本文選題：AKT2　切入點(diǎn)：膠質(zhì)母細(xì)胞瘤　出處：《第二軍醫(yī)大學(xué)》2014年博士論文

【摘要】：成人原發(fā)性中樞神經(jīng)系統(tǒng)腫瘤中，包括多種組織學(xué)類(lèi)型的腦神經(jīng)膠質(zhì)瘤是最為多見(jiàn)的。而惡性級(jí)別最高的要數(shù)膠質(zhì)母細(xì)胞瘤，GBM的標(biāo)準(zhǔn)治療包括早期腫瘤的手術(shù)切除、口服烷化劑替莫唑胺（TMZ）化療及輔助性放療結(jié)合。不過(guò)，由于這些腫瘤令人難以置信的耐藥和腫瘤頻繁復(fù)發(fā)，膠質(zhì)母細(xì)胞瘤對(duì)外科手術(shù)治療、放化療極其耐受，GBM的中位生存時(shí)間僅限于診斷后約15個(gè)月。除了腫瘤細(xì)胞自身對(duì)放療、化療的抵制，腫瘤與內(nèi)環(huán)境之間的相互作用還通過(guò)參與腫瘤的抗血管生成、組織缺氧和免疫抑制�；熕幬镌趷盒阅z質(zhì)瘤的治療中發(fā)揮重要作用。新一代烷化劑替莫唑胺是目前對(duì)惡性膠質(zhì)瘤的標(biāo)準(zhǔn)化療藥物，并已被證明有著良好的臨床治療效果。替莫唑胺是經(jīng)過(guò)O6-G甲基化誘導(dǎo)的引發(fā)DNA損傷激活的錯(cuò)配修復(fù)機(jī)制，然而，由于化療藥物內(nèi)在性和獲得性耐藥現(xiàn)象還有血腦屏障的存在，很大程度上影響了化療效果。 AKT作為PI3K/AKT傳導(dǎo)途徑中的關(guān)鍵因子，在促成腫瘤細(xì)胞生長(zhǎng)增殖、血管的生成、增加侵襲轉(zhuǎn)移的發(fā)生、抑制腫瘤的凋亡和抵制放化療中起著非常關(guān)鍵的作用。大量文獻(xiàn)報(bào)導(dǎo)AKT在腫瘤的發(fā)生發(fā)展及轉(zhuǎn)移中扮演了重要角色，更深入的研究AKT作用的分子生物學(xué)機(jī)理并發(fā)現(xiàn)其與惡性癌癥的相關(guān)性，有可能為癌癥的基因治療、抗腫瘤藥物的開(kāi)發(fā)研究提供新的思路和作用靶點(diǎn)。我科前期實(shí)驗(yàn)通過(guò)GBM基因芯片研究發(fā)現(xiàn)了29條基因和替莫唑胺化療耐藥相關(guān)且影響患者生存期，AKT2基因就是其中一個(gè)。近年來(lái)，一些實(shí)驗(yàn)研究已經(jīng)表明PI3K/AKT信號(hào)傳導(dǎo)途徑與癌癥化療療效關(guān)系比較密切。AKT活化還可能參與腫瘤放射治療的抵抗，研究結(jié)果表明AKT2似乎是一個(gè)有效的克服膠質(zhì)瘤治療抵抗重要靶標(biāo)。根據(jù)既往實(shí)驗(yàn)結(jié)果我們初步推測(cè)沉默AKT2基因表達(dá)可能成為臨床上增加膠質(zhì)瘤化療敏感性的一種可行性的方法。本實(shí)驗(yàn)將分四部分研究AKT2基因沉默在膠質(zhì)母細(xì)胞瘤中對(duì)替莫唑胺化療療效的改變及其作用機(jī)理。第一部分，RNA沉默AKT2的表達(dá)對(duì)膠質(zhì)瘤U251細(xì)胞株替莫唑胺療效的影響；第二部分，RNA沉默AKT2的表達(dá)對(duì)膠質(zhì)瘤裸鼠移植瘤的替莫唑胺療效的影響；第三部分，膠質(zhì)瘤替莫唑胺化療耐藥細(xì)胞株的建立及其特性鑒定；第四部分，AKT2在膠質(zhì)瘤細(xì)胞中替莫唑胺化療抵制的作用機(jī)理研究。通過(guò)以上實(shí)驗(yàn)闡明AKT2基因在腦膠質(zhì)瘤莫唑胺化療耐藥中的分子生物學(xué)機(jī)制，在分子水平上尋找和莫唑胺化療耐藥相關(guān)的關(guān)鍵基因及其作用機(jī)制，提高臨床上腦膠質(zhì)瘤患者對(duì)替莫唑胺的化療敏感性。 第一部分RNA干擾AKT2的表達(dá)對(duì)膠質(zhì)瘤U251細(xì)胞株替莫唑胺敏感性的影響目的：研究膠質(zhì)母細(xì)胞瘤U251細(xì)胞中AKT2基因沉默對(duì)替莫唑胺化療效果的改變。方法：AKT2基因慢病毒載體的構(gòu)建，然后在體外條件下將AKT2特異性shRNA表達(dá)載體AKT2-shRNA轉(zhuǎn)染至U251細(xì)胞株中。蛋白印跡、Real-time PCR實(shí)驗(yàn)方法測(cè)定轉(zhuǎn)染shRNA后U251細(xì)胞中AKT2基因表達(dá)水平的變化，應(yīng)用CCK8法檢測(cè)RNA干擾AKT2后U251細(xì)胞對(duì)替莫唑胺敏感性的變化情況。 結(jié)果：轉(zhuǎn)染AKT2-shRNA后U251細(xì)胞AKT2基因表達(dá)程度較U251未轉(zhuǎn)染組和U251轉(zhuǎn)染陰性慢病毒組都顯著下降；替莫唑胺對(duì)U251細(xì)胞的半數(shù)細(xì)胞抑制濃度（IC50）從U251空白對(duì)照組的(39.72±2.41)μg/ml、陰性對(duì)照組的(39.43±2.24)μg/ml降到(27.23±1.93)μg/ml，AKT2干擾組U251細(xì)胞對(duì)TMZ藥物的療效變化有顯著性差異（P＜0.05）。 