本文選題：腦膠質(zhì)瘤 + 白藜蘆醇�。� 參考：《武漢大學(xué)》2014年博士論文

【摘要】：腦膠質(zhì)瘤是原發(fā)性顱內(nèi)腫瘤中最常見的惡性腫瘤。雖然近年來神經(jīng)外科領(lǐng)域已得到長足發(fā)展,但即使是最現(xiàn)代的神經(jīng)外科技術(shù),也難以對腦膠質(zhì)瘤做到病理學(xué)上的完全切除。腦膠質(zhì)瘤的生物學(xué)行為主要表現(xiàn)為局部擴(kuò)散及高度侵襲,因而膠質(zhì)瘤術(shù)后復(fù)發(fā)率高。臨床上迫切需要有效的輔助治療方法,以改善腦膠質(zhì)瘤的預(yù)后。白藜蘆醇(resveratrol, Res)是從虎杖、藜蘆、葡萄等天然植物中提取出來的多酚化合物,具有調(diào)節(jié)脂質(zhì)代謝、抑制血小板聚集、保護(hù)心血管、抗炎等多種生物學(xué)活性。近年研究發(fā)現(xiàn),白藜蘆醇對乳腺癌、前列腺癌、皮膚癌、胃癌等多種腫瘤細(xì)胞的生長均具有顯著的抑制作用,尤其是對腦腫瘤而言,血腦屏障對白藜蘆醇具有良好的通透性,是一種非常有前景的天然抗腫瘤藥物。 第一部分目的探討白藜蘆醇對腦膠質(zhì)瘤細(xì)胞增殖的抑制作用及其可能的機制。方法首先將不同濃度的白藜蘆醇(0、25、50、100μ M)作用于腦膠質(zhì)瘤U87細(xì)胞和U251細(xì)胞,倒置顯微鏡定性觀察白藜蘆醇對腦膠質(zhì)瘤細(xì)胞形態(tài)的影響,臺盼藍(lán)拒染法及MTT增殖抑制試驗定量檢測白藜蘆醇對腦膠質(zhì)瘤細(xì)胞生長的抑制作用,然后再用RT-PCR及Western blot檢測EGFR、LRIG1、磷酸化Akt及ERk核酸水平以及蛋白表達(dá)水平的變化。結(jié)果經(jīng)不同濃度白藜蘆醇分別處理腦膠質(zhì)瘤U87細(xì)胞及U251細(xì)胞后,細(xì)胞活力逐漸下降,貼壁能力差,細(xì)胞開始變圓,細(xì)胞體積變小,一部分細(xì)胞開始懸浮,與瓶底分離。臺盼藍(lán)拒染法及MTT增殖抑制試驗表明,白藜蘆醇能抑制腦膠質(zhì)瘤U251細(xì)胞及U87細(xì)胞的生長,其作用強度呈時間及劑量依賴性。RT-PCR及western blot實驗結(jié)果發(fā)現(xiàn),經(jīng)白藜蘆醇處理后腦膠質(zhì)瘤細(xì)胞中EGFR蛋白表達(dá)水平下降,而其負(fù)性調(diào)節(jié)蛋白LRIG1蛋白的表達(dá)升高,白藜蘆醇對ERK磷酸化的表達(dá)無影響,但能夠明顯抑制Akt磷酸化。結(jié)論白藜蘆醇能夠抑制腦膠質(zhì)瘤細(xì)胞的增殖,其機制可能主要是通過抑制PI3K/Akt信號轉(zhuǎn)導(dǎo)通路來發(fā)揮對腦膠質(zhì)瘤細(xì)胞增殖的抑制作用。 第二部分目的研究白藜蘆醇誘導(dǎo)腦膠質(zhì)瘤細(xì)胞凋亡的作用及其具體機制。方法用DNA瓊脂糖凝膠電泳及Annexin V-FITC凋亡試劑盒檢測白藜蘆醇對腦膠質(zhì)瘤細(xì)胞凋亡的誘導(dǎo)作用,進(jìn)一步用Rhodamine123熒光染料染色后經(jīng)流式細(xì)胞儀檢測,再用Western blot檢測內(nèi)源性凋亡途徑相關(guān)蛋白Cyto C及caspase-9的表達(dá)。結(jié)果經(jīng)25μM、50μM、100μM濃度的白藜蘆醇處理U87細(xì)胞后DNA瓊脂糖凝膠電泳呈現(xiàn)凋亡細(xì)胞的梯級(ladder)格局,而對照組細(xì)胞DNA無斷裂現(xiàn)象；Annexin V-FITC凋亡試劑盒檢測也發(fā)現(xiàn)白藜蘆醇能明顯誘導(dǎo)U87細(xì)胞發(fā)生凋亡；經(jīng)白藜蘆醇處理后,U87細(xì)胞的線粒體膜電位明顯降低,這一作用呈劑量依賴性。Western blot檢測發(fā)現(xiàn)Cyto C及caspase-9蛋白的表達(dá)增強。 結(jié)論白藜蘆醇可以促進(jìn)Cyto C釋放及Caspase-9蛋白表達(dá),通過激活內(nèi)源性細(xì)胞凋亡途徑來發(fā)揮抗腫瘤作用。 第三部分目的探討白藜蘆醇對腦膠質(zhì)瘤細(xì)胞自噬的誘導(dǎo)作用及可能的作用機制。方法用不同濃度白藜蘆醇處理腦膠質(zhì)瘤U87細(xì)胞24h后,在電子顯微鏡下觀察細(xì)胞形態(tài)的變化。應(yīng)用半定量RT-PCR及Realtime PCR分析白藜蘆醇對自噬相關(guān)基因LC-3、Beclin-1、VPS34基因表達(dá)的影響。Western blot檢測白藜蘆醇對LC-3、Beclin-1、VPS34蛋白表達(dá)水平的影響。結(jié)果在電子顯微鏡下可以觀察到典型的自噬性空泡的出現(xiàn),而對照組細(xì)胞未出現(xiàn)明顯自噬空泡。