人结肠癌伊立替康耐药的生物学特性及其机制研究

发布时间：2018-07-05 01:18

本文选题：结肠癌 + 多药耐药　；参考：《南方医科大学》2017年硕士论文

【摘要】：伊立替康(Irinotecan,CPT-11)主要用于出现转移或应用标准的氟尿嘧啶(5-Fluorouracil,5-FU)治疗后失败的进展期结直肠癌患者。然而,仅有30-55%患者对伊立替康的治疗方案有效,且5年存活率小于10%。耐药是限制结直肠癌患者疗效的主要因素之一。Twist1基因是一种高度保守的碱性螺旋-环-螺旋(basic helix-loop-helix,bHLH)转录因子,在细胞的上皮-间充质转化(Epithelial-mesenchymal transition,EMT)、肿瘤干细胞(Cancer stem cells,CSC)、多药耐药(multidrug resistance,MDR)、肿瘤的侵袭转移、细胞增殖分化形成等方面发挥重要的作用。我们前期研究发现,Twist1高表达是结肠癌患者不良预后的独立危险因素之一,并且在结肠癌细胞多药耐药和侵袭转移中发挥重要作用。然而,由Twist1介导的结肠癌伊立替康多药耐药形成及其恶性生物学行为的作用机制仍不清楚。目的1.通过构建结肠癌伊立替康耐药细胞株,分析评价结肠癌耐药细胞的恶性生物学特性;2.探讨Twist1介导结肠癌伊立替康多药耐药形成及其恶性生物学行为的作用机制。方法第一部分人结肠癌伊立替康耐药细胞株的构建、评价及其恶性生物学特性采用药物浓度递增法构建人结肠癌伊立替康耐药模型LoVo/CPT-11R细胞株,光镜下观察细胞形态学改变;CCK-8法检测耐药细胞生存率变化,计算耐药指数(Resistant index,RI),并分别检测细胞耐药前后对不同药物(CPT-11、5-FU、顺铂(Cisplatin,DDP)及姜黄素(Curcumin,Cur))生存率的变化;细胞免疫荧光法检测耐药细胞EMT分子标志物细胞定位;qRT-PCR、Western blot法分别检测耐药细胞多药耐药基因、EMT分子标志物、转录因子、CSC分子标志物在mRNA及蛋白质水平表达变化。第二部分Twist1介导结肠癌伊立替康多药耐药形成及其恶性生物学行为的作用机制采用慢病毒转染法构建Twist1稳定过表达的人结肠癌LoVo/Twist1细胞株;采用siRNA干扰技术抑制LoVo细胞和LoVo/CPT-11R细胞中Twist1的表达。CCK-8法分别检测Twist1过表达前后和抑制表达前后其对伊立替康敏感性变化;Transwell实验检测LoVo/Twist1细胞侵袭迁移能力的变化;同前方法检测EMT、CSC标志物及MMPs、ABC转运蛋白等目的基因表达变化。结果第一部分1.LoVo/CPT-11R细胞能在含70 μg/mL伊立替康的全培中稳定生长,耐药指数为5.79;细胞体积明显增大,长梭形纤维状细胞增多,生长弥散无规律。2.不同浓度CPT-11、5-FU、DDP、Cur处理细胞后,LoVo/CPT-11R细胞的生存率均明显增加;在mRNA和蛋白质水平,ABCB1(P-gp)表达明显上调。3.LoVo/CPT-11R细胞中E-cadherin蛋白更多表达于细胞质,更少表达于细胞膜,位于细胞骨架的Vimentin蛋白表达明显增加;在mRNA和蛋白质水平,EMT标志物中E-cadherin、ZO-1 下调,Vimentin、N-cadherin上调;转录因子中Twist1表达上调;CSC标志物中CD44、CD133表达均上调。第二部分1.过表达Twist1的LoVo细胞对伊立替康的敏感性降低;在蛋白质水平,CD44表达上调,E-cadherin下调。2.过表达Twist1的LoVo细胞侵袭、迁移能力显著增强;MMP2蛋白表达上调。3.抑制Twist1的表达后LoVo细胞对伊立替康的敏感性增加,并逆转LoVo/CPT-11R细胞对伊立替康的耐药。4.抑制Twist1的表达可以下调ABCB1(P-gp)、Vimentin、CD44的表达。结论第一部分1.人结肠癌伊立替康耐药细胞具有多药耐药特性,并且与ABCB1(P-gp)的高表达有关;2.人结肠癌伊立替康耐药细胞发生EMT、CSC样表型改变,且Twist1上调。第二部分1.Twist1介导的EMT、CSC样表型改变诱导结肠癌伊立替康耐药形成;2.Twist1介导ABCB1及MMP2的上调促进结肠癌细胞伊立替康耐药及增强侵袭迁移能力。
[Abstract]:Irinotecan (CPT-11) is mainly used for the onset of advanced colorectal cancer patients who have failed to transfer or apply standard 5-Fluorouracil (5-FU) treatment. However, only 30-55% patients are effective in the treatment of erinotecan, and the 5 year survival rate is less than 10%. Resistance is the main factor limiting the efficacy of colorectal cancer patients. A.Twist1 gene is a highly conserved basic helix basic helix-loop-helix (bHLH) transcription factor, in cell epithelial mesenchymal transition (Epithelial-mesenchymal transition, EMT), tumor stem cells (Cancer stem cells, CSC), multidrug resistance (multidrug), tumor invasion and metastasis, cell proliferation and differentiation Our previous studies have shown that high expression of Twist1 is one of the independent risk factors for the poor prognosis of colon cancer patients and plays an important role in multidrug resistance and invasion and metastasis of colon cancer cells. However, the formation of irinotecan drug resistance and its malignant biological behavior mediated by Twist1 The mechanism of action still remains unclear. Objective 1. to evaluate the malignant biological characteristics of colon cancer resistant cells by constructing irinotecan resistant cell lines of colon cancer; 2. to explore the mechanism of Twist1 mediated formation of irinotecan and its malignant biological behavior in colon cancer. Method first part of the human colon cancer irinotecan resistant cells The construction, evaluation and its malignant biological characteristics were constructed by the method of increasing drug concentration to construct the LoVo/CPT-11R cell line of irinotecan resistant model of human colon