黄芩素促进胶质瘤细胞自噬和凋亡及相关机制研究

发布时间：2018-03-06 10:42

本文选题：BAI　切入点：胶质瘤细胞　出处：《青岛大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:1.讨论黄芩素(Baicalein,BAI)对胶质瘤U251细胞的自噬及在自噬过程中细胞凋亡的情况。2.探讨BAI激活U251细胞自噬的相关通路。方法:1.将Ad-m Cherry-GFP-LC3B腺病毒转染入U251细胞中,观察BAI在不同作用时间条件下,LC3蛋白的表达情况2.Western blot检测不同浓度(0、10、20、40、80μmol/L)BAI对胶质瘤细胞U251作用24 h后LC3、p-AMPK和cleaved caspase-3蛋白的表达情况。3.Western blot检测BAI(80μmol/L)在不同作用时间(0、6、12、24、36、48 h)条件下LC3、p-AMPK和cleaved caspase-3蛋白的表达情况。4.Western blot检测BAI(80μmol/L)单独和联合抑制剂CQ后LC3蛋白的表达情况。5.免疫荧光检测BAI(80μmol/L)在不同作用时间(0、6、24、36 h)后,细胞中LC3蛋白的变化。6.免疫荧光检测BAI(80μmol/L)联合抑制剂CQ条件下,细胞中LC3蛋白的变化。7.BAI(80μmol/L)在不同作用时间(0、24、36、48 h)条件下,DAPI染色观察胶质瘤细胞U251细胞核的碎裂情况和JC-1染色观察细胞中线粒体膜电位的变化。8.BAI(80μmol/L)单独和联合抑制剂compound C后光镜下观察胶质瘤细胞的生长状况,DAPI染色观察细胞核的碎裂情况和JC-1染色观察细胞中线粒体膜电位的变化。9.Western blot检测BAI(80μmol/L)单独和联合抑制剂compound C后LC3、p-AMPK和cleaved caspase-3蛋白的表达情况。10.CCK-8法检测不同浓度的BAI(20、40、80、100、160μmol/L)、不同浓度的替莫唑胺(Temozolomide,TMZ)(100、200、300、400、500μmol/L)在不同作用时间条件下,对U251细胞细胞活力的影响。11.CCK-8法检测BAI(100μmol/L)联合不同浓度的TMZ(100、200、300、400、500μmol/L)在不同作用时间条件下,对U251细胞细胞活力的影响。结果:1.将Ad-m Cherry-GFP-LC3B腺病毒转染入U251细胞中,随着BAI药物作用时间的增加,Ad-m Cherry-GFP-LC3B呈现弥散的黄色荧光——斑点状的黄色荧光——斑点状的红色荧光,表明了BAI作用后LC3蛋白的变化过程。2.Western blot显示LC3蛋白的表达随着BAI浓度和处理时间的增加逐渐增加;加入CQ后LC3蛋白的表达增加;免疫荧光检测LC3蛋白,验证了这个结果。3.DAPI染色观察发现24 h时细胞核发生碎裂,随着时间的增加碎裂情况加剧;JC-1染色观察发现24 h时细胞中线粒体膜电位发生变化,而且具有时间依赖性;western blot检测显示,cleaved caspase-3蛋白表达量与BAI作用时间和BAI浓度呈正相关。4.Western blot检测p-AMPK蛋白发现细胞在发生自噬的同时p-AMPK蛋白含量也增加;加入compound C后细胞生长受到抑制,不仅p-AMPK蛋白表达水平下降,LC3和cleaved caspase-3蛋白水平也呈下降趋势;另外DAPI和JC-1染色显示凋亡情况也得到了缓解。5.CCK-8结果显示BAI和TMZ单独用药U251细胞随着药物浓度和作用时间的增加细胞活力下降;TMZ和BAI联合用药后则随着TMZ浓度的增加和作用时间的延长,细胞活力出现明显的下降。结论:1.BAI可诱导胶质瘤细胞U251细胞发生自噬和凋亡。2.BAI通过激活AMPK参与BAI诱发胶质瘤的自噬和死亡。3.BAI促进TMZ的抗胶质细胞瘤作用。
[Abstract]:Objective: 1. To investigate the effect of baicalein (Baicalein) on autophagy and apoptosis of U251 glioma cells during autophagy. (2) to explore the pathway related to BAI activation of autophagy in U251 cells. Methods: 1. Ad-m Cherry-GFP-LC3B adenovirus was transfected into U251 cells. To observe the expression of BAI in different time groups 2. Western blot was used to detect the expression of LC3 p-AMPK and cleaved caspase-3 protein in human glioma cell U251 24 h after the treatment with different concentrations of 10 ~ (10) ~ 20 ~ (20) ~ 40 ~ (80 渭 mol/L)BAI). 3. Western blot was used to detect the expression of BAI(80 渭 mol / L at different time (61224 ~ 36 ~ 48 h). Expression of BAI(80 渭 mol / L and cleaved caspase-3 protein. 4. Western blot was used to detect the expression of LC3 protein after BAI(80 渭 mol / L alone and combined inhibitor CQ. 5. Immunofluorescence detection of BAI(80 渭 mol / L at different time points (6 ~ 2436 h). Changes of LC3 protein in cells. 