結(jié)論：AKT2-shRNA能夠抑制膠質(zhì)母細(xì)胞瘤細(xì)胞株U251中AKT2表達(dá)，并能增加對(duì)替莫唑胺的敏感性。 第二部分RNA干擾AKT2表達(dá)對(duì)膠質(zhì)瘤裸鼠移植瘤替莫唑胺敏感性的影響 目的：研究RNA沉默AKT2對(duì)腦膠質(zhì)瘤移植瘤的替莫唑胺療效的改變。 方法：首先，構(gòu)建膠質(zhì)瘤裸鼠成瘤模型，瘤內(nèi)注射和腹腔內(nèi)給TMZ藥物，以替莫唑胺化療藥物和AKT2-shRNA表達(dá)載體對(duì)成瘤后的裸鼠瘤體進(jìn)行聯(lián)合干預(yù)治療，進(jìn)而觀察并測(cè)量各組裸鼠腫瘤體積大小。Real-time PCR及免疫組化實(shí)驗(yàn)分別測(cè)定各組裸鼠腫瘤細(xì)胞的AKT2的表達(dá)程度， TUNEL實(shí)驗(yàn)測(cè)定并計(jì)算分析各組裸鼠成瘤后腫瘤組織細(xì)胞的凋亡的變化。 結(jié)果：空白對(duì)照組、替莫唑胺化療組、TMZ+陰性對(duì)照組、TMZ+AKT2干擾組裸鼠瘤體體積分別為：(669.34±98.73) mm3、(399.86±55.26) mm3、(383.81±34.01) mm3、(297.72±41.49) mm3；瘤體質(zhì)量分別為：(1.25±0.26)g、(0.72±0.11)g、(0.69±0.07)g、(0.52±0.07)g。TMZ+AKT2干擾組其腫瘤體積及瘤體質(zhì)量都明顯小于空白對(duì)照組、替莫唑胺組和TMZ+陰性對(duì)照組，有顯著性差異（P＜0.05）。免疫組化和Real-time PCR實(shí)驗(yàn)表明TMZ+AKT2干擾組比其他對(duì)照組AKT2表達(dá)程度顯著降低（P＜0.05）。TUNEL實(shí)驗(yàn)結(jié)果顯示：空白對(duì)照組、替莫唑胺化療組、TMZ+陰性對(duì)照組、TMZ+AKT2干擾組裸鼠瘤體凋亡指數(shù)分別為：(7.15±1.04)%、(25.26±2.71)%、(26.63±3.46)%、(42.81±5.97)%。TMZ+AKT2干擾組其腫瘤凋亡情況明顯高于空白對(duì)照組、替莫唑胺組和TMZ+陰性對(duì)照組，結(jié)果有顯著性差異（P＜0.05）。 結(jié)論: RNA干擾AKT2能夠顯著提高裸鼠膠質(zhì)母細(xì)胞瘤對(duì)TMZ化療的敏感性。 第三部分膠質(zhì)瘤替莫唑胺化療耐藥細(xì)胞株的建立及其特性鑒定 目的：建立U251耐替莫唑胺的穩(wěn)定細(xì)胞株，并對(duì)該細(xì)胞株U251/TMZ的生物學(xué)特性進(jìn)行研究鑒定。 方法：通過(guò)從低藥物濃度開(kāi)始逐步增加U251細(xì)胞培養(yǎng)基中化療藥物替莫唑胺的濃度，建立對(duì)TMZ化療耐藥的膠質(zhì)母細(xì)胞瘤U251/TMZ。采用四甲基偶氮唑藍(lán)(MTT)法，計(jì)算出兩種細(xì)胞株的半數(shù)細(xì)胞抑制濃度(IC50)及耐藥系數(shù)(RI)。流式細(xì)胞儀測(cè)定U251和U251/TMZ兩組細(xì)胞周期的分布情況。利用蛋白芯片技術(shù)對(duì)U251和U251/TMZ兩種細(xì)胞株中1358中常見(jiàn)蛋白進(jìn)行測(cè)定和分析。 結(jié)果：替莫唑胺對(duì)U251細(xì)胞株的IC50為(10.78±0.72)μg/ml，替莫唑胺對(duì)U251/TMZ的IC50為(45.42±3.17) μg/ml，細(xì)胞增殖實(shí)驗(yàn)所測(cè)結(jié)果計(jì)算后的耐藥指數(shù)為4.21(P0.05)。流式細(xì)胞儀分析提示U251/TMZ耐藥株較U251細(xì)胞株的細(xì)胞周期主要發(fā)生G2期阻滯。蛋白芯片測(cè)定結(jié)果顯示bcl-2家族、caspase家族、P53等多種凋亡相關(guān)蛋白和DNA損傷修復(fù)蛋白發(fā)生明顯上調(diào)或下調(diào)。 結(jié)論：成功構(gòu)建了對(duì)替莫唑胺化療耐藥的膠質(zhì)瘤細(xì)胞株U251/TMZ，，耐藥性能十分顯著而且生物學(xué)特性穩(wěn)定，該細(xì)胞株適合用于膠質(zhì)瘤替莫唑胺化療耐藥的研究。 第四部分AKT2在膠質(zhì)瘤細(xì)胞中替莫唑胺化療耐藥的作用機(jī)制研究 目的：研究AKT2基因、多藥耐藥基因、MGMT等DNA損傷修復(fù)蛋白、多藥耐藥相關(guān)蛋白以及bcl-2、survivin、beclin-1、caspase-3等凋亡自噬相關(guān)蛋白，在膠母U251替莫唑胺(TMZ)化療耐藥中的相互關(guān)系及調(diào)控作用。 