奥氮平通过诱导自噬抑制胶质瘤增殖

发布时间：2018-10-20 14:59

【摘要】：目的：本文旨在探讨奥氮平（olanzapine）的抗胶质瘤作用及其可能的作用机制。方法：体外实验选用胶质瘤T98和LN229细胞。MTT法检测不同浓度的奥氮平（0,0.1,0.2,0.4,0.6,0.8,1.0mΜ），作用不同时间后对两个胶质瘤细胞株的增殖的影响。Western blot检测自噬相关蛋白LC3、p62和凋亡相关蛋白Bcl-2的变化，免疫荧光检测自噬荧光颗粒的形成与分布情况。Hoechst33258染色以及AnnexinV-FITC/PI实验检测不同浓度奥氮平处理后，两个细胞凋亡的情况。MTT和集落形成实验分析自噬抑制剂3MA对奥氮平抑瘤作用的影响。LN229细胞建立裸鼠皮下移植瘤模型，口服给以奥氮平，绘制肿瘤体积生长曲线，观察奥氮平抑制肿瘤生长的作用。Western blot检测肿瘤组织中自噬相关蛋白LC3、p62和凋亡相关蛋白Bcl-2的表达的情况。免疫组化分析法分析各组移植瘤中增殖指数Ki-67和凋亡相关蛋白caspase-3的表达情况。结果：MTT结果显示，奥氮平对两株胶质瘤细胞LN229和T98均有抑制作用，且呈剂量依赖性和时间依赖性。LC3、p62的蛋白表达情况以及免疫荧光检测LC3的颗粒形成与分布情况都显示奥氮平能够在这两株细胞中诱导自噬。Western blot分析Bcl-2蛋白表达、Hoechst33258染色实验以及V-FITC/PI染色实验表明，奥氮平能够促进LN229和T98细胞凋亡。MTT和集落形成实验表明，自噬抑制剂3MA减弱了奥氮平对这两株细胞的增殖抑制作用。裸鼠皮下移植瘤体积曲线说明，奥氮平能够在体内抑制胶质瘤的生长。Western blot分析移植瘤的Bcl-2蛋白表达，显示奥氮平能够促进移植瘤凋亡；LC3和p62蛋白表达显示，，高剂量奥氮平在体内皮下移植瘤中诱导自噬。免疫组化分析移植瘤组织中Ki-67和caspase-3阳性表达结果显示，奥氮平抑制裸鼠皮下移植瘤的增殖并且促进其凋亡。结论：奥氮平在体外、体内都能够抑制胶质瘤的生长。其抗胶质瘤作用的机制与自噬诱导相关。自噬的诱导促进了胶质瘤细胞的死亡。
[Abstract]:Objective: to investigate the anti-glioma effect of olanzapine (olanzapine) and its possible mechanism. Methods: glioma T98 and LN229 cells were used in vitro. Different concentrations of olanzapine were detected by MTT method. The proliferation of two glioma cell lines was detected by. Western blot after exposure to different concentrations of olanzapine (0. 1, 0. 2, 0. 4, 0. 6 and 0. 8 渭 m). Western blot was used to detect the changes of autophagy associated protein LC3,p62 and apoptosis-related protein Bcl-2. Immunofluorescence was used to detect the formation and distribution of autophagy particles. Hoechst33258 staining and AnnexinV-FITC/PI assay were used to detect the formation and distribution of autophagy particles treated with different concentrations of olanzapine. The effect of autophagy inhibitor 3MA on antitumor effects of autolytic inhibitor 3MA. LN229 cells were used to establish subcutaneous transplanted tumor model of nude mice and were given olanzapine orally to draw tumor volume growth curve. To observe the inhibitory effect of olanzapine on tumor growth,. Western blot was used to detect the expression of autophagy associated protein (LC3,p62) and apoptosis-related protein (Bcl-2) in tumor tissues. The expression of proliferative index (Ki-67) and apoptosis-related protein (caspase-3) were analyzed by immunohistochemistry. Results: the results of MTT showed that olanzapine could inhibit both LN229 and T98 glioma cells. The protein expression of LC3,p62 and the particle formation and distribution of LC3 detected by immunofluorescence showed that olanzapine could induce autophagy. Western blot to analyze Bcl-2 protein expression and Hoechst33258 staining in these two cell lines. Color experiments and V-FITC/PI staining experiments show that, Olanzapine could promote apoptosis of LN229 and T98 cells. MTT and colony formation assay showed that autophagy inhibitor 3MA could attenuate the inhibitory effect of olanzapine on the proliferation of LN229 and T98 cells. The volume curve of subcutaneous transplanted tumor in nude mice showed that olanzapine could inhibit the growth of glioma in vivo. Western blot analysis showed that olanzapine could promote the apoptosis of transplanted tumor, and the expression of LC3 and p62 protein showed that the expression of olanzapine could promote the apoptosis of transplanted tumor. High-dose olanzapine induces autophagy in vivo subcutaneously transplanted tumor. Immunohistochemical analysis showed that olanzapine inhibited the proliferation and promoted the apoptosis of xenografts in nude mice. Conclusion: olanzapine can inhibit the growth of glioma in vitro. The mechanism of its anti-glioma action is related to autophagy induction. Autophagy induces the death of glioma cells.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

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