SAHA对肺腺癌厄洛替尼耐药细胞的增殖及耐药性的影响

发布时间：2018-06-19 21:36

本文选题：非小细胞肺癌 + 组蛋白去乙酰化酶抑制剂　；参考：《安徽医科大学》2017年硕士论文

【摘要】：背景:表皮生长因子受体(EGFR)酪氨酸激酶(tyrosine kinase,TK)抑制剂已被广泛用于非小细胞肺癌(NSCLC)的治疗,如厄洛替尼(Erlotinib)、吉非替尼(Gefitinib)等。EGFR-TKI在NSCLC的治疗上取得了非常好的效果,特别是在具有EGFR基因突变的NSCLC,几乎所有的患者在EGFR-TKI治疗一年左右后都会发生耐药现象,严重影响了EGFR-TKI的治疗疗效。研究表明,组蛋白去乙酰化酶(Histone Deacetylases inhibitor;HDACI)能够抑制编码如细胞周期抑制、分化、凋亡等相关因子的表达。此外HDACs还可以促进与细胞侵袭和迁移和血管生成相关基因的表达。因此,HDACs在肿瘤的发生、发展中起重要作用。组蛋白去乙酰化酶(Histone Deacetylases;HDAC)抑制剂可以通过改变组蛋白及非组蛋白的乙酰化水平,从而影响基因转录。HDACIs在血液系统肿瘤,以及实体肿瘤的研究中,取得了比较理想的结果。同时研究表明,HDACIs在与放疗以及其他抗肿瘤药物的联合作用中,显示出较强的协同及增强作用。伏立诺他(SAHA)是一种广谱的HDACIs,是第一个被FDA批准用于T细胞淋巴瘤的治疗。本研究通过体外诱导Erlotinib耐药细胞株PC-9/ER,检测其与亲代Erlotinib敏感型细胞PC-9中PTEN表达的差异,结果表明,在PC-9/ER耐药性的形成过程中,PTEN的表达受到抑制。提示PTEN在耐药性的形成过程中起一定的作用。目的:检测SAHA对PC-9/ER细胞的增殖、凋亡及其耐药性的影响。方法:1.通过体外诱导耐药的方法诱导PC-9(Erlotinib敏感型)细胞耐药。开始以10nmol/L Erlotinib培养PC-9细胞48h,弃去旧培养基以及无菌PBS清洗3遍,在无Erlotinib的环境下培养直至细胞生长至80%~90%汇合度时,逐渐升高的Erlotinib的浓度继续培养,直至PC-9细胞能正常生在在含1μmol/L的Erlotinib环境中。2.MTT法检测PC-9,PC-9/ER对Erlotinib的敏感性。3.MTT法检测SAHA单药、Erlotinib单药,以及无毒剂量的SAHA(1μmol/L)联合Erlotinib处理后,PC-9/ER细胞株的增殖抑制情况。4.应用FCM(Flow Cytometry,流式细胞术),Annexin V-FITC/PI双染试剂盒检测SAHA单药、Erlotinib单药,以及无毒剂量的SAHA(1μmol/L)联合Erlotinib处理后,PC-9/ER细胞株的凋亡情况。5.应用WB(Western Blot)法检测PC-9与PC-9/ER细胞中PTEN表达情况。6.应用WB(Western Blot)法检测SAHA单药、Erlotinib单药,以及无毒剂量的SAHA(1μmol/L)联合Erlotinib处理后,PC-9/ER细胞株中PTEN表达的差异。结果:1.体外诱导耐药细胞株PC-9/ER对Erlotinib的敏感性明显低于PC-9细胞。其耐药倍数100,说明耐Erlotinib细胞株的建立是成功的。2.SAHA单药能抑制PC-9/ER的增殖,在无毒计量(1μmol/L)的SAHA与Erlotinib联合作用于PC-9/ER细胞时,能部分逆转PC-9/ER的耐药性,其逆转倍数为3.01,相对逆转率为66.8%。3.SAHA单药即可促进PC-9/ER细胞的凋亡,在无毒剂量(1μmol/L)与Erlotinib联合,更能进一步提高PC-9/ER细胞的凋亡率。4.WB(Western Blot)检测PC-9/ER及PC-9细胞中PTEN的表达情况,结果显示PC-9/ER中PTEN的表达明显下调。5.WB检测经药物处理后的PTEN表达情况,SAHA及SAHA+Erlotinib处理后48H后,PTEN的表达升高,与Erlotinib组及空白组相比,P0.05。结论:1.组蛋白去乙酰化酶抑制SAHA能够部分改善Erlotinib耐药细胞株PC-9/ER的耐药性;2.SAHA能促进PC-9/ER细胞的凋亡;3.PTEN的缺失在Erlotinib耐药的产生中起一定的作用;4.SAHA能够提高PTEN的表达,促进细胞凋亡,来改善Erlotinib的耐药性。
[Abstract]:Background: epidermal growth factor receptor (EGFR) tyrosine kinase (tyrosine kinase, TK) inhibitors have been widely used for the treatment of non-small cell lung cancer (NSCLC), such as erlotinib (Erlotinib), and gefitinib (Gefitinib), and.EGFR-TKI in the treatment of NSCLC, especially in NSCLC with EGFR gene mutations, almost all of them The patients will have resistance to EGFR-TKI after a year or so, which seriously affects the therapeutic effect of EGFR-TKI. The study shows that histone deacetylase (Histone Deacetylases inhibitor; HDACI) can inhibit the expression of phase factors such as cell cycle inhibition, differentiation, apoptosis and so on. In addition, HDACs can also promote the invasion of cells with cell invasion. The expression of genes related to migration and angiogenesis. Therefore, HDACs plays an important role in the development of tumor. Histone deacetylase (Histone Deacetylases; HDAC) inhibitors can affect the level of histone and non histone acetylation, thus affecting the gene transcription of.HDACIs in the blood system tumor, as well as solid tumors. In the study, an ideal result was obtained. The study showed that HDACIs showed strong synergy and enhancement in combination with radiotherapy and other antitumor drugs. SAHA is a broad-spectrum