白藜蘆醇處理前U87細(xì)胞中LC-3、Beclin-1、VPS34基因為低表達(dá),經(jīng)25μM、50μM,100μM濃度的白藜蘆醇處理24h后,LC-3、Beclin-1、VPS34基因mRNA的表達(dá)明顯增強。白藜蘆醇作用后可明顯升高LC-3蛋白的含量,Beclin-1、VPS34的表達(dá)也逐漸升高。結(jié)論自噬過程參與了白藜蘆醇誘導(dǎo)的U87細(xì)胞死亡,LC-3、Beclin-1、VPS34基因表達(dá)上調(diào)是其誘導(dǎo)自噬的可能機制。 第四部分目的腦膠質(zhì)瘤的惡性生物學(xué)行為主要表現(xiàn)為局部擴(kuò)散及高度侵襲轉(zhuǎn)移,本部分研究白藜蘆醇對腦膠質(zhì)瘤細(xì)胞侵襲轉(zhuǎn)移的抑制作用及可能機制。方法通過細(xì)胞劃痕實驗研究白藜蘆醇對腦膠質(zhì)瘤細(xì)胞遷移運動能力的影響,Transwell侵襲小室實驗研究白藜蘆醇對腦膠質(zhì)瘤細(xì)胞侵襲和轉(zhuǎn)移能力的影響,RT-PCR及Western Blot從mRNA表達(dá)及蛋白表達(dá)水平檢測經(jīng)白藜蘆醇作用后,腦膠質(zhì)瘤細(xì)胞中MMP-2、MMP-9、TIMP-1、TIMP-2蛋白水平的變化。結(jié)果白藜蘆醇能明顯抑制腦膠質(zhì)瘤細(xì)胞的遷移運動能力及侵襲、轉(zhuǎn)移能力；能夠降低腦膠質(zhì)瘤U87細(xì)胞中MMP-2、MMP-9的表達(dá),使TIMP-1、TIMP-2的表達(dá)提高,且MMP-2和MMP-9的表達(dá)隨著白黎蘆醇濃度的增加而呈現(xiàn)下降的趨勢。結(jié)論白黎蘆醇可能通過抑制MMP-2及MMP-9的表達(dá)并調(diào)節(jié)TIMP-2/MMP-2,TIMP-1/MMP-9的動態(tài)平衡來抑制腦膠質(zhì)瘤細(xì)胞的遷移和侵襲。 本課題研究表明白藜蘆醇可以通過對腦膠質(zhì)瘤細(xì)胞增殖的抑制、誘導(dǎo)細(xì)胞凋亡及自噬和抑制細(xì)胞侵襲轉(zhuǎn)移等四個方面發(fā)揮其抗腫瘤作用,本研究為白藜蘆醇用于腦膠質(zhì)瘤的臨床治療提供了新的理論依據(jù)。
[Abstract]:Glioma is the most common malignant tumor in primary intracranial tumors. Although the field of Department of neurosurgery has developed rapidly in recent years, even the most modern department of Neurosurgery technique is difficult to complete complete pathological excision of glioma. The biological behavior of glioma is mainly characterized by local diffusion and high invasion. The recurrence rate of glioma after operation is high. It is urgent to use effective adjuvant therapy to improve the prognosis of brain glioma. Resveratrol (Res) is a polyphenol compound extracted from natural plants such as Polygonum cuspidatum, veratris, grape and other natural plants, which can regulate lipid metabolism, inhibit platelet aggregation, protect cardiovascular, anti-inflammatory and so on. In recent years, it has been found that resveratrol has a significant inhibitory effect on the growth of many tumor cells, such as breast cancer, prostate cancer, skin cancer and gastric cancer. Especially for brain tumors, the blood brain barrier has a good permeability to resveratrol. It is a very promising natural antitumor drug.
