【摘要】：背景:替莫唑胺(temozolomide,TMZ)作為目前國內(nèi)外治療惡性膠質(zhì)瘤的一線化療藥物,雖然在膠質(zhì)瘤治療中取得一定療效,但對惡性膠質(zhì)瘤的有效率仍不高,耐藥和快速復(fù)發(fā)是關(guān)鍵原因。膠質(zhì)瘤干細胞(glioma stem cells,GSCs)在膠質(zhì)瘤的發(fā)生發(fā)展、耐藥和復(fù)發(fā)中起關(guān)鍵作用,被認為是膠質(zhì)瘤化療抵抗的根源,已成為治療的重要新靶點。因此,如何提高TMZ的療效、增強TMZ對GSCs的敏感性是亟待解決的問題。最近研究顯示,TMZ可通過激活腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)信號通路并抑制雷帕霉素靶蛋白(mammalian target of rapamycin,m TOR)使膠質(zhì)瘤細胞發(fā)生凋亡;但使用臨床濃度的TMZ可引起內(nèi)源性蛋白激酶B(protein kinase B,Akt)活化,是腫瘤細胞發(fā)生耐藥及逃避TMZ殺傷的一種代償保護性機制。二甲雙胍(metformin,MET)作為一種最廣泛用于治療Ⅱ型糖尿病的藥物,可激活A(yù)MPK,激活的AMPK繼而進一步抑制m TOR活性,從而達到抗腫瘤作用;還可以抑制磷脂酰肌醇-3-激酶/蛋白激酶B(phosphatidylinositol 3-kinase/protein kinase B,PI3K/Akt)信號通路,降低細胞內(nèi)Akt磷酸化水平,從而選擇性殺傷腫瘤干細胞(cancer stem cells,CSCs)。然而,TMZ與MET合用是否能進一步活化AMPK,并抑制Akt的活性,進而協(xié)同抑制或清除GSCs,目前國內(nèi)外尚無此方面研究。目的:本研究的目的是觀察TMZ聯(lián)合MET對膠質(zhì)瘤及其干細胞性能的影響,并探討這些影響的分子機制。方法:1.無血清培養(yǎng)法培養(yǎng)分選GSCs,鏡下觀察神經(jīng)腫瘤球生長形態(tài),免疫熒光法鑒定GSCs,流式細胞術(shù)檢測CD133+GSCs比例。2.TMZ,MET以及TMZ+MET分別作用于膠質(zhì)瘤及其干細胞;CCK-8法測細胞增殖率/抑制率;Calcu Syn軟件分析兩藥聯(lián)合指數(shù)(Combination index,CI);鏡下觀察GSCs自我更新和二次成球能力;流式細胞術(shù)檢測GSCs凋亡率,Western blot檢測凋亡相關(guān)蛋白;Transwell小室觀察GSCs侵襲能力。3.TMZ、MET以及PI3K/m TOR雙重抑制劑NVP-BEZ235三者單獨或聯(lián)合作用于GSCs,Western blot檢測GSCs中PI3K/Akt/m TOR信號通路的表達;流式細胞術(shù)檢測NVP-BEZ235聯(lián)合TMZ和/或MET對GSCs凋亡率的影響。4.TMZ、MET以及AMPK抑制劑Compound C三者單獨或聯(lián)合作用于U87-GSCs、U251-GSCs,Western blot檢測GSCs中AMPK的表達;流式細胞術(shù)檢測Compound C聯(lián)合TMZ和/或MET對GSCs凋亡率的影響。采用SPSS 19.0統(tǒng)計軟件進行Student's t檢驗,P0.05為具有統(tǒng)計學(xué)意義。結(jié)果:1.無血清培養(yǎng)法可培養(yǎng)出穩(wěn)定表達CD133和Nestin的神經(jīng)腫瘤球;神經(jīng)腫瘤球具有自我更新和二次成球能力;在神經(jīng)腫瘤球中,CD133+的U87-GSCs、U251-GSCs比例分別為86.2±6.3%,82.8%±7.1%;將神經(jīng)腫瘤球置于分化培養(yǎng)基中培養(yǎng)7d后,神經(jīng)腫瘤球顯示出多向分化能力,表達GFAP、β-tubulin III等分化標志物,基本不表達CD133、Nestin等神經(jīng)干細胞標志物。2.TMZ與MET單獨應(yīng)用時對膠質(zhì)瘤及其干細胞增殖有抑制作用(P0.05),呈時間-濃度依賴性;與單藥組比較,TMZ與MET聯(lián)合應(yīng)用時對膠質(zhì)瘤及其干細胞抑制率更高(P0.05),且具有協(xié)同作用(CI1);TMZ及MET單獨應(yīng)用時對GSCs的自我更新及二次成球能力有抑制作用(P0.05);與單藥組比較,TMZ與MET聯(lián)合使用時GSCs的自我更新及二次成球能力明顯下降(P0.05);與對照組比較,TMZ、MET和TMZ+MET對U87-GSCs的凋亡率分別為31.0±5.9%(P0.05)、26.8±6.6%(P0.05)和52.3±9.7%(P0.01),對U251-GSCs的凋亡率分別為33.1±6.7%(P0.05)、20.1±4.9%(P0.05)和48.7±9.2%(P0.01);在U87-GSCs和U251-GSCs中,聯(lián)合用藥組的細胞凋亡率均明顯高于單藥組(P0.05);與單藥組比較,聯(lián)合用藥組凋亡抑制蛋白Bcl-2的表達有了明顯的減少,而細胞凋亡相關(guān)蛋白Bax和Cleaved Caspase-3的表達有所升高;與單藥組比較,TMZ與MET聯(lián)合使用時GSCs的侵襲能力明顯下降(P0.05)。以上結(jié)果表明TMZ聯(lián)合MET對GSCs具有協(xié)同清除作用。3.TMZ可引起Akt磷酸化表達水平增高,呈時間依賴性,但其下游信號通路m TOR、4EBP1和S6K磷酸化表達水平降低;MET可引起Akt磷酸化表達水平降低,呈時間-濃度依賴性,其下游信號通路m TOR、4EBP1和S6K磷酸化表達水平隨著Akt的降低而降低;TMZ聯(lián)合MET作用于GSCs后,MET可反轉(zhuǎn)由TMZ引起的Akt活性升高,且m TOR、4EBP1和S6K磷酸化表達水平明顯降低;各組分別加入PI3K/m TOR雙重抑制劑NVP-BEZ235后,不僅Akt、m TOR、4EBP1和S6K磷酸化表達水平均降低,相應(yīng)的細胞凋亡率相比加入NVP-BEZ235前也更高(P0.05)。