本文選題：胃癌干細(xì)胞 + CD133�。� 參考：《山東大學(xué)》2017年博士論文

【摘要】：第一部分胃癌干細(xì)胞的分選與鑒定,以及細(xì)胞中l(wèi)ncRNAROR的表達(dá)情況。背景:胃癌是一種在全世界范圍內(nèi)發(fā)病率位居第四位的惡性腫瘤,在我國(guó)腫瘤發(fā)病率中,胃癌位列第二,并且腫瘤死亡率中,胃癌位列第三,給人們的健康帶來(lái)了很大威脅。胃癌具有術(shù)后轉(zhuǎn)移、容易復(fù)發(fā)、耐藥等特性,而且至今還沒(méi)有研究出其調(diào)控機(jī)制,在這幾年中,腫瘤發(fā)生發(fā)展的相關(guān)研究中,研究學(xué)者提出了腫瘤干細(xì)胞學(xué)說(shuō)。該學(xué)說(shuō)認(rèn)為腫瘤起源于小部分具有自我更新和多種分化潛能的腫瘤細(xì)胞,這部分細(xì)胞稱為腫瘤干細(xì)胞。目前,利用腫瘤干細(xì)胞表面特異的標(biāo)記分子分離和純化腫瘤干細(xì)胞的方法已較為普遍,并且有研究通過(guò)利用特異性的細(xì)胞表面標(biāo)志分子CD133,使用流式細(xì)胞儀從胃癌中成功分選出胃癌干細(xì)胞,并證明了CD133是胃癌干細(xì)胞的表面標(biāo)志物。目的:使用MKN-45細(xì)胞構(gòu)建胃癌干細(xì)胞細(xì)胞系,并檢測(cè)其中l(wèi)ncRNA ROR的功能。方法:人MKN-45胃癌細(xì)胞用RMPI1640培養(yǎng)液常規(guī)培養(yǎng),制作單細(xì)胞懸液,調(diào)整細(xì)胞濃度后加入CD133-FITC抗體進(jìn)行分選,孵育后使用流式細(xì)胞儀收集被CD133-FITC抗體標(biāo)記的細(xì)胞,即為CD133+MKN-45細(xì)胞(簡(jiǎn)稱CD133+);收集余下的未被CD133-FITC抗體標(biāo)記的細(xì)胞,即為CD133-MKN-45細(xì)胞(簡(jiǎn)稱CD133-)。并使用免疫熒光法檢測(cè)CD133+細(xì)胞中CD133與CD44v8-10的共表達(dá)定位情況;之后使用實(shí)時(shí)定量PCR法和免疫印跡法檢測(cè)CD133+和CD133-細(xì)胞中OCT4,SOX2和NANOG等腫瘤干細(xì)胞標(biāo)記物的表達(dá)。之后,我們使用實(shí)時(shí)定量PCR法對(duì)CD133-和CD133+細(xì)胞中l(wèi)ncRNA ROR的表達(dá)進(jìn)行檢測(cè),并取患者胃癌組織標(biāo)本,分析其中l(wèi)ncRNA ROR和CD133表達(dá)量之間的關(guān)系。結(jié)果:免疫熒光法檢測(cè)結(jié)果顯示,分選得到的CD133+細(xì)胞表面標(biāo)記物CD133與CD44v8-10有非常好的共定位,這提示我們分選得到的細(xì)胞為胃癌腫瘤干細(xì)胞;實(shí)時(shí)定量PCR法和免疫印跡法結(jié)果則顯示,與CD133-細(xì)胞相比,CD133+細(xì)胞中腫瘤干細(xì)胞標(biāo)記物OCT4,SOX2和NANOG等出現(xiàn)明顯的高表達(dá),這進(jìn)一步驗(yàn)證了CD133+細(xì)胞具有腫瘤干細(xì)胞的特質(zhì)。同時(shí),CD133+細(xì)胞中l(wèi)ncRNAROR的表達(dá)量較CD133-顯著增多,并且患者的組織標(biāo)本分析也顯示lncRNA ROR與CD133+二者表達(dá)量呈現(xiàn)顯著的正相關(guān)。結(jié)論:通過(guò)流式細(xì)胞分選技術(shù),我們使用MKN-45細(xì)胞成功構(gòu)建了胃癌腫瘤干細(xì)胞細(xì)胞系CD133+,并且測(cè)定胃癌干細(xì)胞中l(wèi)ncRNA ROR表達(dá)顯著增高。第二部分lncRNA ROR對(duì)胃癌干細(xì)胞增殖、侵襲和凋亡能力的影響背景:近年來(lái),lncRNA的研究領(lǐng)域如火如荼,長(zhǎng)度超過(guò)200個(gè)核苷酸的非編碼RNA就稱之為lncRNA。而該類lncRNA是非編碼蛋白質(zhì),所以在原來(lái)的研究中很多人的觀點(diǎn)是其為轉(zhuǎn)錄噪音。不過(guò)隨著研究水平的提高,發(fā)現(xiàn)lncRNA不管是表觀遺傳、翻譯水平、轉(zhuǎn)錄水平等各個(gè)層次中都能夠?qū)虮磉_(dá)產(chǎn)生調(diào)控影響,同時(shí)在多種腫瘤中都有其身影。Loewer等人員還在研究中得出lncRNA ROR與一些干細(xì)胞的關(guān)鍵轉(zhuǎn)錄因子存在著相互作用,在干細(xì)胞的重編程過(guò)程中發(fā)揮重要作用。Dewi、Lagadec等研究則發(fā)現(xiàn)與誘導(dǎo)干細(xì)胞相似,腫瘤干細(xì)胞參與相同的重編程過(guò)程來(lái)維持腫瘤的生長(zhǎng)。KLF4和SOX11這兩個(gè)基因與腫瘤干細(xì)胞的功能有關(guān)聯(lián),并且這兩個(gè)基因與lncRNA ROR密切相關(guān),因此lncRNA ROR可能是胃癌干細(xì)胞一個(gè)新的治療靶點(diǎn)。綜上所述,探究lncRNA ROR在胃癌干細(xì)胞中的功能勢(shì)在必行,這為將lncRNA ROR作為治療靶點(diǎn)提供理論基礎(chǔ)。目的:探究lncRNA ROR對(duì)胃癌干細(xì)胞增殖、侵襲和凋亡能力的影響。方法:為了更好的探究lncRNA ROR在胃癌干細(xì)胞中的功能,我們將成功建立了lncRNA ROR的過(guò)表達(dá)系統(tǒng)以及敲低系統(tǒng)。我們將該過(guò)表達(dá)質(zhì)粒轉(zhuǎn)染至CD133-細(xì)胞中,外源性過(guò)表達(dá)lncRNAROR;同時(shí),將能夠特異性抑制lncRNA ROR的siRNA轉(zhuǎn)染至CD133+細(xì)胞中,敲低內(nèi)源性lncRNA ROR的表達(dá)。然后,在兩組中,我們使用MTT法和EdU摻入法檢測(cè)改變lncRNA ROR表達(dá)對(duì)細(xì)胞增殖能力的影響。隨后,在處理細(xì)胞24 h后,我們使用Transwel1法檢測(cè)改變lncRNA ROR表達(dá)對(duì)細(xì)胞侵襲能力的影響。最后,我們利用流式細(xì)胞術(shù)使用Annexin V-FITC/PI雙染法檢測(cè)改變lncRNA ROR表達(dá)對(duì)細(xì)胞凋亡能力的影響。結(jié)果:MTT法、EdU摻入法、Transwell法以及流式細(xì)胞術(shù)檢測(cè)結(jié)果顯示,在CD133-細(xì)胞中過(guò)表達(dá)lncRNA ROR能夠顯著提高細(xì)胞的增殖和侵襲能力,但對(duì)細(xì)胞凋亡能力影響并不大。與此相反,在CD133+細(xì)胞中抑制內(nèi)源性lncRNAROR則能夠明顯抑制細(xì)胞的增殖和侵襲能力,并且能夠促進(jìn)細(xì)胞的凋亡能力。結(jié)論:內(nèi)源性lncRNA ROR對(duì)胃癌干細(xì)胞增殖、侵襲以及抑制凋亡能力至關(guān)重要。第三部分lncRNA ROR能夠調(diào)控維持胃癌干細(xì)胞特性的轉(zhuǎn)錄因子的表達(dá)背景:研究結(jié)果顯示,干細(xì)胞基因的表達(dá)不正常,和惡性腫瘤發(fā)生有密切關(guān)系。