本文選題：結(jié)腸癌 + 多藥耐藥�。� 參考：《南方醫(yī)科大學(xué)》2017年碩士論文

【摘要】：伊立替康(Irinotecan,CPT-11)主要用于出現(xiàn)轉(zhuǎn)移或應(yīng)用標(biāo)準(zhǔn)的氟尿嘧啶(5-Fluorouracil,5-FU)治療后失敗的進(jìn)展期結(jié)直腸癌患者。然而,僅有30-55%患者對伊立替康的治療方案有效,且5年存活率小于10%。耐藥是限制結(jié)直腸癌患者療效的主要因素之一。Twist1基因是一種高度保守的堿性螺旋-環(huán)-螺旋(basic helix-loop-helix,bHLH)轉(zhuǎn)錄因子,在細(xì)胞的上皮-間充質(zhì)轉(zhuǎn)化(Epithelial-mesenchymal transition,EMT)、腫瘤干細(xì)胞(Cancer stem cells,CSC)、多藥耐藥(multidrug resistance,MDR)、腫瘤的侵襲轉(zhuǎn)移、細(xì)胞增殖分化形成等方面發(fā)揮重要的作用。我們前期研究發(fā)現(xiàn),Twist1高表達(dá)是結(jié)腸癌患者不良預(yù)后的獨(dú)立危險(xiǎn)因素之一,并且在結(jié)腸癌細(xì)胞多藥耐藥和侵襲轉(zhuǎn)移中發(fā)揮重要作用。然而,由Twist1介導(dǎo)的結(jié)腸癌伊立替康多藥耐藥形成及其惡性生物學(xué)行為的作用機(jī)制仍不清楚。目的1.通過構(gòu)建結(jié)腸癌伊立替康耐藥細(xì)胞株,分析評價(jià)結(jié)腸癌耐藥細(xì)胞的惡性生物學(xué)特性;2.探討Twist1介導(dǎo)結(jié)腸癌伊立替康多藥耐藥形成及其惡性生物學(xué)行為的作用機(jī)制。方法第一部分人結(jié)腸癌伊立替康耐藥細(xì)胞株的構(gòu)建、評價(jià)及其惡性生物學(xué)特性采用藥物濃度遞增法構(gòu)建人結(jié)腸癌伊立替康耐藥模型LoVo/CPT-11R細(xì)胞株,光鏡下觀察細(xì)胞形態(tài)學(xué)改變;CCK-8法檢測耐藥細(xì)胞生存率變化,計(jì)算耐藥指數(shù)(Resistant index,RI),并分別檢測細(xì)胞耐藥前后對不同藥物(CPT-11、5-FU、順鉑(Cisplatin,DDP)及姜黃素(Curcumin,Cur))生存率的變化;細(xì)胞免疫熒光法檢測耐藥細(xì)胞EMT分子標(biāo)志物細(xì)胞定位;qRT-PCR、Western blot法分別檢測耐藥細(xì)胞多藥耐藥基因、EMT分子標(biāo)志物、轉(zhuǎn)錄因子、CSC分子標(biāo)志物在mRNA及蛋白質(zhì)水平表達(dá)變化。第二部分Twist1介導(dǎo)結(jié)腸癌伊立替康多藥耐藥形成及其惡性生物學(xué)行為的作用機(jī)制采用慢病毒轉(zhuǎn)染法構(gòu)建Twist1穩(wěn)定過表達(dá)的人結(jié)腸癌LoVo/Twist1細(xì)胞株;采用siRNA干擾技術(shù)抑制LoVo細(xì)胞和LoVo/CPT-11R細(xì)胞中Twist1的表達(dá)。CCK-8法分別檢測Twist1過表達(dá)前后和抑制表達(dá)前后其對伊立替康敏感性變化;Transwell實(shí)驗(yàn)檢測LoVo/Twist1細(xì)胞侵襲遷移能力的變化;同前方法檢測EMT、CSC標(biāo)志物及MMPs、ABC轉(zhuǎn)運(yùn)蛋白等目的基因表達(dá)變化。結(jié)果第一部分1.LoVo/CPT-11R細(xì)胞能在含70 μg/mL伊立替康的全培中穩(wěn)定生長,耐藥指數(shù)為5.79;細(xì)胞體積明顯增大,長梭形纖維狀細(xì)胞增多,生長彌散無規(guī)律。2.不同濃度CPT-11、5-FU、DDP、Cur處理細(xì)胞后,LoVo/CPT-11R細(xì)胞的生存率均明顯增加;在mRNA和蛋白質(zhì)水平,ABCB1(P-gp)表達(dá)明顯上調(diào)。3.LoVo/CPT-11R細(xì)胞中E-cadherin蛋白更多表達(dá)于細(xì)胞質(zhì),更少表達(dá)于細(xì)胞膜,位于細(xì)胞骨架的Vimentin蛋白表達(dá)明顯增加;在mRNA和蛋白質(zhì)水平,EMT標(biāo)志物中E-cadherin、ZO-1 下調(diào),Vimentin、N-cadherin上調(diào);轉(zhuǎn)錄因子中Twist1表達(dá)上調(diào);CSC標(biāo)志物中CD44、CD133表達(dá)均上調(diào)。第二部分1.過表達(dá)Twist1的LoVo細(xì)胞對伊立替康的敏感性降低;在蛋白質(zhì)水平,CD44表達(dá)上調(diào),E-cadherin下調(diào)。2.過表達(dá)Twist1的LoVo細(xì)胞侵襲、遷移能力顯著增強(qiáng);MMP2蛋白表達(dá)上調(diào)。3.抑制Twist1的表達(dá)后LoVo細(xì)胞對伊立替康的敏感性增加,并逆轉(zhuǎn)LoVo/CPT-11R細(xì)胞對伊立替康的耐藥。4.抑制Twist1的表達(dá)可以下調(diào)ABCB1(P-gp)、Vimentin、CD44的表達(dá)。結(jié)論第一部分1.人結(jié)腸癌伊立替康耐藥細(xì)胞具有多藥耐藥特性,并且與ABCB1(P-gp)的高表達(dá)有關(guān);2.人結(jié)腸癌伊立替康耐藥細(xì)胞發(fā)生EMT、CSC樣表型改變,且Twist1上調(diào)。第二部分1.Twist1介導(dǎo)的EMT、CSC樣表型改變誘導(dǎo)結(jié)腸癌伊立替康耐藥形成;2.Twist1介導(dǎo)ABCB1及MMP2的上調(diào)促進(jìn)結(jié)腸癌細(xì)胞伊立替康耐藥及增強(qiáng)侵襲遷移能力。
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.35

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