發(fā)布時間：2019-06-05 08:48

【摘要】：背景:肺癌是全世界常見的惡性腫瘤之一,在我國,男性和女性的惡性腫瘤死亡率排第1位者均為肺癌。雖然對肺癌的治療有很多新的方法,但從目前情況來看,肺癌的治療效果仍然不理想,其5年生存率低于15%。大量研究表明,腫瘤干細胞(cancer stem cells,CSC)雖然在腫瘤細胞中占比很少,但卻是腫瘤發(fā)生、發(fā)展的關(guān)鍵,同時也是形成腫瘤并促成腫瘤不斷生長、侵襲、局部復(fù)發(fā)和遠處轉(zhuǎn)移的根源,也是影響肺癌預(yù)后效果的根本原因。針對性根除CSCs的藥物和治療方法仍在探索中。目的:本研究旨在觀察紫花牡荊素(Casticin)能選擇性地增強TRAIL表達以誘導(dǎo)肺癌干細胞樣細胞凋亡,并對其作用機制進行探討,為Casticin的臨床應(yīng)用提供更加充分的理論依據(jù)。方法:采用干細胞條件培養(yǎng)基在低粘附的6孔板中培養(yǎng)及擴增H446干細胞;用不同濃度Casticin和TRAIL蛋白以及固定濃度比例的Casticin與TRAIL合用將H446干細胞及普通細胞培養(yǎng)分成9組,分別用Casticin或TRAIL作用于這些細胞;第1組為未分選的普通H446細胞,培養(yǎng)基中未加Casticin或TRAIL;第2組為通過干細胞條件培養(yǎng)基分選的的H446干細胞樣細胞,培養(yǎng)基在不加Casticin或TRAIL;第3組為H446干細胞樣細胞,培養(yǎng)基終濃度為1μmol/m L的Casticin;第4組為H446干細胞樣細胞,培養(yǎng)基終濃度為3μmol/m L的Casticin;第5組為H446干細胞樣細胞,培養(yǎng)基終濃度為10μmol/m L的Casticin;第6組為H446干細胞樣細胞,培養(yǎng)基終濃度為1ng/L的TRAIL;第7組為H446干細胞樣細胞,培養(yǎng)基終濃度為10ng/L的TRAIL;第8組為H446干細胞樣細胞,培養(yǎng)基終濃度為100ng/L的TRAIL;第9組為H446干細胞樣細胞,培養(yǎng)基終濃度為100ng/L的TRAIL和3μmo L/L的Casticin;分別培養(yǎng)24h,然后在顯微鏡比較各組的成球大小與數(shù)目,并用ELISA,FACS,Western-bolt等實驗方法檢測其對H446干細胞樣細胞的凋亡作用。用慢病毒過表達與敲除Fox M1的方式來探討Casticin選擇性增強TRAIL誘導(dǎo)H446干細胞凋亡的機制。結(jié)果:利用干細胞條件培養(yǎng)基培養(yǎng)成功獲得H446干細胞樣細胞并通過蛋白質(zhì)免疫印跡法(Western-bolt)和流式細胞儀檢測得到驗證,其干細胞樣細胞標志物UPAR明顯高于普通H446細胞;用不同濃度Casticin和TRAIL蛋白單獨及聯(lián)合作用于H446干細胞樣細胞,結(jié)果顯示,Casticin與TRAIL具有抑制H446干細胞樣細胞成球數(shù)目與成球大小的能力,且Casticin能選擇性增強TRAIL的作用,兩者合用可更加明顯的抑制H446干細胞樣細胞成球能力;通過Western-bolt,ELISA,FACS等實驗方法發(fā)現(xiàn)Casticin可選擇性的增強TRAIL誘導(dǎo)H446干細胞樣細胞的凋亡,抗凋亡蛋白c-Filp,P56等的表達明顯降低;并采用慢病毒的過表達與敲除的方式確定Fox M1是Casticin選擇性增強TRAIL誘導(dǎo)H446干細胞樣細胞凋亡的重要基因。結(jié)論:1、首次證實Casticin具有促進H446干細胞樣細胞凋亡的作用;2、Casticin可通過選擇性增強TRAIL引起H446干細胞樣細胞的凋亡,其機制可能與Fox M1有關(guān)。Fox M1是Casticin引起H446干細胞凋亡的可能靶基因之一。
[Abstract]:BACKGROUND: Lung cancer is one of the most common malignant tumors in the world. Although there are many new methods for the treatment of lung cancer, the therapeutic effect of lung cancer is still not ideal, and the 5-year survival rate is less than 15%. A large number of studies have shown that the tumor stem cells (CSC), while occupying less than a few in tumor cells, are the key to the tumorigenesis and development, and at the same time are the causes of the formation of tumors and the growth, invasion, local recurrence and distant metastasis of the tumor. And also is the root cause of the effect of the prognosis of the lung cancer. The drug and treatment methods of the targeted eradication of CSCs are still in the process of exploration. Objective: The purpose of this study was to observe the ability of Casticin to selectively enhance the expression of TRAIL in order to induce the apoptosis of stem cell-like cells of lung cancer. Methods: H446 stem cells were cultured and amplified in a 6-well plate with low adhesion by stem cell conditioned medium. H446 stem cells and normal cell culture were divided into 9 groups with Castcin and TRAIL protein