纳米氧化石墨烯与TGF-β1对肿瘤上皮—间质细胞转移（EMT）的影响及其机制的研究

发布时间：2018-03-02 20:11

本文选题：纳米氧化石墨烯　切入点：上皮-间质细胞转化EMT　出处：《山东大学》2014年硕士论文　论文类型：学位论文

【摘要】：在材料研究领域,随着对碳纳米材料的不断深入研究,特别是自2004年石墨烯首次被发现以来,它己逐渐成为材料科学界的“超级明星”。二维碳材料结构的石墨烯及其衍生物,由于其拥有独特的物理化学性质,具有超高的疏水表面、较佳的表面修饰和独特的光学性质,目前已经被广泛的应用于生物医学、电子器件和复合材料等研究领域,例如在生物传感器、生物成像、光敏剂与基因输送载体、疏水抗肿瘤药物、光热治疗肿瘤、抗菌材料和组织功能材料等领域均具有非常好的应用前景。这几年来,生物技术与各学科的交叉研究,特别是材料生物学的不断深入研究,在生物医药、食品健康、能源环境和航空航天、信息产业等众多技术领域带来了巨大影响。当纳米材料和技术越来越多的应用于各个领域时,随之而来也会带来未知的影响,这就要求我们不能只注重于纳米材料给我们生活带来的有益效应,也要考虑到它可能会带来的环境和健康方面的潜在影响,特别是纳米材料引起的生物相容性和对机体相关的生理病理学上的影响。其中一个重要的生理病理学上的影响就是肿瘤的转移侵润—上皮间质细胞转化(Epithelial—Mesenchymal transition, EMT),这种过程的发生是具有极性的上皮细胞转换成为具有迁移能力的间质细胞并获得侵袭和迁移能力,它存在于人体多个生理和病理过程中。很多研究表明EMT与肿瘤细胞的侵袭和转移有着密切的关系,我们采用材料界的“明星材料”—石墨烯为研究对象,研究其对细胞EMT的作用影响,从而为今后材料修饰载药等提供靶向输送可能性,同时对其可能引起的肿瘤机制的研究,为抑制肿瘤转移提供新的治疗方法。本文重点研究了小尺寸粒径比(100nm左右)的纳米氧化石墨烯(Grapheneoxide,NGO,GO)与上皮细胞系的直接作用观察其是否引起上皮-间质细胞转化(Epithelial-mesenchymal transition, EMT),以及可能对细胞生长转化因子TGF-β1诱导的肿瘤上皮-间质细胞转化的发生的参与作用。拟用小鼠乳腺上皮细胞(NMuMG)为主要研究对象,探究未经修饰纳米氧化石墨烯(NGO)对上皮细胞系直接和间接作用带来的影响,观察石墨烯对细胞毒性效应和诱导其EMT过程的发生,以期为石墨烯的生物效应机制提供一定的理论基础。 (1)选用纳米氧化石墨烯和多种上皮细胞系,应用浓度梯度(0、5、10、15、20和40μg/ml)和不同时间(24、48和96h)进行处理,筛选低毒性有效作用的剂量和时间和在形态观察发生明显效应的细胞系。我们通过使用TGF-β1诱导建立EMT模型,最后我们筛选出对GO和TGF-β1产生较明显生物效应的细胞系为小鼠乳腺上皮细胞(NMuMG)。 (2)分组情况：对照组、石墨烯处理组、TGF-β1组、石墨烯和TGF-β1共同处理组,分别在形态学和细胞迁移能力检测,处理24h和48h后,经TGF-β1单独处理的细胞均出现一定的形态学变化,向纺锤样间质细胞的形态演变,并且迁移能力最强,表现为典型的EMT。而GO单独处理组的细胞并没有发生形态变化,愈合能力较弱；在用TGF-β1和GO共同处理后,只有极少数发生纤维化现象,我们初步可以得出氧化石墨烯对TGF-β1诱导的细胞纤维化功能起到了一定的抑制作用。 (3)运用Real-time PCR和Western blot技术,分别在基因表达水平和蛋白水平检测。最后发现,NMuMG细胞经TGF-β1诱导培养后,上皮细胞标志物E-cadherin的表达降低,而间质标志物FN的表达增加,此结果同样提示经TGF-β1诱导后,细胞内EMT过程的发生。然而在加入氧化石墨烯后,我们观察到,经TGF-β1诱导后所出现的EMT相关标志物的表达受到不同程度的抑制。从上述相关基因的表达变化,我们初步得出氧化石墨烯也参与了TGF-β1调节的EMT过程。随后根据可能引起EMT的信号通路我们分别从TGF/Smad通路和MAPK(ERK和p38)通路进行验证GO直接和间接参与NMuMG细胞EMT生物效应机制。
[Abstract]:In the field of materials research, with the deepening of research on carbon nano materials, especially since 2004, graphene was first discovered, it has gradually become the field of material science "super star". The graphene and its derivatives two-dimensional structure of carbon material, due to its unique physical and chemical properties, with super hydrophobic surface better, surface modification and unique optical properties, has been widely used in biomedical research in the field of electronic devices, and composite materials, such as biosensors, biological imaging and gene delivery carrier, photosensitizer, hydrophobic anticancer drugs, photothermal therapy of tumor and has a very good application prospect of antibacterial materials and tissue functional materials and other fields.
In recent years, biotechnology research and cross disciplines, especially in-depth study materials in biology, biological medicine, health food, energy and environment, aerospace, information industry has brought tremendous impact to many technical fields. When the nano materials and technology more and more applied in various fields, it also influence will bring unknown, this requires that we can not only focus on the beneficial effects of nano materials to our lives, we should also take into account the potential impact of it may bring environmental and health aspects, especially the influence of nano materials caused by the biocompatibility on the body and related physiological pathology.
