本文選題：BAI　切入點：膠質瘤細胞　出處：《青島大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的:1.討論黃芩素(Baicalein,BAI)對膠質瘤U251細胞的自噬及在自噬過程中細胞凋亡的情況。2.探討B(tài)AI激活U251細胞自噬的相關通路。方法:1.將Ad-m Cherry-GFP-LC3B腺病毒轉染入U251細胞中,觀察BAI在不同作用時間條件下,LC3蛋白的表達情況2.Western blot檢測不同濃度(0、10、20、40、80μmol/L)BAI對膠質瘤細胞U251作用24 h后LC3、p-AMPK和cleaved caspase-3蛋白的表達情況。3.Western blot檢測BAI(80μmol/L)在不同作用時間(0、6、12、24、36、48 h)條件下LC3、p-AMPK和cleaved caspase-3蛋白的表達情況。4.Western blot檢測BAI(80μmol/L)單獨和聯合抑制劑CQ后LC3蛋白的表達情況。5.免疫熒光檢測BAI(80μmol/L)在不同作用時間(0、6、24、36 h)后,細胞中LC3蛋白的變化。6.免疫熒光檢測BAI(80μmol/L)聯合抑制劑CQ條件下,細胞中LC3蛋白的變化。7.BAI(80μmol/L)在不同作用時間(0、24、36、48 h)條件下,DAPI染色觀察膠質瘤細胞U251細胞核的碎裂情況和JC-1染色觀察細胞中線粒體膜電位的變化。8.BAI(80μmol/L)單獨和聯合抑制劑compound C后光鏡下觀察膠質瘤細胞的生長狀況,DAPI染色觀察細胞核的碎裂情況和JC-1染色觀察細胞中線粒體膜電位的變化。9.Western blot檢測BAI(80μmol/L)單獨和聯合抑制劑compound C后LC3、p-AMPK和cleaved caspase-3蛋白的表達情況。10.CCK-8法檢測不同濃度的BAI(20、40、80、100、160μmol/L)、不同濃度的替莫唑胺(Temozolomide,TMZ)(100、200、300、400、500μmol/L)在不同作用時間條件下,對U251細胞細胞活力的影響。11.CCK-8法檢測BAI(100μmol/L)聯合不同濃度的TMZ(100、200、300、400、500μmol/L)在不同作用時間條件下,對U251細胞細胞活力的影響。結果:1.將Ad-m Cherry-GFP-LC3B腺病毒轉染入U251細胞中,隨著BAI藥物作用時間的增加,Ad-m Cherry-GFP-LC3B呈現彌散的黃色熒光——斑點狀的黃色熒光——斑點狀的紅色熒光,表明了BAI作用后LC3蛋白的變化過程。2.Western blot顯示LC3蛋白的表達隨著BAI濃度和處理時間的增加逐漸增加;加入CQ后LC3蛋白的表達增加;免疫熒光檢測LC3蛋白,驗證了這個結果。3.DAPI染色觀察發(fā)現24 h時細胞核發(fā)生碎裂,隨著時間的增加碎裂情況加劇;JC-1染色觀察發(fā)現24 h時細胞中線粒體膜電位發(fā)生變化,而且具有時間依賴性;western blot檢測顯示,cleaved caspase-3蛋白表達量與BAI作用時間和BAI濃度呈正相關。4.Western blot檢測p-AMPK蛋白發(fā)現細胞在發(fā)生自噬的同時p-AMPK蛋白含量也增加;加入compound C后細胞生長受到抑制,不僅p-AMPK蛋白表達水平下降,LC3和cleaved caspase-3蛋白水平也呈下降趨勢;另外DAPI和JC-1染色顯示凋亡情況也得到了緩解。5.CCK-8結果顯示BAI和TMZ單獨用藥U251細胞隨著藥物濃度和作用時間的增加細胞活力下降;TMZ和BAI聯合用藥后則隨著TMZ濃度的增加和作用時間的延長,細胞活力出現明顯的下降。結論:1.BAI可誘導膠質瘤細胞U251細胞發(fā)生自噬和凋亡。2.BAI通過激活AMPK參與BAI誘發(fā)膠質瘤的自噬和死亡。3.BAI促進TMZ的抗膠質細胞瘤作用。
[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.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R739.41

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