發(fā)布時(shí)間：2018-03-02 20:11

  本文選題：納米氧化石墨烯　切入點(diǎn)：上皮-間質(zhì)細(xì)胞轉(zhuǎn)化EMT　出處：《山東大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：在材料研究領(lǐng)域,隨著對(duì)碳納米材料的不斷深入研究,特別是自2004年石墨烯首次被發(fā)現(xiàn)以來(lái),它己逐漸成為材料科學(xué)界的“超級(jí)明星”。二維碳材料結(jié)構(gòu)的石墨烯及其衍生物,由于其擁有獨(dú)特的物理化學(xué)性質(zhì),具有超高的疏水表面、較佳的表面修飾和獨(dú)特的光學(xué)性質(zhì),目前已經(jīng)被廣泛的應(yīng)用于生物醫(yī)學(xué)、電子器件和復(fù)合材料等研究領(lǐng)域,例如在生物傳感器、生物成像、光敏劑與基因輸送載體、疏水抗腫瘤藥物、光熱治療腫瘤、抗菌材料和組織功能材料等領(lǐng)域均具有非常好的應(yīng)用前景。 這幾年來(lái),生物技術(shù)與各學(xué)科的交叉研究,特別是材料生物學(xué)的不斷深入研究,在生物醫(yī)藥、食品健康、能源環(huán)境和航空航天、信息產(chǎn)業(yè)等眾多技術(shù)領(lǐng)域帶來(lái)了巨大影響。當(dāng)納米材料和技術(shù)越來(lái)越多的應(yīng)用于各個(gè)領(lǐng)域時(shí),隨之而來(lái)也會(huì)帶來(lái)未知的影響,這就要求我們不能只注重于納米材料給我們生活帶來(lái)的有益效應(yīng),也要考慮到它可能會(huì)帶來(lái)的環(huán)境和健康方面的潛在影響,特別是納米材料引起的生物相容性和對(duì)機(jī)體相關(guān)的生理病理學(xué)上的影響。 其中一個(gè)重要的生理病理學(xué)上的影響就是腫瘤的轉(zhuǎn)移侵潤(rùn)—上皮間質(zhì)細(xì)胞轉(zhuǎn)化(Epithelial—Mesenchymal transition, EMT),這種過(guò)程的發(fā)生是具有極性的上皮細(xì)胞轉(zhuǎn)換成為具有遷移能力的間質(zhì)細(xì)胞并獲得侵襲和遷移能力,它存在于人體多個(gè)生理和病理過(guò)程中。很多研究表明EMT與腫瘤細(xì)胞的侵襲和轉(zhuǎn)移有著密切的關(guān)系,我們采用材料界的“明星材料”—石墨烯為研究對(duì)象,研究其對(duì)細(xì)胞EMT的作用影響,從而為今后材料修飾載藥等提供靶向輸送可能性,同時(shí)對(duì)其可能引起的腫瘤機(jī)制的研究,為抑制腫瘤轉(zhuǎn)移提供新的治療方法。 本文重點(diǎn)研究了小尺寸粒徑比(100nm左右)的納米氧化石墨烯(Grapheneoxide,NGO,GO)與上皮細(xì)胞系的直接作用觀察其是否引起上皮-間質(zhì)細(xì)胞轉(zhuǎn)化(Epithelial-mesenchymal transition, EMT),以及可能對(duì)細(xì)胞生長(zhǎng)轉(zhuǎn)化因子TGF-β1誘導(dǎo)的腫瘤上皮-間質(zhì)細(xì)胞轉(zhuǎn)化的發(fā)生的參與作用。擬用小鼠乳腺上皮細(xì)胞(NMuMG)為主要研究對(duì)象,探究未經(jīng)修飾納米氧化石墨烯(NGO)對(duì)上皮細(xì)胞系直接和間接作用帶來(lái)的影響,觀察石墨烯對(duì)細(xì)胞毒性效應(yīng)和誘導(dǎo)其EMT過(guò)程的發(fā)生,以期為石墨烯的生物效應(yīng)機(jī)制提供一定的理論基礎(chǔ)。 (1)選用納米氧化石墨烯和多種上皮細(xì)胞系,應(yīng)用濃度梯度(0、5、10、15、20和40μg/ml)和不同時(shí)間(24、48和96h)進(jìn)行處理,篩選低毒性有效作用的劑量和時(shí)間和在形態(tài)觀察發(fā)生明顯效應(yīng)的細(xì)胞系。我們通過(guò)使用TGF-β1誘導(dǎo)建立EMT模型,最后我們篩選出對(duì)GO和TGF-β1產(chǎn)生較明顯生物效應(yīng)的細(xì)胞系為小鼠乳腺上皮細(xì)胞(NMuMG)。 (2)分組情況：對(duì)照組、石墨烯處理組、TGF-β1組、石墨烯和TGF-β1共同處理組,分別在形態(tài)學(xué)和細(xì)胞遷移能力檢測(cè),處理24h和48h后,經(jīng)TGF-β1單獨(dú)處理的細(xì)胞均出現(xiàn)一定的形態(tài)學(xué)變化,向紡錘樣間質(zhì)細(xì)胞的形態(tài)演變,并且遷移能力最強(qiáng),表現(xiàn)為典型的EMT。而GO單獨(dú)處理組的細(xì)胞并沒(méi)有發(fā)生形態(tài)變化,愈合能力較弱；在用TGF-β1和GO共同處理后,只有極少數(shù)發(fā)生纖維化現(xiàn)象,我們初步可以得出氧化石墨烯對(duì)TGF-β1誘導(dǎo)的細(xì)胞纖維化功能起到了一定的抑制作用。 (3)運(yùn)用Real-time PCR和Western blot技術(shù),分別在基因表達(dá)水平和蛋白水平檢測(cè)。最后發(fā)現(xiàn),NMuMG細(xì)胞經(jīng)TGF-β1誘導(dǎo)培養(yǎng)后,上皮細(xì)胞標(biāo)志物E-cadherin的表達(dá)降低,而間質(zhì)標(biāo)志物FN的表達(dá)增加,此結(jié)果同樣提示經(jīng)TGF-β1誘導(dǎo)后,細(xì)胞內(nèi)EMT過(guò)程的發(fā)生。然而在加入氧化石墨烯后,我們觀察到,經(jīng)TGF-β1誘導(dǎo)后所出現(xiàn)的EMT相關(guān)標(biāo)志物的表達(dá)受到不同程度的抑制。從上述相關(guān)基因的表達(dá)變化,我們初步得出氧化石墨烯也參與了TGF-β1調(diào)節(jié)的EMT過(guò)程。隨后根據(jù)可能引起EMT的信號(hào)通路我們分別從TGF/Smad通路和MAPK(ERK和p38)通路進(jìn)行驗(yàn)證GO直接和間接參與NMuMG細(xì)胞EMT生物效應(yīng)機(jī)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.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R318.08

【共引文獻(xiàn)】

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本文編號(hào)：1557996