以上結(jié)果表明,MET反轉(zhuǎn)由TMZ引起的Akt活性升高,并共同抑制m TOR及其下游信號通路是TMZ和MET有協(xié)同殺傷GSCs的重要原因。4.TMZ可引起AMPK磷酸化表達水平增高,呈時間-濃度依賴性;MET可也引起AMPK磷酸化表達水平增高,呈時間-濃度依賴性;TMZ聯(lián)合MET作用于GSCs后,AMPK磷酸化表達水平進一步增高;各組分別加入AMPK抑制劑Compound C后,不論單藥組還是聯(lián)合用藥組AMPK磷酸化表達水平均降低;Compound C可降低由TMZ導(dǎo)致的細胞凋亡率(P0.05),對MET以及TMZ+MET導(dǎo)致的細胞凋亡率無明顯降低(P0.05)。以上結(jié)果表明,TMZ通過激活A(yù)MPK導(dǎo)致GSCs凋亡,但MET引起的AMPK活性升高并不是MET導(dǎo)致GSCs凋亡的主要原因;TMZ聯(lián)合MET雖可共同激活A(yù)MPK信號通路,但此途徑并不是其具有協(xié)同殺傷清除GSCs的主要原因。結(jié)論:1.無血清培養(yǎng)法可培養(yǎng)、分選出性能穩(wěn)定的GSCs,這些GSCs具有自我更新能力和多向分化能力。2.TMZ聯(lián)合MET對膠質(zhì)瘤及其干細胞具有協(xié)同殺傷和清除作用。3.MET能反轉(zhuǎn)由TMZ引起的Akt活性升高并共同抑制m TOR及其下游信號通路是TMZ聯(lián)合MET具有協(xié)同清除GSCs的主要機制。4.TMZ聯(lián)合MET可共同激活GSCs AMPK信號通路,但此通路并不是TMZ聯(lián)合MET具有協(xié)同清除GSCs作用的主要原因。
[Abstract]:Background: temozolomide (TMZ) is a first-line chemotherapy drug for malignant glioma at home and abroad. Although it has achieved a certain therapeutic effect in the treatment of glioma, the effective rate of malignant glioma is still not high. The key reason is the resistance and rapid recurrence. The development of glioma stem cells (glioma stem cells, GSCs) is the development of glioma. The key role of drug resistance and relapse is considered to be the root of chemotherapeutic resistance to glioma and has become an important new target for treatment. Therefore, how to improve the efficacy of TMZ and enhance the sensitivity of TMZ to GSCs is an urgent problem. Recent studies have shown that TMZ can be activated by the activation of adenylate active protein kinase (AMP-activated protein kinase, AMPK) letter. Mammalian target of rapamycin (m TOR) can induce apoptosis of glioma cells, but the use of clinical concentration of TMZ can cause endogenous protein kinase B (protein kinase B, Akt) activation. It is a compensatory protective mechanism for the occurrence of drug resistance and evasion of tumor cells. As one of the most widely used drugs for the treatment of type II diabetes, AMPK can be activated. The activated AMPK then further inhibits the activity of M TOR, thus achieving the antitumor effect, and also inhibits the phosphatidylinositol -3- kinase / protein kinase B (phosphatidylinositol 3-kinase/protein kinase B, PI3K/Akt) signaling pathway and reduces the intracellular Akt phosphorylated water. To selectively kill tumor stem cells (cancer stem cells, CSCs). However, whether TMZ and MET can be used to further activate AMPK, inhibit the activity of Akt, and inhibit or eliminate GSCs, there is no study at home and abroad. Objective: To observe the effect of TMZ combined MET on the performance of glioma and stem cells. To explore the molecular mechanism of these effects. Methods: 1. serum-free culture method was used to cultivate GSCs. The growth morphology of nerve