而且對(duì)于胃癌的出現(xiàn)、發(fā)展、復(fù)發(fā)、轉(zhuǎn)移過(guò)程中都有關(guān)鍵性影響。干細(xì)胞轉(zhuǎn)錄因子會(huì)使得有關(guān)基因簇2(sexdeterminingregionY-box2,SOX2)表達(dá)不正常時(shí),致使胃粘膜上皮細(xì)胞分化失衡紊亂,引發(fā)腫瘤出現(xiàn);在細(xì)胞多能性的特點(diǎn)中,能夠維持該功能的標(biāo)志物是八聚體結(jié)合蛋白-4(octamerbindingfactor4,OCT4),該蛋白能夠和細(xì)胞分化有密切關(guān)系;Nanog與體細(xì)胞實(shí)體瘤的關(guān)系也日益受到關(guān)注,NANOG具有激發(fā)腫瘤形成的干細(xì)胞樣特性。因此,本研究以胃癌干細(xì)胞為研究對(duì)象,改變內(nèi)源性lncRNA ROR表達(dá)后,探討lncRNA ROR對(duì)維持胃癌干細(xì)胞特性的重要轉(zhuǎn)錄因子的作用,為胃癌的臨床治療和預(yù)后判斷提供可能的參考依據(jù)。目的:探究lncRNA ROR對(duì)胃癌干細(xì)胞特性的影響。方法:我們將該過(guò)表達(dá)質(zhì)粒轉(zhuǎn)染至CD133-細(xì)胞中,外源性過(guò)表達(dá)lncRNAROR;同時(shí),將能夠特異性抑制lncRNA ROR的siRNA轉(zhuǎn)染至CD133+細(xì)胞中,敲低內(nèi)源性lncRNAROR的表達(dá)。然后,在兩組中,我們使用實(shí)時(shí)定量PCR法和免疫印跡法檢測(cè)維持胃癌干細(xì)胞特性的關(guān)鍵轉(zhuǎn)錄因子OCT4、SOX2和NANOG的表達(dá)水平。結(jié)果:實(shí)時(shí)定量PCR法和免疫印跡法檢測(cè)結(jié)果顯示,在CD133-細(xì)胞中過(guò)表達(dá)lncRNA ROR能夠有效的上調(diào)OCT4、SOX2和NANOG的表達(dá)水平。與此相反,在CD133+細(xì)胞中抑制內(nèi)源性lncRNA ROR則能夠明顯抑制OCT4、SOX2和NANOG的表達(dá)水平。結(jié)論:胃癌干細(xì)胞中,內(nèi)源性lncRNA ROR的高表達(dá)能夠通過(guò)上調(diào)OCT4、SOX2和NANOG等的表達(dá)水平,有效的維持胃癌干細(xì)胞特性。
[Abstract]:The first part is the separation and identification of gastric cancer stem cells and the expression of lncRNAROR in the cells. Background: gastric cancer is one of the fourth malignant tumors in the world. In the incidence of cancer, gastric cancer ranks second in our country, and the number of gastric cancer is third, which poses a great threat to people's health. Gastric cancer has the characteristics of postoperative metastasis, recurrence, drug resistance, and so far the regulatory mechanism has not been studied. In these years, the researchers have proposed the theory of tumor stem cells in the development of tumor. This theory suggests that the tumor originates in a small part of tumor cells with self renewal and multiple differentiation potential. The cells are called tumor stem cells. At present, the method of separating and purifying cancer stem cells with specific marker molecules on the surface of cancer stem cells is more common, and the research has been done by using the specific cell surface marker molecule CD133, using flow cytometry to separate the gastric cancer stem cells from gastric cancer, and prove that CD133 is the stomach. The surface markers of cancer stem cells. Objective: to construct the cell line of gastric cancer stem cells using MKN-45 cells and detect the function of lncRNA ROR. Methods: human MKN-45 gastric cancer cells were cultured in RMPI1640 culture medium, made single cell suspension, and adjusted the cell concentration after adding CD133-FITC anti body, and then used the flow cytometry to collect the cells. The cells labeled by CD133-FITC, namely, CD133+MKN-45 cells (CD133+), collect the remaining cells that are not labeled with CD133-FITC antibodies, that is, CD133-MKN-45 cells (called CD133-). The co expression of CD133 and CD44v8-10 in CD133+ cells is detected by immunofluorescence; and then the real-time quantitative PCR method and immunoblotting are used. The expression of OCT4, SOX2, NANOG and other tumor stem cell markers in CD133+ and CD133- cells was detected by method. The expression of lncRNA ROR in CD133- and CD133+ cells was detected by real-time quantitative PCR method, and