Casticin选择性的增强TRAIL诱导H446干细胞凋亡

发布时间：2019-06-05 08:48

【摘要】：背景:肺癌是全世界常见的恶性肿瘤之一,在我国,男性和女性的恶性肿瘤死亡率排第1位者均为肺癌。虽然对肺癌的治疗有很多新的方法,但从目前情况来看,肺癌的治疗效果仍然不理想,其5年生存率低于15%。大量研究表明,肿瘤干细胞(cancer stem cells,CSC)虽然在肿瘤细胞中占比很少,但却是肿瘤发生、发展的关键,同时也是形成肿瘤并促成肿瘤不断生长、侵袭、局部复发和远处转移的根源,也是影响肺癌预后效果的根本原因。针对性根除CSCs的药物和治疗方法仍在探索中。目的:本研究旨在观察紫花牡荆素(Casticin)能选择性地增强TRAIL表达以诱导肺癌干细胞样细胞凋亡,并对其作用机制进行探讨,为Casticin的临床应用提供更加充分的理论依据。方法:采用干细胞条件培养基在低粘附的6孔板中培养及扩增H446干细胞;用不同浓度Casticin和TRAIL蛋白以及固定浓度比例的Casticin与TRAIL合用将H446干细胞及普通细胞培养分成9组,分别用Casticin或TRAIL作用于这些细胞;第1组为未分选的普通H446细胞,培养基中未加Casticin或TRAIL;第2组为通过干细胞条件培养基分选的的H446干细胞样细胞,培养基在不加Casticin或TRAIL;第3组为H446干细胞样细胞,培养基终浓度为1μmol/m L的Casticin;第4组为H446干细胞样细胞,培养基终浓度为3μmol/m L的Casticin;第5组为H446干细胞样细胞,培养基终浓度为10μmol/m L的Casticin;第6组为H446干细胞样细胞,培养基终浓度为1ng/L的TRAIL;第7组为H446干细胞样细胞,培养基终浓度为10ng/L的TRAIL;第8组为H446干细胞样细胞,培养基终浓度为100ng/L的TRAIL;第9组为H446干细胞样细胞,培养基终浓度为100ng/L的TRAIL和3μmo L/L的Casticin;分别培养24h,然后在显微镜比较各组的成球大小与数目,并用ELISA,FACS,Western-bolt等实验方法检测其对H446干细胞样细胞的凋亡作用。用慢病毒过表达与敲除Fox M1的方式来探讨Casticin选择性增强TRAIL诱导H446干细胞凋亡的机制。结果:利用干细胞条件培养基培养成功获得H446干细胞样细胞并通过蛋白质免疫印迹法(Western-bolt)和流式细胞仪检测得到验证,其干细胞样细胞标志物UPAR明显高于普通H446细胞;用不同浓度Casticin和TRAIL蛋白单独及联合作用于H446干细胞样细胞,结果显示,Casticin与TRAIL具有抑制H446干细胞样细胞成球数目与成球大小的能力,且Casticin能选择性增强TRAIL的作用,两者合用可更加明显的抑制H446干细胞样细胞成球能力;通过Western-bolt,ELISA,FACS等实验方法发现Casticin可选择性的增强TRAIL诱导H446干细胞样细胞的凋亡,抗凋亡蛋白c-Filp,P56等的表达明显降低;并采用慢病毒的过表达与敲除的方式确定Fox M1是Casticin选择性增强TRAIL诱导H446干细胞样细胞凋亡的重要基因。结论:1、首次证实Casticin具有促进H446干细胞样细胞凋亡的作用;2、Casticin可通过选择性增强TRAIL引起H446干细胞样细胞的凋亡,其机制可能与Fox M1有关。Fox M1是Casticin引起H446干细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.
【学位授予单位】：湖南师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R734.2

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