本文關(guān)鍵詞： EphA2 鼻咽癌 化療耐藥性 P-gp PI3-K/Akt 自噬作用 紫杉醇　出處：《中南大學》2014年博士論文　論文類型：學位論文

【摘要】：目的：鼻咽癌紫杉醇耐藥性的形成是一復雜調(diào)控機制,鑒于經(jīng)典耐藥理論中P-gp蛋白發(fā)揮的重要作用以及近年來自噬作用在腫瘤化療耐藥領(lǐng)域的新興熱點地位,本研究在發(fā)現(xiàn)EphA2可對鼻咽癌細胞紫杉醇耐藥性起調(diào)控作用的前期研究基礎(chǔ)上,旨在進一步探討EphA2調(diào)控鼻咽癌細胞紫杉醇耐藥性的P-gp相關(guān)及自噬相關(guān)分子機制。 方法：1.利用Western Blot分別檢測鼻咽癌細胞中慢病毒介導的EphA2沉默及過表達組與陰性對照組(轉(zhuǎn)染無義shRNA或無義cDNA)和空白對照組(轉(zhuǎn)染空載體)內(nèi)P-gp蛋白的表達情況,探討EphA2是否具有調(diào)控鼻咽癌細胞P-gp表達的能力； 2.利用siRNA干擾技術(shù)在鼻咽癌細胞中下調(diào)P-gp的表達,同時給予空白對照組(原始細胞)、陰性對照組(轉(zhuǎn)染無義序列)和P-gp表達下調(diào)組0.1nM/L的紫杉醇(該濃度為前期研究所得的原始細胞的IC30濃度,結(jié)合文獻,考慮到P-gp表達下調(diào)后的紫杉醇增敏作用,故選擇該濃度)刺激,48小時后CCK-8法檢測吸光度,得到各組的存活率,以說明P-gp蛋白的表達改變是否影響鼻咽癌細胞的紫杉醇耐藥行為； 3.利用siRNA干擾技術(shù)在EphA2過表達鼻咽癌細胞中下調(diào)P-gp的表達,同時給予空白對照組(EphA2過表達細胞)、陰性對照組(EphA2過表達并轉(zhuǎn)染無義序列細胞)和P-gp表達下調(diào)組(EphA2過表達并P-gp表達下調(diào)細胞)5nM/L的紫杉醇(前期研究基礎(chǔ)中該濃度下實驗組與對照組存活率差異最大)刺激,48小時后CCK-8法檢測吸光度,得到各組的存活率,以探討在EphA2過表達細胞中,調(diào)控P-gp表達對鼻咽癌細胞紫杉醇耐藥性的影響； 4.鑒于文獻報道PI3-K/Akt通路參與P-gp蛋白表達調(diào)控以及前期研究發(fā)現(xiàn)EphA2通過PI3-K/Akt通路影響鼻咽癌紫杉醇耐藥性,本研究在EphA2過表達鼻咽癌細胞中,分別加入0nM/L和20nM/L的PI3-K/Akt抑制劑LY294002,再給予5nM/L的紫杉醇刺激,48小時后利用Western Blot檢測各組PI3-K/Akt信號通路蛋白Akt和p-Akt,以及P-gp的表達水平,以探討在EphA2過表達細胞中,PI3-K/Akt通路是否參與P-gp蛋白的表達調(diào)控； 5.給予實驗組(EphA2過表達鼻咽癌細胞)及對照組(轉(zhuǎn)染無義cDNA)5nM/L紫杉醇刺激,48小時后利用Western Blot檢測自噬標志性LC3B-Ⅱ蛋白在實驗組及對照組中的表達差異,初步探討EphA2調(diào)控鼻咽癌紫杉醇耐藥性的可能機制(自噬作用)。 結(jié)果：1.EphA2沉默組中P-gp蛋白較兩對照組表達下調(diào),EphA2過表達組中P-gp蛋白較兩對照組表達上調(diào)； 2.紫杉醇作用于空白對照組、陰性對照組和P-gp表達下調(diào)組的存活率分別是68.9±4.0%,72.4±6.2%v.s.52.1±3.7%,P-gp表達下調(diào)組對紫杉醇的耐藥性較兩對照組降低(P0.05)； 3.在EphA2過表達鼻咽癌細胞中,紫杉醇作用于空白對照組、陰性對照組和P-gp表達下調(diào)組的存活率分別是57.9±6.2%,58.2±5.8%v.s.36.1±5.1%,P-gp表達下調(diào)組對紫杉醇的耐藥性較兩對照組降低(P0.05)； 4.在EphA2過表達鼻咽癌細胞中,給予通路抑制劑組較對照組Akt表達基本不變,而磷酸化的Akt表達下降,P-gp表達下降； 5. EphA2過表達鼻咽癌細胞株中自噬標志性LC3B-Ⅱ蛋白的表達較對照組顯著上調(diào)。 結(jié)論：1.EphA2具有調(diào)控鼻咽癌細胞P-gp表達的能力； 2.下調(diào)P-gp蛋白表達可降低鼻咽癌細胞紫杉醇耐藥性； 3.下調(diào)P-gp蛋白表達可逆轉(zhuǎn)EphA2介導的鼻咽癌紫杉醇耐藥性； 4.在EphA2過表達細胞中,PI3-K/Akt通路參與P-gp蛋白的表達調(diào)控； 5.在經(jīng)紫杉醇作用的鼻咽癌細胞中,EphA2表達的改變能影響自噬相關(guān)蛋白的表達水平,提示EphA2也可能經(jīng)白噬途徑調(diào)控鼻咽癌細胞的紫杉醇耐藥性。 綜上所述,EphA2可通過介導P-gp蛋白表達,進而調(diào)控鼻咽癌細胞紫杉醇耐藥性,PI3-K/Akt通路可能參與這一過程；此外,EphA2還可能經(jīng)自噬途徑調(diào)控鼻咽癌細胞紫杉醇耐藥性。EphA2有望成為鼻咽癌紫杉醇增敏靶點。
[Abstract]:Objective: the formation of taxol resistance is a complex regulatory mechanism, in view of the important role of P-gp protein in the theory of classical resistance and apoptosis in play in tumor chemotherapy resistance in the field of emerging hot status, this study found that EphA2 in paclitaxel resistance in nasopharyngeal carcinoma cells based on the regulation of early research, aimed at further to explore the P-gp and autophagy related molecular mechanism of EphA2 regulation of paclitaxel resistance in nasopharyngeal carcinoma cells.
