本文選題：非小細胞肺癌 + Notch3�。� 參考：《安徽醫(yī)科大學》2017年碩士論文

【摘要】：目的:肺癌是目前人類高發(fā)的腫瘤之一,其中非小細胞肺癌(non-small-cell lung cancer,NSCLC)是其主要類型。雖然有手術(shù),多種化療藥物及靶向治療應用于其治療,但是其預后仍然很差。Notch3蛋白在NSCLC中表達明顯高于正常肺組織,并且與NSCLC的發(fā)生發(fā)展及預后密切相關(guān)。肺癌患者因個體化差異對常規(guī)化學藥物治療具有一定的耐藥性,例如紫杉醇和吉西他濱等。而且,Notch信號在肺癌細胞的化學耐藥性中是至關(guān)重要的。因此,本研究探索Notch3蛋白在非小細胞肺癌中是否與紫杉醇敏感性有關(guān),并且探討Notch3抑制劑是否通過激活凋亡途徑提高紫杉醇的化療敏感性。方法:本研究培養(yǎng)A549和H1299細胞,分別檢測兩種細胞對紫杉醇的敏感性,根據(jù)其對紫杉醇的敏感性,分別加入含一定不同濃度的紫杉醇長時間培養(yǎng)后,用Western blot的方法分別檢測兩種細胞中Notch3蛋白的表達,與未處理細胞進行比較。同時采用MTT方法檢測Notch3-siRNA和特殊抑制劑GSI(gamma-secretase inhibitor,GSI)協(xié)同紫杉醇較單純應用紫杉醇及對照組對兩種細胞增殖的影響。繼而用流式細胞儀檢測兩種抑制劑協(xié)同紫杉醇對這兩種肺癌細胞的凋亡作用。繼而繼續(xù)應用克隆形成的方法分別檢測兩種細胞經(jīng)不同的預處理后細胞的克隆形成能力。同時,分別用兩種抑制劑處理細胞后,經(jīng)Westernblot的方法分別檢測兩種細胞中Notch3蛋白和NICD3(Notch intracellular domain 3,NICD3)以及凋亡相關(guān)蛋白的表達水平,并比較不同組之間的表達水平。結(jié)果:Notch3蛋白在兩種細胞系中顯著過表達,并且在紫杉醇處理后Notch3表達升高,表明Notch信號傳導途徑的活化。經(jīng)Notch3-siRNA處理后,紫杉醇的IC50較對照組明顯下降并且經(jīng)GSI處理后,兩種細胞較單純加入紫杉醇組在24h,48h及72h后,細胞增殖明顯減少。經(jīng)兩種抑制劑處理后細胞的凋亡作用明顯增加及克隆形成能力明顯下降。同時,兩種抑制劑明顯調(diào)控凋亡蛋白的表達從而影響了紫杉醇對細胞的作用。Notch3 siRNA和GSI明顯降低了Notch3蛋白和NICD3的表達水平。GSI或siRNA處理后伴隨著Bcl-2表達下調(diào)和Bax表達水平上調(diào)。結(jié)論:這些結(jié)果表明Notch3蛋白特異性抑制劑調(diào)控凋亡途徑,從而改變紫杉醇誘導的NSCLC細胞中Notch3蛋白的增加而導致的化學抗性。這種結(jié)合Notch3蛋白特異性抑制和紫杉醇的方法將可能應用于NSCLC的治療。Notch3蛋白抑制劑明顯提高紫杉醇對非小細胞肺癌的化療敏感性,為將來治療非小細胞肺癌提供有效的治療方法。
[Abstract]:Objective: lung cancer is one of the most common tumors in human. Non-small cell lung cancer (NSCLC) is the main type of lung cancer. Although surgery, various chemotherapeutic drugs and targeted therapy were used in NSCLC, the expression of Notch3 protein in NSCLC was significantly higher than that in normal lung tissue, and was closely related to the occurrence, development and prognosis of NSCLC. Patients with lung cancer are resistant to conventional chemotherapeutic agents such as paclitaxel and gemcitabine because of individual differences. Moreover, Notch signaling is critical to chemoresistance in lung cancer cells. Therefore, this study was to investigate whether Notch3 protein is related to paclitaxel sensitivity in non-small cell lung cancer and whether Notch3 inhibitors can enhance the chemosensitivity of paclitaxel by activating apoptosis pathway. Methods: A549 and H1299 cells were cultured in this study. The sensitivity of the two cells to paclitaxel was detected. According to their sensitivity to paclitaxel, paclitaxel was added with different concentrations of paclitaxel for a long time. The expression of Notch3 protein was detected by Western blot and compared with that of untreated cells. MTT assay was used to detect the effects of taxol combined with Notch3-siRNA and GSIGMA-secretase inhibitor GSI on the proliferation of two kinds of cells compared with paclitaxel alone and the control group. Then the apoptotic effects of two inhibitors combined with paclitaxel on lung cancer cells were detected by flow cytometry. Then the clone forming ability of two kinds of cells after different pretreatment was determined by clone forming method. At the same time, the expression levels of Notch3 protein and NICD3tNotch intracellular domain 3nICD3) and apoptosis-related protein were detected by Western blot after treated with two inhibitors, and the expression levels of Notch3 and NICD3 were compared between different groups. Results the expression of Notch3 protein was significantly overexpressed in two cell lines, and increased after paclitaxel treatment, indicating the activation of Notch signal transduction pathway. After treated with Notch3-siRNA, the IC50 of paclitaxel was significantly lower than that of control group. After treated with GSI, the proliferation of the two kinds of cells was significantly decreased after 48 h and 72 h treatment with paclitaxel alone. After treatment with two inhibitors, the apoptotic effect and clone formation ability were significantly increased. At the same time, the two inhibitors significantly regulated the expression of apoptotic protein, which affected the effect of paclitaxel. Notch3 siRNA and GSI significantly decreased the expression level of Notch3 protein and NICD3. GSI or siRNA was associated with down-regulation of Bcl-2 expression and up-regulation of Bax expression. Conclusion: these results suggest that Notch3 protein specific inhibitors regulate apoptotic pathways and thus alter the chemical resistance induced by the increase of Notch3 protein in paclitaxel-induced NSCLC cells. The combination of Notch3 protein specific inhibition and paclitaxel may be used in the treatment of NSCLC. The chemosensitivity of paclitaxel to non-small cell lung cancer can be significantly improved, which provides an effective treatment for NSCLC in the future.
【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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