Notch3蛋白抑制剂通过激活固有凋亡途径与紫杉醇在非小细胞肺癌中的协同效应

发布时间：2018-06-21 21:07

本文选题：非小细胞肺癌 + Notch3　；参考：《安徽医科大学》2017年硕士论文

【摘要】：目的:肺癌是目前人类高发的肿瘤之一,其中非小细胞肺癌(non-small-cell lung cancer,NSCLC)是其主要类型。虽然有手术,多种化疗药物及靶向治疗应用于其治疗,但是其预后仍然很差。Notch3蛋白在NSCLC中表达明显高于正常肺组织,并且与NSCLC的发生发展及预后密切相关。肺癌患者因个体化差异对常规化学药物治疗具有一定的耐药性,例如紫杉醇和吉西他滨等。而且,Notch信号在肺癌细胞的化学耐药性中是至关重要的。因此,本研究探索Notch3蛋白在非小细胞肺癌中是否与紫杉醇敏感性有关,并且探讨Notch3抑制剂是否通过激活凋亡途径提高紫杉醇的化疗敏感性。方法:本研究培养A549和H1299细胞,分别检测两种细胞对紫杉醇的敏感性,根据其对紫杉醇的敏感性,分别加入含一定不同浓度的紫杉醇长时间培养后,用Western blot的方法分别检测两种细胞中Notch3蛋白的表达,与未处理细胞进行比较。同时采用MTT方法检测Notch3-siRNA和特殊抑制剂GSI(gamma-secretase inhibitor,GSI)协同紫杉醇较单纯应用紫杉醇及对照组对两种细胞增殖的影响。继而用流式细胞仪检测两种抑制剂协同紫杉醇对这两种肺癌细胞的凋亡作用。继而继续应用克隆形成的方法分别检测两种细胞经不同的预处理后细胞的克隆形成能力。同时,分别用两种抑制剂处理细胞后,经Westernblot的方法分别检测两种细胞中Notch3蛋白和NICD3(Notch intracellular domain 3,NICD3)以及凋亡相关蛋白的表达水平,并比较不同组之间的表达水平。结果:Notch3蛋白在两种细胞系中显著过表达,并且在紫杉醇处理后Notch3表达升高,表明Notch信号传导途径的活化。经Notch3-siRNA处理后,紫杉醇的IC50较对照组明显下降并且经GSI处理后,两种细胞较单纯加入紫杉醇组在24h,48h及72h后,细胞增殖明显减少。经两种抑制剂处理后细胞的凋亡作用明显增加及克隆形成能力明显下降。同时,两种抑制剂明显调控凋亡蛋白的表达从而影响了紫杉醇对细胞的作用。Notch3 siRNA和GSI明显降低了Notch3蛋白和NICD3的表达水平。GSI或siRNA处理后伴随着Bcl-2表达下调和Bax表达水平上调。结论:这些结果表明Notch3蛋白特异性抑制剂调控凋亡途径,从而改变紫杉醇诱导的NSCLC细胞中Notch3蛋白的增加而导致的化学抗性。这种结合Notch3蛋白特异性抑制和紫杉醇的方法将可能应用于NSCLC的治疗。Notch3蛋白抑制剂明显提高紫杉醇对非小细胞肺癌的化疗敏感性,为将来治疗非小细胞肺癌提供有效的治疗方法。
[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.
【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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