本文選題：膠質瘤 + 替莫唑胺��； 參考：《山東大學》2017年碩士論文

【摘要】：腦膠質細胞瘤是由于大腦中的膠質細胞癌變所引起的,在原發(fā)性的顱內腫瘤中最為常見,約占顱內所有惡性腫瘤的50%,發(fā)病率為3～10/10萬,占全身惡性腫瘤的1%～3%,病人死亡率較高。目前膠質瘤主要通過手術切除,隨后放射治療和替莫唑胺(TMZ)化療聯(lián)合治療。TMZ是新一代的烷化劑類抗腫瘤藥物,能夠穿過血腦屏障并通過產生引發(fā)DNA錯配修復途徑的無效循環(huán)的甲基加合物而誘導腫瘤細胞凋亡。目前臨床使用TMZ治療膠質瘤的效果并不能達到令人滿意的程度,主要的原因是部分膠質瘤患者對烷化劑化療藥物存在耐藥性。近來有研究表明,導致烷化劑類藥物耐藥主要的原因是DNA修復酶06-甲基鳥嘌呤DNA 甲基轉移酶(06-methylguanine-DNA methyltransferase,MGMT)的存在。MGMT通過將烷基從DNA鳥嘌呤的06位置轉移到其半胱氨酸殘基來逆轉DNA烷基化過程,恢復腫瘤細胞的繁殖增生能力,膠質瘤具有升高的MGMT活性時通常顯示對TMZ治療的化學抗性。一些研究已經證實,當MGMT啟動子處于甲基化狀態(tài)時,能夠使MGMT基因處于靜止狀態(tài),進而阻止其所介導的DNA修復功能和從而增強膠質瘤對烷化劑的敏感性。MicroRNA(miRNA)通過與靶基因的非編碼區(qū)結合而導致靶mRNA的降解或翻譯過程的抑制,進而調控基因的表達,參與一系列生物學行為。研究目的:檢測MGMT在膠質瘤病例標本中的表達水平,分析MGMT表達水平與膠質瘤級別、膠質瘤大小之間的關系,探尋能夠影響MGMT表達水平的MiRNA,并為緩解部分膠質瘤患者對烷化劑類藥物耐藥尋找新的作用靶點。研究方法:山東大學齊魯醫(yī)院隨機選擇47例于2015年7月至2015年11月接受手術切除的患者神經膠質瘤組織樣本。所有樣品通過獨立的病理學檢查鑒定為神經膠質瘤。每例樣本分為兩部分,一部分用于組織切片進行免疫組織化學染色檢測MGMT表達量,另一部分用于提取RNA。根據(jù)WHO中樞神經系統(tǒng)腫瘤分級標準(2007年),將取得的膠質細胞瘤標本分為Ⅰ-Ⅳ級,其中低惡性組包含Ⅰ級與Ⅱ級,高惡性組包含Ⅲ級與Ⅳ級。選用患者顱腦磁共振橫斷位液體衰減反轉恢復(fluidattenuated inversion recovery,FLAIR)序列成像測量最大直徑。免疫組化中采用H評分法對MGMT表達情況進行定量。使用SPSS18.0統(tǒng)計學軟件對數(shù)據(jù)進行統(tǒng)計分析,MGMT表達陽性率與膠質瘤級別及大小的關系采用χ2檢驗。miRNA及mRNA表達水平計算采用2^-ΔΔCT方法計算,實驗組與對照組兩組間的比較采用t-檢驗法,p0.05為具有顯著統(tǒng)計學差異。利用miRNA數(shù)據(jù)庫(TargetScan database;http://www.targetscan.org/vert_61/)找出在 MGMT 3'UTR 存在結合位點的miRNA,并進行細胞水平驗證。對膠質瘤組織中MGMT與miRNA的表達量采用Westen blot及Real-Time PCR方法檢測,明確兩者表達關系。構建MGMT 3'UTR熒光素酶野生型及突變型載體,進行雙熒光素酶檢測,明確兩者調控關系。細胞水平驗證,選取MGMT表達相對較高的膠質瘤細胞系轉入miRNA mimics及inhibitor,檢測MGMT表達變化情況。功能驗證,對轉入miRNA mimics及inhibitor的細胞進行耐藥性實驗,利用cck-8,凋亡蛋白表達情況及流式細胞術檢測細胞增殖及凋亡情況。探討miRNA-4539對MGMT的調控作用,及這種作用在膠質瘤中的表達與替莫唑胺(TMZ)耐藥之間的關系。結果:1.MGMT在膠質瘤組織中的表達量與膠質瘤病理級別及膠質瘤大小沒有相關關系;2.MGMT與MiRNA-4539在膠質瘤組織中表達呈負相關關系;3.MiRNA-4539能夠作用于MGMT 3'UTR,抑制其表達;4.在體外細胞水平,轉入 MiRNA-4539 mimics 時 MGMT 表達下降,轉入 MiRNA-4539 inhibitor 時MGMT表達升高;5.在體外細胞水平,轉入MiRNA-4539 mimics時,膠質瘤細胞對TMZ的耐藥性減低,而轉入MiRNA-4539 inhibitor時則相反,TMZ耐藥性增加。結論:MiRNA-4539在體外試驗中可以抑制MGMT表達并改善膠質瘤細胞對TMZ的耐藥性。
[Abstract]:Glioblastoma is caused by glial carcinogenesis in the brain. It is the most common in the primary intracranial tumor, accounting for about 50% of all the malignant tumors of the brain. The incidence is 3 to 10/10 million, which accounts for 1% to 3% of the malignant tumors of the whole body. The mortality of the patients is high. Amine (TMZ) chemotherapy combined with.TMZ is a new generation of alkylating antitumor drugs, which can pass through the blood brain barrier and induce the apoptosis of tumor cells by producing an ineffective cyclic methyl adduct that causes DNA mismatch repair. The main reason for the clinical use of TMZ in the treatment of gliomas is not satisfactory. Some glioma patients have resistance to alkylating agents. Recent studies have shown that the main cause of drug resistance of alkylating agents is the existence of the DNA repair enzyme 06- methyl guanine DNA methyltransferase (06-methylguanine-DNA methyltransferase, MGMT) in the presence of.MGMT by transferring alkyl from the 06 position of DNA guanine to its half. Cystine residues reversion the DNA alkylation process and restore the proliferative ability of tumor cells. The MGMT activity of glioma usually shows chemical resistance to TMZ treatment. Some studies have shown that when the MGMT promoter is in the methylation state, the MGMT gene can be kept in a state of rest, thereby preventing its mediated DNA repair. Compound function and thus enhance the sensitivity of glioma to alkylating agent.MicroRNA (miRNA) by combining with the non coding region of the target gene to lead to the degradation