Methods: 1. Blot were detected by Western in nasopharyngeal carcinoma cells with lentivirus mediated EphA2 silencing and overexpression group and negative control group (transfected with shRNA or nonsense nonsense cDNA) and control group (transfected empty vector) expression of P-gp protein, and investigate whether EphA2 has the ability to control the expression of P-gp in nasopharyngeal carcinoma cells;
2. expression by siRNA interference down-regulation of P-gp in nasopharyngeal carcinoma cells, while giving the blank control group (original cells), negative control group (transfected antisense sequence) downregulation of P-gp and 0.1nM/L group (the concentration of paclitaxel for preliminary research the original cell concentration of IC30, combined with the literature, considering the expression of P-gp after the decline of paclitaxel sensitization, the choice of the concentration) stimulation, CCK-8 method was used to detect the absorbance after 48 hours, get the survival rate of each group, to illustrate the expression of P-gp protein changes effect of paclitaxel resistance in nasopharyngeal carcinoma cells;
3. by siRNA interference expression in nasopharyngeal carcinoma cells down-regulation of P-gp in EphA2, while giving the control group (EphA2 cells), negative control group (transfected with EphA2 overexpression and antisense sequence of cells) and P-gp expression group (EphA2 overexpression and P-gp expression down regulated cells of paclitaxel (5nM/L) the basis of the preliminary study in the concentration of the experimental group and the control group survival rate difference) stimulation, CCK-8 method was used to detect the absorbance after 48 hours, get the survival rate of each group, in order to explore the expression of EphA2 in cells, regulate the expression of P-gp on paclitaxel resistance in nasopharyngeal carcinoma cells alcohol;
In view of the 4. reported that PI3-K/Akt pathway is involved in regulation of P-gp protein expression and the previous study found that EphA2 through PI3-K/Akt signaling pathway of taxol resistance in this study, overexpression of EphA2 in nasopharyngeal carcinoma cells, the PI3-K/Akt inhibitor LY294002 were added to 0nM/L and 20nM/L, then given 5nM/L paclitaxel stimulation, 48 hours after the use of Western Blot to detect the PI3-K/Akt signal pathway protein Akt and p-Akt, and the expression level of P-gp, to investigate the expression of EphA2 in cells, regulating the expression of PI3-K/Akt pathway P-gp protein;
5. given the experimental group (EphA2 overexpression in nasopharyngeal carcinoma cells) and control group (transfection nonsense cDNA 5nM/L paclitaxel) stimulation, 48 hours after the use of Western Blot to detect marker expression difference of LC3B- protein in the experimental group and control group, to discuss the possible mechanism of EphA2 regulation of nasopharyngeal carcinoma paclitaxel resistance (autophagy).