The first part is to investigate the inhibitory effect of resveratrol on the proliferation of glioma cells and its possible mechanism. Methods first, the resveratrol (0,25,50100) of resveratrol (resveratrol) of different concentrations was used to act on the U87 cells and U251 cells of glioma, and the effect of resveratrol on the morphology of the glioma cells was observed by inverted microscope, and trypan blue staining method was used to determine the effect of resveratrol on the cell morphology of brain glioma. The inhibitory effect of resveratrol on the growth of glioma cells was detected by MTT proliferation inhibition test and then RT-PCR and Western blot were used to detect the changes of EGFR, LRIG1, phosphorylated Akt, ERk nucleic acid level and protein expression level. Results after different concentrations of resveratrol in the glioma U87 and U251 cells, the cells live The force gradually declined, the adhesion ability was poor, the cell began to circle, the cell volume became smaller, some cells began to suspend and separated from the bottle bottom. Trypan blue exclusion assay and MTT proliferation inhibition test showed that resveratrol could inhibit the growth of U251 and U87 cells in glioma, and its action intensity was time and dose dependent.RT-PCR and Western blot experiments The results showed that the expression level of EGFR protein in the glioma cells after resveratrol treatment decreased, but the expression of its negative regulatory protein LRIG1 protein increased, and resveratrol had no effect on the expression of ERK phosphorylation, but could obviously inhibit the phosphorylation of Akt. Conclusion resveratrol can inhibit the proliferation of glioma cells, and the mechanism may be main. It inhibits the proliferation of glioma cells by inhibiting PI3K/Akt signaling pathway.
The second part aims to study the effect of resveratrol on the apoptosis of glioma cells induced by resveratrol and its specific mechanism. Methods the induction of resveratrol on the apoptosis of glioma cells was detected by DNA agarose gel electrophoresis and Annexin V-FITC apoptosis kit, and then detected by flow cytometry after staining with Rhodamine123 fluorescent dye, and then Wes The expression of endogenous apoptotic pathway related protein Cyto C and caspase-9 was detected by tern blot. The results showed the cascade (ladder) pattern of apoptotic cells by DNA agarose gel electrophoresis after 25 u M, 50 mu M, and 100 u M concentration of resveratrol. The apoptosis of U87 cells could be induced by resveratol. After resveratrol treatment, the mitochondrial membrane potential of U87 cells decreased significantly. The effect of.Western blot detection showed that the expression of Cyto C and caspase-9 protein was enhanced.
Conclusion resveratrol can promote the release of Cyto C and the expression of Caspase-9 protein, and play an anti-tumor role by activating the endogenous apoptotic pathway.
The third part aims to explore the induction and mechanism of resveratrol on autophagy in glioma cells. Methods the morphologic changes were observed under the electron microscope after treating glioma U87 cell 24h with resveratrol with different concentrations. The autophagy related gene LC-3 was analyzed by semi quantitative RT-PCR and Realtime PCR. The effect of Beclin-1, VPS34 gene expression on.Western blot to detect the effect of resveratrol on the expression of LC-3, Beclin-1, and VPS34 protein. Results the typical autophagic vacuoles can be observed under the electron microscope, while the cells in the control group have no obvious autophagic vacuoles. LC-3, Beclin-1, VPS34 base in the U87 cells before resveratrol treatment The expression of LC-3, Beclin-1, VPS34 gene mRNA was obviously enhanced after low expression of resveratrol at 25 M, 50 M and 100 micron M. The content of LC-3 protein was obviously increased after resveratrol action, and the expression of Beclin-1 and VPS34 increased gradually. Conclusion autophagy was involved in the death of resveratrol induced U87 cells. The up-regulated expression is a possible mechanism for its induction of autophagy.
In the fourth part, the malignant biological behavior of brain glioma is mainly characterized by local diffusion and high invasion and metastasis. This part studies the inhibitory effect of resveratrol on the invasion and metastasis of glioma cells and its possible mechanism. Methods the effect of resveratrol on the migration and movement of brain glioma cells was studied by cell scratch test, Transwel The effect of resveratrol on the invasion and metastasis of glioma cells, and the changes in the level of MMP-2, MMP-9, TIMP-1, TIMP-2 protein in glioma cells after resveratrol were detected by resveratrol on the invasion and metastasis of glioma cells in the L invasion experiment. The results showed that resveratrol could significantly inhibit glioma cells in the brain glioma cells after resveratrol action. Migration ability, invasion and metastasis ability, can reduce the expression of MMP-2 and MMP-9 in glioma U87 cells, improve the expression of TIMP-1 and TIMP-2, and the expression of MMP-2 and MMP-9 decreases with the increase of the concentration of resveratol. Conclusion resveratol may inhibit the expression of MMP-2 and MMP-9 and regulate TIMP-2/MMP-2, TIMP The dynamic balance of -1/MMP-9 inhibits the migration and invasion of glioma cells.
This study shows that veratrol can inhibit the proliferation of glioma cells, induce apoptosis, autophagy and inhibit cell invasion and metastasis in four aspects. This study provides a new theoretical basis for the clinical treatment of resveratrol for brain glioma.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R739.41

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本文編號：1958107