at different concentrations and a fixed concentration ratio of Castcin and TRAIL, and the cells were treated with Castcin or TRAIL, respectively. The first group was the unsorted normal H446 cells, and no Castcin or TRAIL was found in the culture medium; the second group was H446 stem cell-like cells sorted by the stem cell conditioned medium, and the medium was not added with Castcin or TRAIL; the third group was H446 stem cell-like cells, and the final concentration of the culture medium was 1. m The fourth group was the H446 stem cell-like cell, the final concentration of the medium was 3. m u.mol/ m L of Casticin, the fifth group was the H446 stem cell-like cell, the final concentration of the medium was 10. m u.mol/ m L, and the sixth group was the H446 stem cell-like cell, the final concentration of the medium was 1 ng/ L of TRAIL, and the seventh group was H446 stem cell-like cells, The final concentration of the medium was 10 ng/ L of TRAIL, the 8th group was H446 stem cell-like cells, the final concentration of the medium was 100 ng/ L of TRAIL, the 9th group was H446 stem cell-like cells, the final concentration of the medium was 100 ng/ L of TRAIL and 3. m The apoptosis of H446 cell-like cells was detected by ELISA, FACS, Western-blot and other experimental methods. The mechanism of the selective enhancement of TRAIL-induced apoptosis of H446 stem cells was discussed with lentiviral overexpression and the way of knock-out Fox M1. Results: H446 stem cell-like cells were successfully obtained by the culture of stem cell conditioned medium, and the cell-like cell marker UPAR was significantly higher than that of normal H446 cells by Western-blot and flow cytometry. Castcin and TRAIL were used alone and in combination on H446 stem cell-like cells. The results showed that Castcin and TRAIL had the ability to inhibit the number of cells of H446 cell-like cells and the size of the ball, and Castcin can selectively enhance the effect of TRAIL. By Western-bolt, ELISA, FACS and other experimental methods, Castcin can selectively enhance the apoptosis of H446 stem cell-like cells, and the expression of anti-apoptotic protein c-Filp, P56 and so on is obviously reduced. And the overexpression and knockout of the lentivirus is used to determine that the Fox M1 is an important gene for the selective enhancement of TRAIL to induce the apoptosis of the H446 stem cell-like cell. Conclusion:1. The first time that Castcin has the effect of promoting the apoptosis of H446 stem cell-like cells, and 2, Castcin can induce the apoptosis of H446 stem cell-like cells by selective enhancement of TRAIL, and the mechanism may be related to Fox M1. Fox M1 is one of the possible target genes for the apoptosis of H446 stem cells.
【學(xué)位授予單位】：湖南師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R734.2

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