6. Immunofluorescence detection of BAI(80 渭 mol / L combined with inhibitor CQ, Changes of LC3 protein in cells .7.The changes of LC3 protein in U251 cells were observed by DAPI staining and mitochondrial membrane potential of U251 cells were observed by JC-1 staining under different action time (80 渭 mol / L) and compound C alone and in combination with the inhibitor compound C (80 渭 mol 路L ~ (-1)). The growth of glioma cells was observed under light microscope. Nuclear fragmentation was observed by DAPI staining and mitochondrial membrane potential was observed by JC-1 staining. 9. Western blot was used to detect the expression of BAI(80 渭 mol / L in LC3p-AMPK and cleaved caspase-3 protein after compound C alone and in combination with compound C. 10. CCK-8 method was used to detect the effects of different concentrations of BAIZO20, 40,80FU, 100 渭 mol / L, Temozolomide, Temozolomide, Temozolomide, Temozolomide, and temozolomide, in the presence of 400 渭 mol / L, 500 渭 mol / L, at different time points. Effects of CCK-8 method on the viability of U251 cells. The effect of BAI(100 渭 mol / L) combined with different concentrations of TMZN 100m / L and 400渭 mol / L) on the viability of U251 cells at different time. Results: 1. The adenovirus of Ad-m Cherry-GFP-LC3B was transfected into U251 cells. With the increase of the time of action of BAI, Ad-m Cherry-GFP-LC3B showed a diffuse yellow fluorescence, a yellow fluorescent spot and a red red fluorescence. 2. Western blot showed that the expression of LC3 protein increased with the increase of BAI concentration and treatment time, the expression of LC3 protein increased after adding CQ, and the expression of LC3 protein was detected by immunofluorescence. The results were verified by .3.DAPI staining showed that nuclear fragmentation occurred at 24 h, and the fragmentation increased with time. The changes of mitochondrial membrane potential were observed at 24 h after observation with JC-1 staining. The expression of p-AMPK protein was positively correlated with the time of BAI treatment and the concentration of BAI. 4. Western blot detection of p-AMPK protein showed that the level of p-AMPK protein was also increased while autophagy occurred. After adding compound C, cell growth was inhibited, not only the expression level of p-AMPK protein decreased, but also the levels of cleaved caspase-3 and p-AMPK protein decreased. In addition, DAPI and JC-1 staining showed that apoptosis was alleviated. 5. The results of CCK-8 showed that the cell viability of U251 cells treated with BAI and TMZ alone decreased with the increase of drug concentration and time. After combined treatment with TMZ and BAI, the cell viability increased with the concentration of TMZ. The extension of the additive action time, Conclusion: 1. Bai can induce autophagy and apoptosis in U251 glioma cells. 2. Bai participates in the anti-glioma effect of TMZ by activating AMPK. 3. Bai can promote the anti-glioma effect of TMZ.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41

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