The effect of an important physiological pathology on the metastasis of epithelial - mesenchymal transition is tumor (Epithelial - Mesenchymal transition, EMT), the occurrence of this process is the polarity of epithelial cells into mesenchymal cells with mobility and the invasion and migration ability, it exists in the human body a number of physiological and pathological processes. Many studies showed that EMT and tumor cell invasion and metastasis are closely related, we use the materials sector "star material" - graphene as the research object, the research on EMT cells by influence, so as to provide future materials modified drug targeting delivery possibility at the same time, the research on the mechanism of tumor may be caused by the inhibition of tumor metastasis, to provide a new treatment method.
This paper focuses on the research of small size particle diameter ratio (100nm) of the nano graphene oxide (Grapheneoxide, NGO, GO) direct interaction with epithelial cell line to observe whether the induced epithelial mesenchymal transition (Epithelial-mesenchymal transition, EMT), and TGF- on cell growth factor beta 1 induced epithelial tumor transformation mesenchymal transition occurred in rats. The mouse mammary epithelial cells (NMuMG) as the main research object, explore the unmodified nano graphene oxide (NGO) impact on epithelial cells in direct and indirect effect, observation of graphene on cytotoxicity and induction of the EMT process, in order to to provide a theoretical basis for the mechanism of the biological effects of graphene.
(1) nano graphene oxide and a variety of epithelial cell lines, using concentration gradient (0,5,10,15,20 and 40 g/ml) and different time (24,48 and 96h) were treated with low toxicity screening of effective dose and time and observe obvious effect in cell morphology. We use the TGF- beta 1 was established by EMT the model, finally we screened for GO and TGF- beta 1 have significantly biological effects of cell lines of mouse mammary epithelial cells (NMuMG).
(2) group: control group, treatment group of graphene, graphene TGF- beta 1 group, and TGF- beta 1 treatment group, respectively in morphology and cell migration detection, 24h and 48h, by TGF- beta 1 alone treated cells showed morphological changes, to fusiform mesenchymal cell morphology evolution and migration ability was the strongest, is characterized by EMT. and GO treatment group and no single cell morphological changes, healing ability is weak; and in the 1 GO treatment with TGF- beta, only a handful of fibrosis phenomenon, we can infer the graphene oxide of TGF- beta 1 induced fibrosis function has played a certain effect.
(3) the use of Real-time PCR and Western blot technology to detect the expression of gene and protein level in NMuMG cells respectively. Finally, by TGF- beta 1 cultured epithelial cell marker E-cadherin expression decreased, while mesenchymal marker FN expression increased, the results also suggest that the TGF- beta 1 after induction EMT cellular processes occur. However, in adding graphene oxide, we observed that the expression of EMT related markers TGF- beta 1 induced the inhibition of different degrees. From the expression of these genes, we initially obtained graphene oxide is also involved in the regulation process of EMT TGF- beta 1 then according to the signal pathway of EMT may cause we were from the TGF/Smad pathway and MAPK pathway (ERK and p38) to verify the GO directly and indirectly involved in the NMuMG cell EMT biological effect mechanism.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R318.08

【共引文献】