方法：利用實(shí)驗(yàn)的第三部分建立的耐替莫唑胺的U251/TMZ，通過(guò)蛋白芯片對(duì)U251細(xì)胞株和U251/TMZ耐藥細(xì)胞株分別進(jìn)行檢測(cè)相關(guān)蛋白的變化，并用計(jì)算機(jī)軟件進(jìn)行以AKT2為中心的相關(guān)蛋白的singal-net分析。流式細(xì)胞儀測(cè)定沉默AKT2基因表達(dá)后各組細(xì)胞凋亡的改變情況。AKT2-shRNA干擾膠質(zhì)母細(xì)胞瘤細(xì)胞株U251的AKT2表達(dá)，同時(shí)采用了Realtime-PCR、Western Blot實(shí)驗(yàn)方法，來(lái)進(jìn)一步分析和評(píng)價(jià)AKT2與MDR-1、MRP-1、MGMT、bcl-2、caspase-3、P53等相關(guān)蛋白表達(dá)情況和相互調(diào)控關(guān)系。 結(jié)果: singal-net分析提示U251耐藥株中AKT2基因靶向調(diào)節(jié)了bcl-2、survivin、caspase-3、beclin-1、 P53等凋亡、自噬相關(guān)蛋白，AKT2-shRNA干擾U251后其凋亡明顯增加，Real-time PCR和蛋白印跡實(shí)驗(yàn)結(jié)果提示bcl-2、survivin、MGMT明顯下調(diào)，而caspase-3、PTEN、P53、beclin-1明顯上調(diào)。 結(jié)論：AKT2可能參與調(diào)控bcl-2、caspase-3、P53、survivin、beclin1等凋亡自噬相關(guān)蛋白及DNA損傷修復(fù)蛋白MGMT的表達(dá)，從而介導(dǎo)膠質(zhì)母細(xì)胞瘤細(xì)胞株U251對(duì)替莫唑胺化療抵抗。
[Abstract]:Adult primary central nervous system tumors, including glioma various histological types are the most common. While the malignant degree of the highest number of glioblastoma resection, standard treatment for GBM including early tumor surgery, oral alkylating agent temozolomide (TMZ) chemotherapy and adjuvant radiotherapy combined with these tumors. However, due to the incredible resistance and frequent recurrence of tumor, the surgical treatment of glioblastoma, chemotherapy is tolerance, the median survival time of GBM is only about 15 months after diagnosis. In addition to tumor cells to radiotherapy, chemotherapy resistance, the interaction between the tumor and the environment also by participating in tumor angiogenesis, tissue hypoxia and immune suppression. Chemotherapy play an important role in the treatment of malignant glioma. A new generation of alkylating agent temozolomide is currently the standard for malignant glioma Chemotherapy drugs, and has been shown to have a good clinical effect. Temozolomide is through O6-G methylation induced by mismatch repair mechanism, activation of the DNA damage however, due to the existence of intrinsic and acquired drug resistance and blood brain barrier, has great influence on the effect of chemotherapy.