HDACIs, the first FDA approved treatment for T cell lymphoma. This study induced Erlotinib in vitro. PC-9/ER, a drug resistant cell line, was used to detect the difference in the expression of PTEN in the Erlotinib sensitive cell PC-9 of the parents. The results showed that the expression of PTEN was inhibited during the formation of PC-9/ER resistance. It suggested that PTEN plays a role in the formation of drug resistance. Objective: to detect the effect of SAHA on the proliferation, apoptosis and drug resistance of PC-9/ER cells. Methods: 1. the drug resistance of PC-9 (Erlotinib sensitive) cells was induced by induction of drug resistance in vitro. PC-9 cell 48h was cultured with 10nmol/L Erlotinib, the old medium was abandoned and the aseptic PBS was cleaned 3 times, and the concentration of Erlotinib increased gradually when the cell grew to the 80%~90% confluence in the environment without Erlotinib. Until PC-9 cells can be normal in the Erlotinib environment containing 1 mu mol/L,.2.MTT method is used to detect PC-9, PC-9/ER to Erlotinib sensitivity.3.MTT method to detect SAHA single drug, Erlotinib single drug, and non toxic dose SAHA (1 micron mol/L). Annexin V-FITC/PI double staining kit was used to detect SAHA single drug, Erlotinib single drug, and non toxic dose SAHA (1 u mol/L) combined with Erlotinib treatment. The apoptosis of PC-9/ER cell lines was detected by WB (Western Blot) method. And the difference of PTEN expression in PC-9/ER cell lines after Erlotinib treatment with nontoxic dose of SAHA (1 mu mol/L). Results: 1. the sensitivity of PC-9/ER to Erlotinib in vitro was significantly lower than that of PC-9 cells. The multidrug resistance was 100, indicating that the establishment of a Erlotinib resistant cell line is a successful.2.SAHA single drug that inhibits PC-9/ER proliferation. When SAHA and Erlotinib are combined with Erlotinib in PC-9/ER cells, the drug resistance can partly reverse the resistance of PC-9/ER, and the reversal multiplier is 3.01. The relative reversal rate of 66.8%.3.SAHA single agent can promote the apoptosis of PC-9/ER cells, combined with Erlotinib (1 mu mol/L) and Erlotinib, and can further increase the.4.WB apoptosis rate of PC-9/ER cells (West) (West) (West) can further increase the.4.WB apoptosis rate of PC-9/ER cells (West) (West). (West),.4.WB (West) (West) can further increase the apoptotic rate of PC-9/ER cells (West) (West). (West),.4.WB (West) (West) can further enhance the apoptosis rate of PC-9/ER cells. The expression of PTEN in PC-9/ER and PC-9 cells was detected by ERN Blot. The results showed that the expression of PTEN in PC-9/ER obviously decreased the PTEN expression of.5.WB detected by drug treatment. After SAHA and SAHA+Erlotinib after 48H, the expression of PTEN was increased. Enough to improve the drug resistance of the Erlotinib resistant cell line PC-9/ER; 2.SAHA can promote the apoptosis of PC-9/ER cells; the deletion of 3.PTEN plays a role in the production of Erlotinib resistance; 4.SAHA can improve the expression of PTEN, promote apoptosis, and improve the resistance of Erlotinib.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

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