兰州大学机构库 >数学与统计学院
HMSC-bm与A549肺癌细胞共培养后增殖分化及遗传稳定性研究
Alternative TitleStudy on malignant transformation and genetic stability of HMSC-bm in A549 lung cancer cell microenvironment
刘永琦
Thesis Advisor谢小冬
2014-06-06
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name博士
Keyword肺癌细胞 HMSC-bm 共培养 增殖 遗传稳定性
Abstract目的:本文通过Transwell小室建立人骨髓间充质干细胞(human mesenchymal stem cells-bone marrow,HMSC-bm)与肺腺癌细胞株A549共培养体系,研究可能诱发HMSC-bm异常增殖分化的条件;观察A549共培养肿瘤微环境中HMSC-bm(CO-HMSC-bm)形态、生长、细胞周期的变化;研究CO-HMSC-bm黏附、迁移、侵袭能力及MMP-2、MMP-9蛋白表达的改变;检测CO-HMSC-bm细胞骨架、克隆增殖及多向分化性的变化;探讨CO-HMSC-bm染色体核型、癌/抑癌基因表达及ERK1/2相关信号通路的的影响;分析CO-HMSC-bm组蛋白去乙酰化酶活性(HDAC4)及组蛋白乙酰化水平等的变化,以期为MSCs的临床应用提供科学依据。 方法:第一部分:HMSC-bm与A549肺癌微环境共培养体系建立及条件优化研究。利用A549 条件培养液孵育MSCs,同时采用6孔板结合Transwell小室建立HMSC-bm和A549细胞的共培养体系;分别以A549 条件培养液孵育MSCs组、共培养HMSC-bm组为实验组。A549 条件培养液孵育MSCs组依次按25%、50%、75%、100% 加入A549细胞条件培养液,培养72h后进行HMSC-bm细胞增殖活性及形态学分析;共培养组于3天、7天后终止培养,收集细胞移到培养瓶中继续培养,分别传代至第3代(P3)、第5代(P5)的细胞;另设单独培养的HMSC-bm、A549为对照组。通过镜下观察细胞形态、MTT法分析生长曲线、流式细胞术检测周期等的变化,研究A549可能诱发MSCs异常增殖分化的条件。 第二部分: A529肺癌微环境对HMSC-bm粘附、迁移、侵袭及相关分子表达的影响研究。通过细胞基质粘附实验、Transwell 模型法检测共培养7d的P3、P5HMSC-bm(CO-HMSC-bm)细胞体外粘附、迁移、侵袭能力;利用Western blot及RT-PCR检测共培养7d的P5 HMSC-bm(CO-HMSC-bm 7d-P5)细胞迁移相关MMP-2、MMP-9 蛋白、mRNA表达的变化。 第三部分:A549肺癌微环境对HMSC-bm细胞骨架、克隆增殖及多向分化性的影响研究。利用平板集落形成实验、MTT法检测细胞的增殖能力及流式细胞术检测CO-HMSC-bm 7d-P5细胞周期;采用激光共聚焦显微镜观察细胞骨架;分别用成骨细胞诱导液、β-巯基乙醇诱导后,观察碱性磷酸酶的沉积、矿化结节或神经突触形成等多向分化潜力。 第四部分:A549肺癌微环境对HMSC-bm染色体核型、癌/抑癌基因表达及相关信号通路的影响研究。通过倒置相差显微镜下观察CO-HMSC-bm 7d-P5细胞的形态变化;对数生长期细胞加秋水仙素作用、冰玻片滴片,Giemsa染色显带分析;提取CO-HMSC-bm 7d-P5及对照组细胞蛋白,采用Western blot技术检测抑癌基因p53、原癌基因Ras及ERK信号传导通路蛋白p-ERK、NF-κB、Capase-3蛋白的表达变化。 第五部分: A549肺癌微环境对HMSC-bm组蛋白乙酰化水平的影响研究。提取CO-HMSC-bm 7d-P5及对照组细胞蛋白,采用Western blot技术检测各组细胞HDAC4及H3K9、H4K5、H4K8等乙酰化修饰位点的变化。 结果:HMSC-bm在25%、50%、75%、100%浓度的A549条件培养液中培养,其生长停滞,各浓度组与对照组比较均有统计学意义(P<0.05)。HMSC-bm与A549细胞通过Transwell小室共培养,发现随着诱导时间的延长及传代代次的增加,实验组细胞形态逐渐发生显著变...
Other AbstractObjective:In this study, to establish the co-culture system which contains human mesenchymal stem cells-bone marrow(HMSC-bm) and lung adenocarcinoma cell line A549 through transwell technique, screen the the abnormal proliferation and differentiation of HMSC-bm, observe the change of HMSC-bm in morphology, growth and cell cycle in tumor microenvironment, detect the adhesion, migration and invasion of HMSC-bm and expression of MMP-2, MMP-9 protein, study multi-direction differentiation characteristics of MSCs in tumor microenvironment, cytoskeleton changes, chromosome heteronuclear rate and the expression of apoptosis associated protein, observe the expression of oncogene, tumor suppressor gene and key molecule in ERK1/2 related signaling pathway, analyze activities of histone deacetylase and changes of histone acetylation, to provide the scientific basis for the clinical application of MSCs. Method: Part I: Research on the effect of A549 lung cancer microenvironment on morphology and proliferation characteristics of bone marrow mesenchymal stem cell. MSCs were incubated with A549 conditional medium. 6 hole plate combined with the Transwell was applied to build co-culture system between HMSC-bm and A549 cells. A549 conditional medium incubated MSCs group, and HMSC-bm co-culture group were observation groups. Using 25%, 50%, 75%, 100% A549 conditioned medium for co-culture incubation, proliferation activity and morphology of HMSC-bm cell were analyzed after 72h of co-culture. Co-culture groups in 3 days, 7 days, respectively, were terminated co-culture and cells were collected into culture bottle for passage into P3 and P5 generation. There were also HMSC-bm and A549 control groups. Cell morphology were observed through microscopic observation. Growth curve was analyzed by MTT. Flow cytometry was used to detect cell cycle change to select appropriate conditions of abnormal proliferation and differentiation of MSCs induced by A549. Part II: Research on the effects of A549 lung cancer microenvironment on the abilities of adhesion, migration, invasion of HMSC-bm and the expression of related molecules. Adhesion, migration and invasion of P3 and P5 HMSC-bm (CO-HMSC-bm) cells co-cultured for 7D were detected by cell matrix adhesion experiment and Transwell model method.Western blot and RT-qPCR were applied to detect MMP-2, MMP-9 protein, mRNA expression of P5 HMSC-bm co-cultured for 7d, respectively. Part III: Research on the effect of A549 lung cancer microe...
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Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/225697
Collection数学与统计学院
Recommended Citation
GB/T 7714
刘永琦. HMSC-bm与A549肺癌细胞共培养后增殖分化及遗传稳定性研究[D]. 兰州. 兰州大学,2014.
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