AKT is a key factor in the PI3K/AKT pathway, contributed to the growth of tumor cell proliferation, angiogenesis, invasion and metastasis increased the occurrence of apoptosis and inhibition of tumor resistance plays a key role in chemotherapy. A large number of reported AKT in tumor development and metastasis plays an important role in molecular biology mechanism study on the role of AKT further and found its correlation with malignant cancer, possibly for cancer gene therapy, to provide ideas and targets of new research and development of antitumor drugs. Our previous experiments by GBM gene chip research found 29 genes and temozolomide resistance and influence the survival of the patients, the AKT2 gene is one of them. In recent years, some experimental studies have shown that the close relationship between PI3K/AKT signaling pathway and cancer chemotherapy may also be involved in the activation of.AKT tumor The resistance of radiotherapy, the results show that AKT2 appears to be an effective treatment for glioma resistance to overcome an important target. According to previous experimental results we presumed that silencing of AKT2 gene expression may become a feasible method to increase the sensitivity of glioma chemotherapy in clinic. This experiment will be divided into four parts of AKT2 gene silencing on the changes and the mechanism of temozolomide chemotherapy for glioblastoma. The first part, RNA inhibited the expression of AKT2 on glioma U251 cells to temozolomide efficacy; the second part, the expression of RNA silencing AKT2 on glioma xenografts in nude mice of temozolomide effect; the third part glioma for establishment and characterization of temozolomide chemotherapy resistant cell lines; the fourth part, AKT2 for the study of mechanism of temozolomide chemotherapy resistance in glioma cells. Through the above. Objective to elucidate the molecular biological mechanism of AKT2 gene in glioma mozolomide chemoresistance, and find molecular key genes related to resistance to mozolomide chemotherapy and its action mechanism at molecular level, so as to improve chemosensitivity of glioma patients to temozolomide in clinic.
The first part of the expression of RNA AKT2 interference effect on glioma U251 cells temozolomide Objective: To study the AKT2 glioblastoma cells U251 gene silencing on temozolomide effect change. Methods: Construction of AKT2 gene lentiviral vector, then the expression of AKT2 in vitro specific shRNA plasmid AKT2-shRNA was transfected into U251 cells. Western blot, the expression of AKT2 gene in U251 cells Real-time PCR experimental method to determine the shRNA after transfection was detected by CCK8 RNA interference AKT2 on changes of temozolomide sensitivity of U251 cells.
Results: U251 cells AKT2 gene expression level is U251 transfected group and U251 transfected negative lentivirus group were significantly decreased after transfection of AKT2-shRNA; temozolomide half cells on U251 cell inhibitory concentration (IC50) from U251 in the control group (39.72 + 2.41) g/ml, negative control group (39.43 + 2.24 g/ml (27.23) reduced to + 1.93) g/ml, AKT2 and U251 cells of interference group have difference in efficacy to TMZ drugs (P < 0.05).
Conclusion: AKT2-shRNA can inhibit the expression of AKT2 in glioblastoma cell line U251 and increase the sensitivity to temozolomide.
Effect of the second part of RNA interference AKT2 expression on the sensitivity of timozolamine in glioma xenografts in nude mice
Objective: To study the effect of RNA silencing AKT2 on the effect of temozolomide on the tumor of brain glioma.
Method: first, construct glioma in nude mice model, intratumoral injection and intraperitoneal injection of TMZ drugs with temozolomide chemotherapy and AKT2-shRNA expression vector of tumors of nude mice tumor were treated, and then observed and measured expression level of the tumor volume of size.Real-time PCR and immunohistochemical assay. The tumor cells were measured in each group of AKT2, and the determination of the calculation and analysis of the nude mice changes of apoptosis of tumor cells in the tumor after TUNEL experiment.
緇撴灉錛氱┖鐧藉鐓х粍,鏇胯帿鍞戣兒鍖栫枟緇

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