CN112034183A - 影响mdk基因调节胶质瘤迁移和侵袭的分子机制及应用 - Google Patents
影响mdk基因调节胶质瘤迁移和侵袭的分子机制及应用 Download PDFInfo
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Abstract
本发明涉及了影响MDK基因调节胶质瘤迁移和侵袭的分子机制及应用。1)Western blot分析胶质母细胞瘤细胞系MDK表达，3)过表达MDK基因对胶质瘤细胞迁移和侵袭的影响，2)慢病毒介导敲除MDK基因对胶质瘤细胞迁移和侵袭的影响，4)对干扰MDK基因表达后的细胞系中P‑PI3K，PI3K，Akt，p‑ERK及vimentin，beta‑catenin进行Western blot分析。本发明提供了一种胶质瘤治疗的新思路，为研究胶质母细胞瘤药物治疗靶点提供了新途径。
Description
技术领域
本发明属于基因工程技术领域，它涉及一种影响MDK基因以调节胶质瘤迁移和侵袭的分子机制及其应用。
背景技术
胶质瘤是脑和脊髓最常见的原发肿瘤，即使在治疗胶质瘤上取得了重大的进展，胶质瘤的死亡率仍居高不下。研究表明高侵袭性是胶质瘤生存率低的重要原因之一[Quinn,T,Ostrom,etal.CBTRUSStatistical Report:PrimaryBrainandCentralNervousSystemTumorsDiagnosedin theUnitedStatesin2008-2012.[J].NeuroOncology,2015.]。因此，寻找介导胶质瘤迁移和侵袭的机制将会为治疗胶质瘤提供新的见解。MDK编码一种肝素结合生长因子，MDK编码蛋白在肿瘤发生过程中会促进细胞生长[HoribaM,KadomatsuK,NakamuraE,etal.Neointimaformationinarestenosismodelissuppressedin midkine-deficientmice[J].J.clin.invest,2000,105(4):489-95.]、迁移[MuramatsuH,ZouK,SakaguchiN,etal.LDLreceptor-relatedprotein asacomponentofthemidkinereceptor.[J].BiochemBiophysRes Commun,2000,270(3):936-941.]和血管生成[JuanL,Qi-HuiL,FanW,etal.ExosomalmiR-9inhibitsangiogenesisbytargetingMDKand regulatingPDK/AKTpathwayinnasopharyngealcarcinoma[J].Journal ofExperimental&ClinicalCancerResearch,2018,37(1):1-12.]。MDK能与NOTCH2相互作用，从而导致HES1和STAT3之间的相互作用促进细胞上皮间充质转化[HuangY,HoqueMO,WuF,etal.Midkineinducesepithelial-mesenchymaltransitionthrough Notch2/Jak2-Stat3signalinginhumankeratinocytes[J].CellCycle,2008,7(11):1613-1622.]。同样地，该基因可以编码多个异构体的剪接转录体[SakaguchiN,MuramatsuH,Ichihara-TanakaK,etal.Receptor-type proteintyrosinephosphatasezetaasacomponentofthesignalingreceptorcomplexformidkine-dependentsurvivalofembryonic neurons.[J].NeuroenceResearch,2003,45(2):219-224.]。MDK在许多肿瘤中均有过表达的情况，包括非小细胞肺癌、甲状腺癌和低级别胶质瘤等[MDKProteinOverexpressionCorrelateswiththeMalignantStatus andPrognosisofNon-smallCellLungCancer[J].ArchivesofMedicalResearch,2015,46(8):635-641.ChoiYW,KimYH,LeeJ,etal.Strong immunoexpressionofmidkineisassociatedwithmultiplelymphnode metastasesinBRAFV600Epapillarythyroidcarcinoma[J].Human Pathology,2015,46(10):1557-1565.]。研究发现MDK能够激活激活磷酸肌醇3激酶(PI3K)和丝裂原活化蛋白激酶(MAPK)[OhuchidaT,OkamotoK,AkahaneK,etal.Midkineprotectshepatocellular carcinomacellsagainstTRAIL-mediatedapoptosisthrough down-regulationofcaspase-3activity[J].Cancer,2004.]，进而诱导肿瘤细胞增殖，增强血管生成和抗凋亡活性。因此MDK基因常作为治疗多种不同疾病的靶点。然而，MDK影响胶质瘤细胞迁移和侵袭的机制尚未明确。
