CN114032309A - Mdk基因或mdk蛋白作为生物标志物在肺癌诊断中的应用 - Google Patents
Mdk基因或mdk蛋白作为生物标志物在肺癌诊断中的应用 Download PDFInfo
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Abstract
本发明公开了一种MDK基因或MDK蛋白作为生物标志物在肺癌诊断中的应用。本发明通过荧光定量PCR方法和免疫组织化学方法检测MDK在肺癌组织中的mRNA和蛋白表达，结果显示无论是mRNA水平还是蛋白水平，MDK在肺癌中的表达都是异常的，MDK在肺癌患者组织中的表达量显著高于癌旁组织。本发明提示MDK基因或MDK蛋白可以作为一种特异性的生物标志物在肺癌的辅助诊断中发挥重要作用。
Description
技术领域
本发明属于生物技术领域，具体涉及MDK基因或MDK蛋白作为生物标志物在肺癌诊断中的应用。
背景技术
肺癌作为一类高发病率及高死亡率的恶性肿瘤，因其严重威胁了人类的生命并增加了社会的负担，已成为威胁中国及世界的严重公共卫生问题。根据我国2008年临床数据调查结果显示，在所有的肿瘤中，肺癌的发病率和死亡率均处于首位，其发病率为33.5/100000，死亡率高达28.7/1000000，且在未来20年将呈逐年上升趋势。目前，肺癌的治疗主要采用多学科相结合的治疗策略，包括手术治疗，新辅助放、化疗，分子靶向治疗及细胞生物治疗，但因为肺癌起病隐匿，约有70％的肺癌患者确诊时即为晚期，常失去手术机会，其5年生存率仅为15％。因此，开发一种可以在肺癌诊断和治疗中具有应用价值的分子标志物，可以在临床治疗中及早发现肿瘤细胞、有效减轻患者的肿瘤负荷、缓解患者症状、改善患者生活质量以及延长患者的生命。
中期因子(Midkne，MDK)是1988年由日本学者Kadomasu等发现的一种新型肝素结合生长因子(HBGF)，与多效生长因子(Pleiotrophin，PTN)共同构成新型肝素蛋白家族。MDK因最初被发现在妊娠中期小鼠胚胎肾脏组织中表达广泛而得名，但出生后表达水平则明显降低。近年来的研究发现MDK具有诱导细胞恶性转化、抗细胞凋亡、促血管生成等促进肿瘤的生长和演进的作用，其在肿瘤领域越来越引起研究者们的关注。目前较多研究证明，MDK在肝细胞癌、肺癌、膀胱癌、前列腺癌、口腔癌、乳腺癌及神经母细胞瘤等多种肿瘤组织中均呈高表达。MDK无论在肺癌早期还是晚期都呈高表达，与肺癌发生、进展及预后密切相关，有可能成为一种新型的肺癌诊断标志物及提示肺癌细胞侵袭的标志物，并有可能成为肺癌治疗的新的分子靶点。
本发明旨在利用MDK基因或MDK蛋白作为生物标志物可以用于肺癌患者的诊断，并以MDK为靶点设计治疗肺癌的新的靶向药物。
发明内容
本发明所要解决的技术问题是提供一种可用于肺癌诊断的MDK基因或 MDK蛋白生物标志物，MDK基因或MDK蛋白生物标志物与肺癌发病率呈正相关，在肺癌组织中呈高表达，其特异性高，针对性强。
本发明解决上述技术问题所采用的技术方案为：一种用于肺癌诊断的 MDK基因或MDK蛋白生物标志物，所述MDK基因或MDK蛋白生物可用于检测肿瘤患者组织中的MDK的基因或蛋白表达。
一种可用于肺癌诊断的MDK基因或MDK蛋白生物标志物的应用，该肺癌诊断的MDK基因或MDK蛋白生物标志物可用于肺癌辅助诊断或治疗指导。
进一步地，
(1)肺癌患者癌组织与癌旁组织MDK基因表达的检测；评估其作为肺癌诊断候选生物标志物，用于肺癌检测的可能性；
(2)肺癌患者癌组织与癌旁组织MDK蛋白表达的检测；评估其作为肺癌诊断候选生物标志物，用于肺癌检测的可能性；
(3)验证MDK基因或MDK蛋白与肺癌患者临床特征的相关性。
本发明的优点在于：本发明首次公开了可用于肺癌诊断的MDK基因或 MDK蛋白生物标志物及其应用，MDK基因或MDK蛋白生物标志物在肺癌组织中呈高表达，与肺癌的发生呈正相关，并且参与调控肺癌的进展和预后，是潜在的肺癌诊断的生物标志物。MDK基因或MDK蛋白生物标志物的开发，有望成为肺癌辅助诊断和靶向治疗的一种新手段。
附图说明
图1为MDK基因在肺癌组织及癌旁组织中表达水平。
图2为MDK基因在肺癌组织中表达的ROC曲线。
图3为免疫组织化学法检测MDK蛋白在肺癌和癌旁组织中的表达。
图4为MDK蛋白在肺癌组织及癌旁组织中表达水平。
图5为MDK蛋白在肺癌组织中表达的ROC曲线。
具体实施方式
以下结合附图实施例对本发明作进一步详细描述。
实施例1：
荧光定量PCR法检测MDK基因在肺癌组织及癌旁组织中表达水平
(1)样本收集
