时不同同位素标签的同一肽段产生不同的质荷比(114-117)的峰。因此，根据波峰的高度和面积，可获得样品间相同肽段的定量信息，再经过软件处理得到肽段和蛋白质的定量信息。
实施例2
临床组织标本实验
本实施例在临床组织标本中研究GATA4与“炎癌转化”的关系。
1方法
取来自于上海市第一人民医院的7个胰腺癌旁，39个胰腺癌样本，分析GATA4在该临床样本中的表达：在临床胰腺癌样本中行RT-PCR、Western-blot及免疫组化的进一步验证GATA4表达情况，免疫荧光检测GATA4的水平与CD68水平；
2结果
利用RT-PCR技术分析7个胰腺癌旁，39个胰腺癌样本时，我们发现相对正常胰腺组织，GATA4 RNA水平表达上调(P<0.05)，Western-blot及免疫组化的结果也显示在7对配对的胰腺癌样本中GATA4在胰腺癌组织中表达上调。免疫荧光检测发现GATA4的水平与CD68(肿瘤相关巨噬细胞标志物)水平正相关，从而提示GATA4可能参与胰腺癌发展过程中的炎症调节(图2)。
实施例3
细胞实验
本实施例在细胞中研究GATA4与“炎癌转化”的关系。
(1)GATA4的水平与CD68(巨噬细胞标记物)水平相关性：在胰腺癌组织中免疫荧光检测发现两者能共表达，从而提示GATA4可能参与胰腺癌发展过程中的炎症调节；
(2)LMCM对胰腺癌细胞促进作用：在诱导THP-1分化的巨噬细胞培养基中加入不同浓度的脂多糖LPS(0、50、100ng/ml)，观察并记录结果。
(3)GATA4在胰腺癌细胞中表达情况：用RT-PCR及WB检测各种胰腺癌细胞株中GATA4表达情况，观察并记录结果。
(4)干扰GATA4观察不同LPS浓度的LMCM对胰腺癌细胞增殖、迁移和侵袭能力的影响。
(5)过表达GATA4观察不同LPS浓度的LMCM诱导下对胰腺癌细胞的增殖、迁移和侵袭能力的影响。
2结果
(1)用加入不同浓度LPS(0、50、100ng/ml)的诱导THP-1分化的巨噬细胞培养基LMCM来诱导培养胰腺癌细胞，结果显示炎症能够促进胰腺癌细胞的增殖、迁移和侵袭(图3)。
(2)RT-PCR及WB检测发现GATA4在不同胰腺癌细胞中也高表达，并且用加入不同浓度的LPS的LMCM诱导培养胰腺癌细胞能够促进GATA4水平上调，呈浓度依赖性(图4)。
(3)干扰GATA4能够明显削弱不同LPS浓度LMCM对胰腺癌细胞增殖活性以及克隆形成的促进，呈浓度依赖性(图5)，同样地，干扰GATA4也能够明显抑制不同LPS浓度的LMCM诱导下胰腺癌细胞的迁移和侵袭能力(图6、图7)。
(4)过表达GATA4能够明显促进不同LPS浓度的LMCM诱导下胰腺癌细胞的增殖、迁移和侵袭能力(图8)。
实施例4
动物模型实验
本实施例旨在通过动物模型研究GATA4对胰腺癌发生发展及“炎癌转化”过程的影响。
1方法
SCID小鼠原位胰腺癌模型的建立以研究GATA4对胰腺成瘤的影响：
1)构建SCID小胰腺原位癌模型：将上述GATA4的干扰组、过表达组及对照组的胰腺癌细胞(细胞用荧光素酶基因标记)原位注射入SCID小鼠胰腺内，建立胰腺癌原位模型，每组6只。每隔两周用Caliper IVIS成像系统检测荧光素酶的表达，用以观察各组成瘤情况；
2)标本制备：一定时间内处死SCID小鼠，取瘤块组织及静脉血，血液检测CA199浓度，测量瘤块重量及大小，并提取RNA及蛋白，部分组织福尔马林固定后石蜡包埋制片，观察并记录结果。
2结果
GATA4的干扰组、过表达组胰腺炎症能够明显促进胰腺癌细胞的增殖、迁移和侵袭。对照组则无此现象。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明原理的前提下，还可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。
Claims (2)
1.GATA4作为诊断标志物在制备鉴别胰腺炎或胰腺癌炎症调节试剂或试剂盒中的应用。
2.根据权利要求1所述的应用，其特征在于，所述胰腺炎包括慢性胰腺炎和急性胰腺炎。
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| US20130157272A1 (en) * | 2011-09-13 | 2013-06-20 | Alex Adai | Methods and compositions involving mir-135b for distinguishing pancreatic cancer from benign pancreatic disease |
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| YAN GONG 等: "GATA4 inhibits cell differentiation and proliferation in pancreatic cancer", PLOS ONE, vol. 13, no. 8, 24 August 2018 (2018-08-24), pages 1 - 2 * |
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