综上所述，我们发现MDK能促进胶质瘤细胞迁移和侵袭，但是尚无研究分析探索其具体的机制，因此分析MDK促进胶质瘤细胞迁移和侵袭的机制能为治疗胶质瘤提供理论基础。因此我们的研究团队以人U87-MG和A172为细胞来源，建立了自己的实验技术平台及方法，进一步优化了MDK序列设计构建慢病毒质粒的方法，并鉴定其对胶质瘤迁移和侵袭的影响，通过在Transwell、Matrigel-Transwell实验过程中优化其干扰胶质瘤迁移和侵袭的功能，并取得了可喜的研究成果，为MDK应用于基因工程、肿瘤学及在临床上胶质瘤的诊断指标和治疗靶标提供可靠的理论依据。解决现存的问题将会为MDK在胶质瘤进展中的作用提供了新的见解，并有助于确定该疾病的潜在治疗靶点。
发明内容
针对现存的技术问题，本发明提供了一种影响MDK基因以调节胶质瘤迁移和侵袭的分子机制及其应用。
本发明是这样实现的，一种影响MDK基因以调节胶质瘤迁移和侵袭的分子机制及其应用的方法包括：
第一步：Westernblot分析胶质瘤细胞系MDK表达；
第二步：慢病毒介导MDK过表达；
第三步：慢病毒介导MDK敲除；
第四步：Transwell、Matrigel-Transwel检测干扰MDK基因表达后，胶质瘤细胞的迁移和侵袭情况观察；
第五步：通过Westernblot分析检测干扰MDK基因表达后的细胞系中P-PI3K，PI3K，Akt，p-ERK及vimentin，beta-catenin的表达水平。PI3K/AKT通路是MDK影响胶质瘤迁移和侵袭的分子机制。
Westernblot分析的方法包括：
用兔抗人MDK抗体和小鼠抗β-肌动蛋白抗体。用RIPA缓冲液溶解组织，其中含有蛋白酶抑制剂。将裂解物样品在8-12％SDS-PAGE凝胶上分离，转移到聚偏氟乙烯膜上，在4℃下与初级抗体孵育过夜，然后与辣根过氧化物酶结合的二级抗体孵育。一个ECL试剂盒用于检测结合抗体。
利用pMSCV-IRES-GFP载体构建了标记MDK的表达质粒，并将该质粒或相应的空载体(NC)转染293T细胞。利用这些细胞的重组逆转录病毒多重感染感染A172细胞，获得稳定感染过表达MDK基因(MDK-OE)的细胞系或对照细胞系。
根据引物设计原则，设计3个能够特异敲除MDK的引物，制备重组MDK-shRNA慢病毒和阴性对照(NC)慢病毒。在U87-MG细胞感染敲除MDK基因的慢病毒载体(MDK-KD)，获得稳定感染敲除MDK表达的细胞系和对照细胞系。
Transwell和Matrigel-Transwell方法包括：
细胞被镀在Transwell分析插入物的上室，在无血清RPMI1640培养基中加入Matrigel基质液并加入下室。用FBS清洗。24小时后用无菌棉签擦洗插入物的顶层，以去除剩余的细胞。用0.1％结晶紫染色底层侵入细胞1小时，用数字显微镜检查、计数和成像。对每个腔室的五个随机视野的细胞数进行计数和平均。
综上所述，本发明的优点及积极效果在于：
MDK被证实是治疗多种不同疾病的靶点。本发明提供一种调控胶质瘤迁移和侵袭的MDK基因在预警胶质瘤的应用。为研究胶质瘤药物治疗靶点提供了新途径。
附图说明
图1是本发明实施提供的一种影响MDK基因以调节胶质瘤迁移和侵袭的分子机制及其应用的流程图
图2是本发明实施提供的过表达MDK后，胶质瘤的迁移和侵袭情况
图3是本发明实施提供的敲除MDK后，胶质瘤的迁移和侵袭情况
图4是本发明实施提供的干扰MDK基因表达后的细胞系中P-PI3K，PI3K，Akt，p-ERK及vimentin，beta-catenin的表达水平
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进行进一步详细说明。此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
现有技术中，MDK促进胶质瘤迁移和侵袭的具体机制尚不明确。为解决上述技术问题，下面结合具体分析对本发明作详细描述。对于MDK促进胶质瘤迁移和侵袭的具体机制尚未见明确报道。因此，本发明选择MDK基因为主要分析内容。本发明初步确定MDK在胶质瘤中表达，在此基础上，本发明拟通过如下实验进一步阐明MDK如何影响胶质瘤迁移和侵袭：
人胶质瘤细胞系A172细胞用于构建过表达MDK基因的细胞系，人胶质瘤细胞系U87-MG用于构建敲除MDK基因的细胞系，观察干扰MDK基因表达后胶质瘤细胞迁移和侵袭情况。