65例原发肺癌患者的手术切除肺癌组织和癌旁组织来源于本院病理科。所有癌组织均术后病理证实为肺癌，全部肺癌患者术前均未行放、化疗，全部病例临床资料完整。
(2)组织RNA的提取
用Trizol法提取各组织中的RNA，紫外分光光度计对所提RNA进行浓度和纯度测定，要求OD260/280在1.8～2.2之间。
(3)荧光定量PCR检测MDK基因的表达
RNA的逆转录采用TAKARA公司的PrimeScriptTM RT Master Mix(Perfect RealTime)试剂盒，所有步骤按照说明书要求进行操作。荧光定量PCR采用 TAKARA公司的TB
Premix Ex TaqTM II(Tli RNaseH Plus)试剂盒，所有步骤按照说明书要求进行操作。PCR反应结束后，读取Ct值并根据MDK和内参基因的表达量计算出相对表达量，结果显示，MDK基因在癌组织中的表达量显著高于癌旁组织(结果见图1)。
(4)MDK基因在肺癌中表达的ROC曲线绘制
根据肺癌患者癌组织和癌旁组织中，MDK基因的表达量，使用SPSS软件计算MDK基因在肺癌中表达的ROC曲线，结果MDK基因在肺癌患者组织中的AUC为0.750(置信区间0.664-0.836)，敏感性为66.2％，特异性为80.0％(结果见图2)。
(5)肺癌组织中MDK基因表达与临床特征的相关性分析
根据MDK基因在肺癌组织中的表达情况，结合其患者的临床资料，对肺癌组织中MDK基因的表达与患者临床特征进行相关性分析。分析采用SPSS21.0 软件进行操作，采用秩和检验法对各组进行统计学分析，P值小于0.05为数据有统计学意义，结果显示，MDK基因的表达与肺癌患者的临床分期具有显著的相关性，MDK基因的表达量越高其临床分期越高，即MDK基因的表达越高提示肺癌的恶性程度越高(结果见附表1)。
表1肺癌组织中MDK基因表达与临床特征的相关性分析
实施例2：
免疫组织化学法检测MDK蛋白在肺癌组织及癌旁组织中表达水平
(1)样本收集
85例原发肺癌患者的手术切除癌组织和癌旁组织来源于上海芯超生物科技有限公司上海国家生物芯片工程中心生物库，组织芯片号为HLugA180Su011。所有癌组织均术后病理证实为肺癌，全部肺癌患者术前均未行放、化疗，全部病例临床资料完整。
(2)组织芯片制备
组织芯片是制作直径为0.6mm的孔以保存HCC组织。在石蜡块上选择肿瘤及其邻近组织，从排列的石蜡块上切下0.66μm的连续切片，并放入玻片中。最后，通过HE染色对组织芯片进行验证。
(3)免疫组织化学检测MDK蛋白的表达
将组织芯片从冰箱中取出并放置在玻片中重新加温，在65℃下加热组织芯片约1小时，以融化密封蜡。将载玻片在二甲苯中浸泡两次10分钟，然后将其放置在无水乙醇中8分钟，再放置95％乙醇5分钟，最后将载玻片在70％乙醇中浸泡10分钟。在室温下，在柠檬酸盐缓冲液(pH 6.0)中进行抗原修复30分钟，以增加免疫反应性。载玻片与兔单克隆抗体[EP1143Y]to Midkine(Abcam 公司)孵育，然后与HRP标记的抗兔的二抗抗体进行孵育。所有染色组织由专用病例组织扫描仪扫面后进行分析。MDK染色强度评分为1(弱)，2(中)，3(强)，MDK阳性的百分比分数为5～100％。分析结果显示，MDK蛋白在肺癌组织中的表达显著高于癌旁组织(结果见图3和图4)。
(4)MDK蛋白在肺癌中表达的ROC曲线绘制
根据肺癌患者癌组织和癌旁组织中，MDK蛋白的表达量，使用SPSS软件计算MDK蛋白在肺癌中表达的ROC曲线，结果MDK蛋白在肺癌患者组织中的AUC为0.878(置信区间0.825-0.93)，敏感性为96.7％，特异性为72.2％(结果见附图5)。
(5)肺癌组织中MDK蛋白表达与临床特征的相关性分析
根据MDK蛋白在肺癌组织中的表达情况，结合其患者的临床资料，对肺癌组织中MDK蛋白的表达与患者临床特征进行相关性分析。分析采用SPSS21.0 软件进行操做，采用秩和检验法对各组进行统计学分析，P值小于0.05为数据有统计学意义。数据分析结果显示，MDK蛋白的表达与肺癌患者的TNM分期和生存显著相关，即MDK蛋白的表达量越高其患者的TNM分期越高，恶性程度越高，并且其预后也越差(结果见附表2)。
表2肺癌组织中MDK蛋白表达与临床特征的相关性分析
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 (5)
1.一种MDK基因或MDK蛋白作为生物标志物在肺癌诊断中的应用；所述MDK蛋白由MDK基因编码。
2.如权利要求1所述的肺癌生物标志物MDK基因的荧光定量PCR引物，其特征为：所述MDK基因的荧光定量PCR的引物序列如下：