Results: the expression of P-gp protein in the 1.EphA2 silencing group was lower than that in the two control group, and the expression of P-gp protein in the EphA2 overexpression group was up up compared with the two control group.
2. paclitaxel played a role in the blank control group. The survival rate of the negative control group and the P-gp expression downregulation group was 68.9 + 4%, 72.4 + 6.2%v.s.52.1 + 3.7%, and the P-gp expression down-regulation group had lower resistance to paclitaxel than the two control group (P0.05).
3., in EphA2 overexpressing nasopharyngeal carcinoma cells, paclitaxel acted on the blank control group. The survival rates of the negative control group and the P-gp expression downregulation group were 57.9 + 6.2%, 58.2 + 5.8%v.s.36.1 + 5.1%, and the P-gp expression downregulation group was lower than that of the two control group (P0.05).
4. in EphA2 over expression of nasopharyngeal carcinoma cells, the expression of Akt in the given pathway inhibitor group was less than that of the control group, but the expression of phosphorylated Akt decreased and the expression of P-gp decreased.
The expression of autophagic marker LC3B- II protein in 5. EphA2 overexpressed nasopharyngeal carcinoma cell lines was significantly higher than that in the control group.
Conclusion: 1.EphA2 has the ability to regulate the expression of P-gp in nasopharyngeal carcinoma cells.
2. down regulation of P-gp protein could reduce the drug resistance of taxol in nasopharyngeal carcinoma cells.
3. the expression of P-gp protein could reverse the drug resistance of taxol in nasopharyngeal carcinoma (NPC) mediated by EphA2.
4. in EphA2 overexpressed cells, PI3-K/Akt pathway participates in the regulation of the expression of P-gp protein.
5., in the paclitaxel treated nasopharyngeal carcinoma cells, the change of EphA2 expression can affect the expression level of autophagy related protein, suggesting that EphA2 may also regulate the paclitaxel resistance of nasopharyngeal carcinoma cells through the white channel.
In conclusion, EphA2 can guide the protein expression of P-gp mediated by, and regulation of paclitaxel resistant nasopharyngeal carcinoma cells, PI3-K/Akt pathway may be involved in this process; in addition, EphA2 may also through the autophagy pathway regulation of paclitaxel resistance in nasopharyngeal carcinoma cells.EphA2 is expected to become the target of taxol sensitization.

【學位授予單位】：中南大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R739.63

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