本发明旨在从体外细胞水平探索MDK促进胶质瘤迁移和侵袭的具体机制，确认MDK作为一种新型标志物在胶质瘤的迁移和侵袭的临床意义，也为今后以MDK为靶点设计药物，对胶质瘤增殖的靶向治疗提供依据。
下面结合具体方案对本发明作进一步描述。
如图1所示，本发明实施例提供的一种影响MDK基因以调节胶质瘤迁移和侵袭的分子机制及其应用：
1)Westernblot分析胶质瘤细胞系MDK表达
a.用兔抗人MDK抗体和小鼠抗β-肌动蛋白抗体。用RIPA缓冲液溶解组织，其中含有蛋白酶抑制剂。将裂解物样品在8-12％SDS-PAGE凝胶上分离，转移到聚偏氟乙烯膜上，在4℃下与初级抗体孵育过夜，然后与辣根过氧化物酶结合的二级抗体孵育。一个ECL试剂盒用于检测结合抗体。
2)细胞学水平分析过表达MDK基因对胶质瘤细胞迁移和侵袭的影响：
a.利用pMSCV-IRES-GFP载体构建了标记MDK的表达质粒，并将该质粒或相应的空载体(NC)转染293T细胞。利用这些细胞的重组逆转录病毒多重感染感染A172细胞，使细胞感染过表达MDK基因的慢病毒载体(MDK-OE)，以获得稳定感染过表达MDK基因的细胞系和对照细胞系。
b.细胞被镀在Transwell分析插入物的上室，在无血清RPMI1640培养基中加入Matrigel基质液并加入下室。用FBS清洗。24小时后用无菌棉签擦洗插入物的顶层，以去除剩余的细胞。用0.1％结晶紫染色1小时底层侵入细胞，用数字显微镜检查、计数和成像。对每个腔室的五个随机场中的细胞数进行计数和平均。
3)细胞学水平分析敲除MDK基因对胶质瘤细胞迁移和侵袭的影响：
a.根据引物设计原则，设计3个能够特异敲除MDK的引物，。制备重组MDK-shRNA慢病毒和阴性对照(NC)慢病毒。在U87-MG细胞感染敲除MDK基因的慢病毒载体(MDK-KD)，获得稳定感染的敲除MDK细胞系和对照细胞系。
b.细胞被镀在Transwell分析插入物的上室，在无血清RPMI1640培养基中加入Matrigel基质液并加入下室。用FBS清洗。24小时后用无菌棉签擦洗插入物的顶层，以去除剩余的细胞。用0.1％结晶紫染色1小时底层侵入细胞，用数字显微镜检查、计数和成像。对每个腔室的五个随机视野的细胞数进行计数和平均。
4)干扰MDK基因表达后，对细胞系中的P-PI3K，PI3K，P-Akt，Akt，p-ERK及vimentin，beta-catenin进行Westernblot分析。
下面结合效果对本发明作进一步描述。
在本发明中，图2所示，细胞水平过表达MDK基因可促进胶质瘤细胞的迁移和侵袭。
图3所示，在细胞水平敲除MDK基因可减少胶质瘤细胞的迁移和侵袭。
在图4所示，干扰MDK基因表达后，对胶质瘤细胞系中P-PI3K，PI3K，Akt，p-ERK及vimentin，beta-catenin进行Westernblot分析。发现PI3K/AKT通路是MDK影响胶质瘤迁移和侵袭的分子机制。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。
Claims (1)
1.影响MDK基因调节胶质瘤迁移和侵袭的分子机制及应用，包括以下步骤：
(1)Westernblot分析胶质瘤细胞系MDK表达
Westernblot分析的方法如下：
用兔抗人MDK抗体和小鼠抗β-肌动蛋白抗体。用含有蛋白酶抑制剂的RIPA缓冲液溶解组织。将裂解物样品在8-12％SDS-PAGE凝胶上分离，随后转移到聚偏氟乙烯膜上，并在4℃下与初级抗体孵育过夜。然后与辣根过氧化物酶结合的二级抗体孵育。通过ECL试剂盒检测结合抗体。
(2)慢病毒介导MDK过表达，方法如下：
使用pMSCV-IRES-GFP载体构建了标记MDK的表达质粒，并将该质粒或相应的空载体(NC)转染293T细胞。利用这些细胞的重组逆转录病毒多重感染感染A172细胞，获得稳定感染过表达MDK基因(MDK-OE)的细胞系或对照细胞系。
(3)慢病毒载体介导MDK基因敲除，方法如下：
根据引物设计原则，设计3个能够特异敲除MDK的引物，制备重组MDK-shRNA慢病毒和阴性对照(N C)慢病毒。在U87-MG细胞中感染敲除MDK基因(MDK-KD)的慢病毒载体，获得稳定感染敲除MDK表达的细胞系和对照细胞系。
(4)Transwell、Matrigel-Transwel检测干扰MDK基因表达对胶质瘤细胞迁移和侵袭能力的影响，方法如下：
细胞被镀在Transwell分析插入物的上室，在无血清RPMI1640培养基中加入Matrigel基质液，并将混合物加入下室，用FBS清洗。24小时后通过用无菌棉签擦洗插入物的顶层，去除剩余的细胞。用0.1％结晶紫染色底层侵入细胞1小时，用数字显微镜检查、计数和成像。对每个腔室的五个随机场中的细胞数进行计数和平均。
(5)通过Westernblot分析检测干扰MDK基因表达后的细胞系中P-PI3K，PI3K，Akt，p-ERK及vimentin，beta-catenin的表达水平。
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