正向引物：5’-GAAGGAGTTTGGAGCCGACT-3’；
反向引物：5’-TTCCCTTTCTTGGCTTTGG-3’。
3.如权利要求1所述的肺癌生物标志物MDK蛋白的特异性抗体，其特征为：所述MDK蛋白的抗体产品为：兔单克隆抗体[EP1143Y]to Midkine。
4.如权利要求2所述的肺癌生物标志物MDK基因的荧光定量PCR引物，其特征为：所述的检测MDK基因的荧光定量PCR产品包括逆转录试剂盒和荧光定量PCR试剂盒。
5.如权利要求3所述的肺癌生物标志物MDK蛋白的特异性抗体，其特征为：所述的检测MDK蛋白的产品包括免疫组织化学相关试剂产品。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011093675A2 (ko) * | 2010-01-28 | 2011-08-04 | (주) 더바이오 | 혈액으로부터 직접 폐암 진단 및 폐암의 서브타입 진단이 가능한 마커를 선별하는 시스템 생물학적 방법 및 이로부터 선별된 혈액으로부터 직접 폐암 진단 및 폐암 서브타입 진단이 가능한 마커 |
| US20120225954A1 (en) * | 2010-09-05 | 2012-09-06 | University Health Network | Methods and compositions for the classification of non-small cell lung carcinoma |
| CN109234391A (zh) * | 2018-09-13 | 2019-01-18 | 昆明医科大学第附属医院 | 作为肺腺癌生物标志物的anxa3基因或anxa3蛋白 |
-
2021
- 2021-11-24 CN CN202111406297.8A patent/CN114032309A/zh active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011093675A2 (ko) * | 2010-01-28 | 2011-08-04 | (주) 더바이오 | 혈액으로부터 직접 폐암 진단 및 폐암의 서브타입 진단이 가능한 마커를 선별하는 시스템 생물학적 방법 및 이로부터 선별된 혈액으로부터 직접 폐암 진단 및 폐암 서브타입 진단이 가능한 마커 |
| US20120225954A1 (en) * | 2010-09-05 | 2012-09-06 | University Health Network | Methods and compositions for the classification of non-small cell lung carcinoma |
| CN109234391A (zh) * | 2018-09-13 | 2019-01-18 | 昆明医科大学第附属医院 | 作为肺腺癌生物标志物的anxa3基因或anxa3蛋白 |
Non-Patent Citations (5)
| Title |
|---|
| KEHUA YUAN等: "MDK Protein Overexpression Correlates with the Malignant Status andPrognosis of Non-small Cell Lung Cancer", vol. 46, no. 8, pages 635 - 641, XP029394166, DOI: 10.1016/j.arcmed.2015.11.006 * |
| 李龙: "早期肝细胞癌患者肝切除术后血清miR-375、miR-122、MDK、AFP和Ku86抗体水平的分析", vol. 47, no. 11, pages 1399 - 1402 * |
| 潘正洲: "中期因子（Midkine，MDK）对甲状腺癌增殖和迁移的功能影响", 《中国优秀硕士论文全文数据库 医药卫生科技辑》, pages 3 * |
| 潘正洲: "中期因子（Midkine,MDK）对甲状腺癌增殖和迁移的功能影响", no. 2021, pages 3 * |
| 石秀换等: "非小细胞肺癌组织中期因子蛋白表达及其临床意义的研究", 《中华肿瘤防治杂志》, vol. 15, no. 12, pages 3 * |
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