本文選題：BAI　切入點(diǎn)：膠質(zhì)瘤細(xì)胞　出處：《青島大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的:1.討論黃芩素(Baicalein,BAI)對(duì)膠質(zhì)瘤U251細(xì)胞的自噬及在自噬過(guò)程中細(xì)胞凋亡的情況。2.探討B(tài)AI激活U251細(xì)胞自噬的相關(guān)通路。方法:1.將Ad-m Cherry-GFP-LC3B腺病毒轉(zhuǎn)染入U(xiǎn)251細(xì)胞中,觀察BAI在不同作用時(shí)間條件下,LC3蛋白的表達(dá)情況2.Western blot檢測(cè)不同濃度(0、10、20、40、80μmol/L)BAI對(duì)膠質(zhì)瘤細(xì)胞U251作用24 h后LC3、p-AMPK和cleaved caspase-3蛋白的表達(dá)情況。3.Western blot檢測(cè)BAI(80μmol/L)在不同作用時(shí)間(0、6、12、24、36、48 h)條件下LC3、p-AMPK和cleaved caspase-3蛋白的表達(dá)情況。4.Western blot檢測(cè)BAI(80μmol/L)單獨(dú)和聯(lián)合抑制劑CQ后LC3蛋白的表達(dá)情況。5.免疫熒光檢測(cè)BAI(80μmol/L)在不同作用時(shí)間(0、6、24、36 h)后,細(xì)胞中LC3蛋白的變化。6.免疫熒光檢測(cè)BAI(80μmol/L)聯(lián)合抑制劑CQ條件下,細(xì)胞中LC3蛋白的變化。7.BAI(80μmol/L)在不同作用時(shí)間(0、24、36、48 h)條件下,DAPI染色觀察膠質(zhì)瘤細(xì)胞U251細(xì)胞核的碎裂情況和JC-1染色觀察細(xì)胞中線粒體膜電位的變化。8.BAI(80μmol/L)單獨(dú)和聯(lián)合抑制劑compound C后光鏡下觀察膠質(zhì)瘤細(xì)胞的生長(zhǎng)狀況,DAPI染色觀察細(xì)胞核的碎裂情況和JC-1染色觀察細(xì)胞中線粒體膜電位的變化。9.Western blot檢測(cè)BAI(80μmol/L)單獨(dú)和聯(lián)合抑制劑compound C后LC3、p-AMPK和cleaved caspase-3蛋白的表達(dá)情況。10.CCK-8法檢測(cè)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(shí)間條件下,對(duì)U251細(xì)胞細(xì)胞活力的影響。11.CCK-8法檢測(cè)BAI(100μmol/L)聯(lián)合不同濃度的TMZ(100、200、300、400、500μmol/L)在不同作用時(shí)間條件下,對(duì)U251細(xì)胞細(xì)胞活力的影響。結(jié)果:1.將Ad-m Cherry-GFP-LC3B腺病毒轉(zhuǎn)染入U(xiǎn)251細(xì)胞中,隨著B(niǎo)AI藥物作用時(shí)間的增加,Ad-m Cherry-GFP-LC3B呈現(xiàn)彌散的黃色熒光——斑點(diǎn)狀的黃色熒光——斑點(diǎn)狀的紅色熒光,表明了BAI作用后LC3蛋白的變化過(guò)程。2.Western blot顯示LC3蛋白的表達(dá)隨著B(niǎo)AI濃度和處理時(shí)間的增加逐漸增加;加入CQ后LC3蛋白的表達(dá)增加;免疫熒光檢測(cè)LC3蛋白,驗(yàn)證了這個(gè)結(jié)果。3.DAPI染色觀察發(fā)現(xiàn)24 h時(shí)細(xì)胞核發(fā)生碎裂,隨著時(shí)間的增加碎裂情況加劇;JC-1染色觀察發(fā)現(xiàn)24 h時(shí)細(xì)胞中線粒體膜電位發(fā)生變化,而且具有時(shí)間依賴(lài)性;western blot檢測(cè)顯示,cleaved caspase-3蛋白表達(dá)量與BAI作用時(shí)間和BAI濃度呈正相關(guān)。4.Western blot檢測(cè)p-AMPK蛋白發(fā)現(xiàn)細(xì)胞在發(fā)生自噬的同時(shí)p-AMPK蛋白含量也增加;加入compound C后細(xì)胞生長(zhǎng)受到抑制,不僅p-AMPK蛋白表達(dá)水平下降,LC3和cleaved caspase-3蛋白水平也呈下降趨勢(shì);另外DAPI和JC-1染色顯示凋亡情況也得到了緩解。5.CCK-8結(jié)果顯示BAI和TMZ單獨(dú)用藥U251細(xì)胞隨著藥物濃度和作用時(shí)間的增加細(xì)胞活力下降;TMZ和BAI聯(lián)合用藥后則隨著TMZ濃度的增加和作用時(shí)間的延長(zhǎng),細(xì)胞活力出現(xiàn)明顯的下降。結(jié)論:1.BAI可誘導(dǎo)膠質(zhì)瘤細(xì)胞U251細(xì)胞發(fā)生自噬和凋亡。2.BAI通過(guò)激活A(yù)MPK參與BAI誘發(fā)膠質(zhì)瘤的自噬和死亡。3.BAI促進(jìn)TMZ的抗膠質(zhì)細(xì)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(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R739.41

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