cancer. The morphological changes of cells were observed under light microscope, the change of the survival rate of drug-resistant cells was detected by CCK-8 method, the resistance index (Resistant index, RI) was calculated, and the difference of the drug resistance before and after the cell resistance was detected respectively. Changes in the survival rate of drugs (CPT-11,5-FU, Cisplatin, DDP) and curcumin (Curcumin, Cur)); cell immunofluorescence assay for the detection of EMT molecular marker cell location in drug-resistant cells; qRT-PCR, Western blot methods to detect multidrug resistance genes, EMT sub markers, transcription factors, CSC molecular markers in mRNA and protein levels, respectively. Expression changes. Second part Twist1 mediates the formation of irinotecan drug resistance in colon cancer and the mechanism of its malignant biological behavior, the Twist1 stable overexpressed human colon cancer LoVo/Twist1 cell line is constructed by the slow virus transfection method, and the siRNA interference technique is used to inhibit the expression of Twist1 in LoVo cells and LoVo/ CPT-11R cells, respectively. The changes in the susceptibility to eritecan before and after Twist1 overexpression were detected, and the changes in the invasion and migration of LoVo/Twist1 cells were detected by Transwell. The expression of EMT, CSC markers, MMPs, ABC transporter and other target genes were detected by the same method. Results the first part of 1.LoVo/CPT-11R cells in the first part of the cell was able to contain 70 u g/mL irinott. The drug resistance index was 5.79, the cell volume was 5.79, the cell volume increased obviously, the long spindle shaped fibroid cells increased, the growth and dispersion of different concentrations of CPT-11,5-FU, DDP, Cur treated cells, and the survival rate of LoVo/CPT-11R cells increased obviously; the expression of ABCB1 (P-gp) in mRNA and protein level was obviously up regulation of.3.LoVo/CPT-11R cells. E-cadherin protein was more expressed in cytoplasm, less expressed in cell membrane, Vimentin protein expression in cytoskeleton increased obviously; in mRNA and protein level, E-cadherin, ZO-1 down regulation, Vimentin, N-cadherin up regulation in EMT markers, Twist1 expression in the transcription factor up regulation, CSC marker CD44, up regulation of CD133 expression. Second The sensitivity of LoVo cells with 1. over expression of Twist1 to erinotecan decreased; at the protein level, the expression of CD44 was up-regulated, and E-cadherin down regulated the LoVo cell invasion of.2. overexpressed Twist1, and the migration ability was significantly enhanced; MMP2 protein expression increased the.3. inhibitory Twist1 expression and increased sensitivity of LoVo cells to irinotecan, and reversed LoVo/CPT-11R fines. The expression of irinotecan resistant.4. inhibited the expression of Twist1 can down regulate the expression of ABCB1 (P-gp), Vimentin, CD44. Conclusion the first part of the 1. human colon cancer irinotecan resistant cells has multiple drug resistance characteristics, and is related to the high expression of ABCB1 (P-gp); 2. human colon cancer irinotecan resistant cells occur EMT, CSC like phenotypic changes, and Twist1 on Second 1.Twist1 mediated EMT, CSC like phenotype changes induce erinecan resistance formation in colon cancer; 2.Twist1 mediated up-regulation of ABCB1 and MMP2 promotes irinotecan resistance and enhanced invasion and migration of colon cancer cells.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.35

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