本文選題：非小細胞肺癌 + 組蛋白去乙酰化酶抑制劑�。� 參考：《安徽醫(yī)科大學》2017年碩士論文

【摘要】：背景:表皮生長因子受體(EGFR)酪氨酸激酶(tyrosine kinase,TK)抑制劑已被廣泛用于非小細胞肺癌(NSCLC)的治療,如厄洛替尼(Erlotinib)、吉非替尼(Gefitinib)等。EGFR-TKI在NSCLC的治療上取得了非常好的效果,特別是在具有EGFR基因突變的NSCLC,幾乎所有的患者在EGFR-TKI治療一年左右后都會發(fā)生耐藥現(xiàn)象,嚴重影響了EGFR-TKI的治療療效。研究表明,組蛋白去乙�；�(Histone Deacetylases inhibitor;HDACI)能夠抑制編碼如細胞周期抑制、分化、凋亡等相關因子的表達。此外HDACs還可以促進與細胞侵襲和遷移和血管生成相關基因的表達。因此,HDACs在腫瘤的發(fā)生、發(fā)展中起重要作用。組蛋白去乙�；�(Histone Deacetylases;HDAC)抑制劑可以通過改變組蛋白及非組蛋白的乙�；�,從而影響基因轉錄。HDACIs在血液系統(tǒng)腫瘤,以及實體腫瘤的研究中,取得了比較理想的結果。同時研究表明,HDACIs在與放療以及其他抗腫瘤藥物的聯(lián)合作用中,顯示出較強的協(xié)同及增強作用。伏立諾他(SAHA)是一種廣譜的HDACIs,是第一個被FDA批準用于T細胞淋巴瘤的治療。本研究通過體外誘導Erlotinib耐藥細胞株PC-9/ER,檢測其與親代Erlotinib敏感型細胞PC-9中PTEN表達的差異,結果表明,在PC-9/ER耐藥性的形成過程中,PTEN的表達受到抑制。提示PTEN在耐藥性的形成過程中起一定的作用。目的:檢測SAHA對PC-9/ER細胞的增殖、凋亡及其耐藥性的影響。方法:1.通過體外誘導耐藥的方法誘導PC-9(Erlotinib敏感型)細胞耐藥。開始以10nmol/L Erlotinib培養(yǎng)PC-9細胞48h,棄去舊培養(yǎng)基以及無菌PBS清洗3遍,在無Erlotinib的環(huán)境下培養(yǎng)直至細胞生長至80%~90%匯合度時,逐漸升高的Erlotinib的濃度繼續(xù)培養(yǎng),直至PC-9細胞能正常生在在含1μmol/L的Erlotinib環(huán)境中。2.MTT法檢測PC-9,PC-9/ER對Erlotinib的敏感性。3.MTT法檢測SAHA單藥、Erlotinib單藥,以及無毒劑量的SAHA(1μmol/L)聯(lián)合Erlotinib處理后,PC-9/ER細胞株的增殖抑制情況。4.應用FCM(Flow Cytometry,流式細胞術),Annexin V-FITC/PI雙染試劑盒檢測SAHA單藥、Erlotinib單藥,以及無毒劑量的SAHA(1μmol/L)聯(lián)合Erlotinib處理后,PC-9/ER細胞株的凋亡情況。5.應用WB(Western Blot)法檢測PC-9與PC-9/ER細胞中PTEN表達情況。6.應用WB(Western Blot)法檢測SAHA單藥、Erlotinib單藥,以及無毒劑量的SAHA(1μmol/L)聯(lián)合Erlotinib處理后,PC-9/ER細胞株中PTEN表達的差異。結果:1.體外誘導耐藥細胞株PC-9/ER對Erlotinib的敏感性明顯低于PC-9細胞。其耐藥倍數(shù)100,說明耐Erlotinib細胞株的建立是成功的。2.SAHA單藥能抑制PC-9/ER的增殖,在無毒計量(1μmol/L)的SAHA與Erlotinib聯(lián)合作用于PC-9/ER細胞時,能部分逆轉PC-9/ER的耐藥性,其逆轉倍數(shù)為3.01,相對逆轉率為66.8%。3.SAHA單藥即可促進PC-9/ER細胞的凋亡,在無毒劑量(1μmol/L)與Erlotinib聯(lián)合,更能進一步提高PC-9/ER細胞的凋亡率。4.WB(Western Blot)檢測PC-9/ER及PC-9細胞中PTEN的表達情況,結果顯示PC-9/ER中PTEN的表達明顯下調。5.WB檢測經(jīng)藥物處理后的PTEN表達情況,SAHA及SAHA+Erlotinib處理后48H后,PTEN的表達升高,與Erlotinib組及空白組相比,P0.05。結論:1.組蛋白去乙酰化酶抑制SAHA能夠部分改善Erlotinib耐藥細胞株PC-9/ER的耐藥性;2.SAHA能促進PC-9/ER細胞的凋亡;3.PTEN的缺失在Erlotinib耐藥的產(chǎn)生中起一定的作用;4.SAHA能夠提高PTEN的表達,促進細胞凋亡,來改善Erlotinib的耐藥性。
[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.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R734.2

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