tumor ball was observed under the microscope, GSCs was detected by immunofluorescence, CD133+GSCs ratio.2.TMZ was detected by flow cytometry, MET and TMZ+MET were used in glioma and stem cells respectively. The cell proliferation rate / inhibition rate was measured by CCK-8 method, and Calcu Syn software was used. The combined index of two drugs (Combination index, CI), the self renewal and two ball forming ability of GSCs were observed under the microscope, the apoptosis rate of GSCs was detected by flow cytometry, and Western blot was used to detect the apoptosis related protein; Transwell chamber was used to observe the GSCs invasion ability.3.TMZ, MET and PI3K/m inhibitory agents. Ot detection of the expression of PI3K/Akt/m TOR signaling pathway in GSCs; flow cytometry detection of the effect of NVP-BEZ235 combined TMZ and / or MET on the apoptosis rate of GSCs,.4.TMZ, MET and AMPK inhibitor Compound, three The effect of MET on the apoptosis rate of GSCs. Using SPSS 19 statistical software for Student's t test, P0.05 was of statistical significance. Results: 1. serum-free culture method can produce a stable expression of CD133 and Nestin in neural tumor balls; neural tumor balls have self renewal and two times of spherical energy; CD133+ U87-GSCs, U251-GS in neural tumor balls The proportion of Cs was 86.2 + 6.3% and 82.8% + 7.1%, respectively. After the nerve tumor ball was placed in the differentiation medium for 7d, the nerve tumor ball showed the ability to differentiate, expressed GFAP, beta -tubulin III and other differentiation markers, basically did not express CD133. Nestin and other neural stem cell markers,.2.TMZ and MET were used separately for the proliferation of glioma and stem cells. Inhibitory effect (P0.05) was time dependent; compared with the single drug group, the inhibition rate of TMZ and MET was higher (P0.05) and synergistic action (CI1) when combined with MET, and TMZ and MET were used alone to inhibit the self renewal and two ball forming ability of GSCs (P0.05); when compared with the single drug group, TMZ and MET were used together. The self renewal and two ball forming ability of GSCs decreased significantly (P0.05). Compared with the control group, the apoptosis rates of TMZ, MET and TMZ+MET were 31 + 5.9% (P0.05), 26.8 + 6.6% (P0.05) and 52.3 + 9.7% (P0.01). The apoptosis rate of U251-GSCs was 33.1 + 6.7% (P0.05), 20.1 + 4.9% (P0.05) and 48.7 + 9.2%. The apoptosis rate of the combined drug group was significantly higher than that in the single drug group (P0.05). Compared with the single drug group, the expression of apoptosis inhibitory protein Bcl-2 in the combination group was significantly reduced, while the expression of apoptosis related protein Bax