the relationship between lncRNA ROR and the amount of expression was analyzed. Results: immunofluorescence assay The results showed that the selected CD133+ cell surface markers CD133 and CD44v8-10 were well Co located, which suggested that the selected cells were cancer stem cells of gastric cancer. Real-time quantitative PCR and immunoblotting results showed that the markers of OCT4, SOX2 and NANOG in CD133+ cells were compared with CD133- cells. Obvious high expression, which further verified the characteristics of CD133+ cells with tumor stem cells. At the same time, the expression of lncRNAROR in CD133+ cells was significantly higher than that of CD133-, and the tissue specimen analysis of the patients showed that the expression of lncRNA ROR was positively correlated with the expression of CD133+ two. Conclusion: we use flow cytometry, we use MKN-45 cells successfully constructed the cancer stem cell line CD133+, and the expression of lncRNA ROR in gastric cancer stem cells was significantly increased. Second the impact of lncRNA ROR on the proliferation, invasion and apoptosis of gastric cancer stem cells: in recent years, the research field of lncRNA is like a raging fire, and the non coded RNA with a length of more than 200 nucleotides is known as a non coded RNA. It is lncRNA. and this class of lncRNA is a non coding protein, so in the previous study, many people have the view that they are transcriptional noise. However, as the level of research is raised, it is found that lncRNA can regulate the expression of genes at all levels, regardless of epigenetic, translation and transcriptional levels, as well as in many kinds of tumors. .Loewer and other people have also found that lncRNA ROR has interaction with key transcription factors of some stem cells, plays an important role in the reprogramming of stem cells,.Dewi, Lagadec and other studies have found that the stem cells are similar to induced stem cells, and the tumor stem cells participate in the same reprogramming process to maintain the growth of the tumor.KL The two genes of F4 and SOX11 are associated with the function of cancer stem cells, and these two genes are closely related to lncRNA ROR. Therefore, lncRNA ROR may be a new target for the treatment of gastric cancer stem cells. To sum up, it is imperative to explore the function of lncRNA ROR in gastric cancer stem cells, which provides the theoretical basis for the lncRNA ROR as a target for treatment. Objective: To explore the effect of lncRNA ROR on the proliferation, invasion and apoptosis of gastric cancer stem cells. Methods: in order to better explore the function of lncRNA ROR in gastric cancer stem cells, we will successfully establish the overexpression system of lncRNA ROR and the knockout system. We transfect the overexpressed particles into CD133- cells and express the exogenous lncRNA. In the two groups, we used MTT and EdU incorporation to detect the effect of lncRNA ROR expression on cell proliferation ability in the two groups. Then, after processing cell 24 h, we used the method to detect the change of siRNA after 24 h. In the two groups, we used the MTT method and EdU incorporation method to detect the effect of the expression of lncRNA ROR on the cell proliferation. The effect of OR expression on cell