of target mRNA or the inhibition of translation process, and then regulate gene expression and participate in a series of biological behavior. Objective: to detect the expression level of MGMT in the specimens of glioma and analyze MGMT The relationship between the level of expression and glioma size, the size of glioma, the exploration of MiRNA that can affect the MGMT expression level, and to find new targets for alleviating the drug resistance of alkylating agents in patients with partial glioma. A randomized selection of 47 cases of surgical excision from July 2015 to November 2015 in Qilu Hospital of Shandong University. All samples were identified as gliomas by independent pathological examination. Each sample was divided into two parts. Part of the sample was used to detect MGMT expression by immunohistochemical staining in tissue sections, and the other was used to extract RNA. according to the WHO central deity system tumor grading standard (2007). The glioma specimens were divided into grade I and IV, of which the low malignant group included grade I and grade II, and the high malignant group consisted of grade III and IV. The maximum diameter was measured by the fluidattenuated inversion recovery, FLAIR sequence imaging in the patients with craniocerebral magnetic resonance transverse declines. The H score was used in the immunohistochemical method for the expression of MGMT. SPSS18.0 statistics software was used to analyze the data. The relationship between the positive rate of MGMT expression and the grade and size of glioma was calculated by the 2^- Delta Delta CT method using the chi 2 test,.MiRNA and mRNA, and the comparison between the experimental group and the control group was compared with the t- test, and P0.05 had significant statistical difference. MiRN was used in miRN. The A database (TargetScan database; http://www.targetscan.org/vert_61/) identified the miRNA of the presence of the binding site of MGMT 3'UTR and tested the cell level. The expression of MGMT and miRNA in glioma tissues was detected by Westen blot and Real-Time methods. The mutant vector was tested by double luciferase, and the regulation relationship was clear. The cell level was verified by selecting the relatively high MGMT expression of glioma cell lines into miRNA mimics and inhibitor to detect the changes of MGMT expression. Expression and flow cytometry to detect cell proliferation and apoptosis. The role of miRNA-4539 in the regulation of MGMT and the relationship between the expression of this effect in glioma and the resistance to temozolomide (TMZ). Results: the expression of 1.MGMT in glioma tissues is not related to the level of gliomatosis and the size of glioma; 2.MGMT The expression of MiRNA-4539 was negatively correlated with the expression of MiRNA-4539 in glioma tissue, and 3.MiRNA-4539 could act on MGMT 3'UTR and inhibit its expression. The expression of MGMT decreased at the level of cell at MiRNA-4539 mimics in vitro, and the expression of MGMT increased at MiRNA-4539 inhibitor; 5. the glioma cells were transferred to MiRNA-4539 mimics, at the cell level in vitro. Resistance to TMZ decreased, while MiRNA-4539 inhibitor was reversed and TMZ resistance increased. Conclusion: MiRNA-4539 can inhibit the expression of MGMT and improve the drug resistance of glioma cells to TMZ in vitro.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R739.41

【參考文獻】

相關期刊論文 前1條

1 宣自學;袁守軍;王維;俞佳;;MGMT基因甲基化檢測在神經膠質瘤治療中的應用[J];腫瘤學雜志;2016年10期

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