and Cleaved Caspase-3 increased. Compared with the single drug group, the invasion ability of GSCs was significantly lower than that of TMZ and MET. (P0.05). The above results show that the synergistic scavenging effect of TMZ combined with MET on GSCs can cause the increase of Akt phosphorylation level, which is time dependent, but the downstream signal pathway m TOR, 4EBP1 and S6K phosphorylation level decrease, MET can cause the decrease of Akt phosphorylation level and time concentration dependence. The level of phosphorylation of OR, 4EBP1 and S6K decreased with the decrease of Akt, and MET could reverse the increase of Akt activity caused by TMZ and m TOR, and the expression level of phosphorylation was significantly reduced after the action of TMZ MET on GSCs. Decrease, the corresponding apoptosis rate was higher than before NVP-BEZ235 (P0.05). The above results showed that the Akt activity induced by TMZ was increased and the m TOR and its downstream signal pathway were the important cause of TMZ and MET co killing GSCs, and.4.TMZ could cause the increase of AMPK phosphorylation level and time concentration dependence; The expression level of AMPK phosphorylation was increased in time concentration dependence, and the expression level of phosphorylation of AMPK was further increased after the action of TMZ combined with MET on GSCs. After AMPK inhibitor Compound C was added to each group, the average amount of AMPK phosphorylation in the single drug group and the combination group decreased, and Compound C could reduce the cell withering caused by TMZ. The apoptosis rate of MET and TMZ+MET was not significantly reduced (P0.05). The above results showed that TMZ induced GSCs apoptosis by activating AMPK, but the increase of AMPK activity caused by MET was not the main cause of GSCs apoptosis by MET; TMZ combined TMZ+MET can jointly stimulate the signaling pathway, but this approach is not a synergistic killing. The main reasons for scavenging GSCs conclusion: 1. the serum-free culture method can be cultivated and separate the stable GSCs. These GSCs have self-renewal ability and multidirectional differentiation ability..2.TMZ combined with MET has synergistic killing and scavenging effects on glioma and stem cells..3.MET can reverse the increase of Akt activity caused by TMZ and co inhibit m TOR and its downstream. The signal pathway is TMZ combined with MET, the main mechanism for synergistic scavenging of GSCs,.4.TMZ combined MET can co activate GSCs AMPK signaling pathways, but this pathway is not the main reason for the synergy of TMZ associated MET to scavenging GSCs.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R739.41

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