invasiveness. Finally, we used Annexin V-FITC/PI double staining to detect the effect of lncRNA ROR expression on the cell apoptosis ability. Results: MTT, EdU incorporation, Transwell and flow cytometry showed that the overexpression of lncRNA ROR in CD133- cells could be significantly improved. Cell proliferation and invasion ability, but not to cell apoptosis, in contrast, the inhibition of endogenous lncRNAROR in CD133+ cells can significantly inhibit cell proliferation and invasion ability, and can promote cell apoptosis. Conclusion: endogenous lncRNA ROR proliferation, invasion and inhibition of apoptosis in gastric cancer stem cells The third part, the third part, can regulate the expression background of the transcription factors that maintain the characteristics of gastric cancer stem cells. The results show that the expression of stem cell genes is abnormal and has a close relationship with the occurrence of malignant tumors. It also has a critical effect on the occurrence, development, recurrence and metastasis of gastric cancer. Stem cell transcription factors will be important. When the expression of the gene cluster 2 (sexdeterminingregionY-box2, SOX2) is abnormal, the differentiation of the epithelial cells of the gastric mucosa is unbalanced and causes the appearance of the tumor. In the characteristics of cell pluripotent, the marker that can maintain the function is the eight polymer binding protein -4 (octamerbindingfactor4, OCT4), which can be closely related to the differentiation of cells. The relationship between Nanog and somatic solid tumor is becoming more and more concerned, and NANOG has the stem cell like characteristics to stimulate the formation of tumor. Therefore, this study takes gastric cancer stem cells as the research object, changes the endogenous lncRNA ROR expression, and explores the role of lncRNA ROR in maintaining the important transcription factors of gastric cancer stem cell specificity for the clinical treatment of gastric cancer. Objective: to provide a possible reference for the treatment and prognosis. Objective: To explore the effect of lncRNA ROR on the characteristics of gastric cancer stem cells. Methods: We transfected the overexpressed plasmid into CD133- cells and overexpressed lncRNAROR; meanwhile, we transfected the siRNA of lncRNA ROR to CD133+ cells and knocked down the expression of low endogenous lncRNAROR. Then, in the two groups, we used real-time quantitative PCR and immunoblotting to detect the expression level of key transcription factors, OCT4, SOX2 and NANOG, to maintain the characteristics of gastric cancer stem cells. Results: real-time quantitative PCR and Western blot detection results showed that the overexpression of lncRNA ROR in CD133- cells could effectively increase the OCT4, SOX2 and NANOG tables. In contrast, inhibition of endogenous lncRNA ROR in CD133+ cells can significantly inhibit the expression level of OCT4, SOX2 and NANOG. Conclusion: the high expression of endogenous lncRNA ROR in gastric cancer stem cells can effectively maintain the characteristics of gastric cancer stem cells by up regulation of the expression level of OCT4, SOX2 and NANOG.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.2

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本文編號(hào)：2097527