替莫唑胺联合二甲双胍对胶质瘤及其干细胞性能的影响及机制的研究

发布时间：2018-07-29 07:31

【摘要】：背景:替莫唑胺(temozolomide,TMZ)作为目前国内外治疗恶性胶质瘤的一线化疗药物,虽然在胶质瘤治疗中取得一定疗效,但对恶性胶质瘤的有效率仍不高,耐药和快速复发是关键原因。胶质瘤干细胞(glioma stem cells,GSCs)在胶质瘤的发生发展、耐药和复发中起关键作用,被认为是胶质瘤化疗抵抗的根源,已成为治疗的重要新靶点。因此,如何提高TMZ的疗效、增强TMZ对GSCs的敏感性是亟待解决的问题。最近研究显示,TMZ可通过激活腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)信号通路并抑制雷帕霉素靶蛋白(mammalian target of rapamycin,m TOR)使胶质瘤细胞发生凋亡;但使用临床浓度的TMZ可引起内源性蛋白激酶B(protein kinase B,Akt)活化,是肿瘤细胞发生耐药及逃避TMZ杀伤的一种代偿保护性机制。二甲双胍(metformin,MET)作为一种最广泛用于治疗Ⅱ型糖尿病的药物,可激活AMPK,激活的AMPK继而进一步抑制m TOR活性,从而达到抗肿瘤作用;还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)信号通路,降低细胞内Akt磷酸化水平,从而选择性杀伤肿瘤干细胞(cancer stem cells,CSCs)。然而,TMZ与MET合用是否能进一步活化AMPK,并抑制Akt的活性,进而协同抑制或清除GSCs,目前国内外尚无此方面研究。目的:本研究的目的是观察TMZ联合MET对胶质瘤及其干细胞性能的影响,并探讨这些影响的分子机制。方法:1.无血清培养法培养分选GSCs,镜下观察神经肿瘤球生长形态,免疫荧光法鉴定GSCs,流式细胞术检测CD133+GSCs比例。2.TMZ,MET以及TMZ+MET分别作用于胶质瘤及其干细胞;CCK-8法测细胞增殖率/抑制率;Calcu Syn软件分析两药联合指数(Combination index,CI);镜下观察GSCs自我更新和二次成球能力;流式细胞术检测GSCs凋亡率,Western blot检测凋亡相关蛋白;Transwell小室观察GSCs侵袭能力。3.TMZ、MET以及PI3K/m TOR双重抑制剂NVP-BEZ235三者单独或联合作用于GSCs,Western blot检测GSCs中PI3K/Akt/m TOR信号通路的表达;流式细胞术检测NVP-BEZ235联合TMZ和/或MET对GSCs凋亡率的影响。4.TMZ、MET以及AMPK抑制剂Compound C三者单独或联合作用于U87-GSCs、U251-GSCs,Western blot检测GSCs中AMPK的表达;流式细胞术检测Compound C联合TMZ和/或MET对GSCs凋亡率的影响。采用SPSS 19.0统计软件进行Student's t检验,P0.05为具有统计学意义。结果:1.无血清培养法可培养出稳定表达CD133和Nestin的神经肿瘤球;神经肿瘤球具有自我更新和二次成球能力;在神经肿瘤球中,CD133+的U87-GSCs、U251-GSCs比例分别为86.2±6.3%,82.8%±7.1%;将神经肿瘤球置于分化培养基中培养7d后,神经肿瘤球显示出多向分化能力,表达GFAP、β-tubulin III等分化标志物,基本不表达CD133、Nestin等神经干细胞标志物。2.TMZ与MET单独应用时对胶质瘤及其干细胞增殖有抑制作用(P0.05),呈时间-浓度依赖性;与单药组比较,TMZ与MET联合应用时对胶质瘤及其干细胞抑制率更高(P0.05),且具有协同作用(CI1);TMZ及MET单独应用时对GSCs的自我更新及二次成球能力有抑制作用(P0.05);与单药组比较,TMZ与MET联合使用时GSCs的自我更新及二次成球能力明显下降(P0.05);与对照组比较,TMZ、MET和TMZ+MET对U87-GSCs的凋亡率分别为31.0±5.9%(P0.05)、26.8±6.6%(P0.05)和52.3±9.7%(P0.01),对U251-GSCs的凋亡率分别为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,联合用药组的细胞凋亡率均明显高于单药组(P0.05);与单药组比较,联合用药组凋亡抑制蛋白Bcl-2的表达有了明显的减少,而细胞凋亡相关蛋白Bax和Cleaved Caspase-3的表达有所升高;与单药组比较,TMZ与MET联合使用时GSCs的侵袭能力明显下降(P0.05)。以上结果表明TMZ联合MET对GSCs具有协同清除作用。3.TMZ可引起Akt磷酸化表达水平增高,呈时间依赖性,但其下游信号通路m TOR、4EBP1和S6K磷酸化表达水平降低;MET可引起Akt磷酸化表达水平降低,呈时间-浓度依赖性,其下游信号通路m TOR、4EBP1和S6K磷酸化表达水平随着Akt的降低而降低;TMZ联合MET作用于GSCs后,MET可反转由TMZ引起的Akt活性升高,且m TOR、4EBP1和S6K磷酸化表达水平明显降低;各组分别加入PI3K/m TOR双重抑制剂NVP-BEZ235后,不仅Akt、m TOR、4EBP1和S6K磷酸化表达水平均降低,相应的细胞凋亡率相比加入NVP-BEZ235前也更高(P0.05)。以上结果表明,MET反转由TMZ引起的Akt活性升高,并共同抑制m TOR及其下游信号通路是TMZ和MET有协同杀伤GSCs的重要原因。4.TMZ可引起AMPK磷酸化表达水平增高,呈时间-浓度依赖性;MET可也引起AMPK磷酸化表达水平增高,呈时间-浓度依赖性;TMZ联合MET作用于GSCs后,AMPK磷酸化表达水平进一步增高;各组分别加入AMPK抑制剂Compound C后,不论单药组还是联合用药组AMPK磷酸化表达水平均降低;Compound C可降低由TMZ导致的细胞凋亡率(P0.05),对MET以及TMZ+MET导致的细胞凋亡率无明显降低(P0.05)。以上结果表明,TMZ通过激活AMPK导致GSCs凋亡,但MET引起的AMPK活性升高并不是MET导致GSCs凋亡的主要原因;TMZ联合MET虽可共同激活AMPK信号通路,但此途径并不是其具有协同杀伤清除GSCs的主要原因。结论:1.无血清培养法可培养、分选出性能稳定的GSCs,这些GSCs具有自我更新能力和多向分化能力。2.TMZ联合MET对胶质瘤及其干细胞具有协同杀伤和清除作用。3.MET能反转由TMZ引起的Akt活性升高并共同抑制m TOR及其下游信号通路是TMZ联合MET具有协同清除GSCs的主要机制。4.TMZ联合MET可共同激活GSCs AMPK信号通路,但此通路并不是TMZ联合MET具有协同清除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.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R739.41

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