本文選題：腫瘤放射治療 + 放療增敏劑　； 參考：《南昌大學》2016年博士論文

【摘要】：第一部分:新型WNT通路抑制劑LGK974放射增敏作用和機制研究目的:探討WNT信號傳導通路抑制劑LGK974在腫瘤細胞中的放療增敏作用和機制。方法:1.通過克隆形成實驗分別觀察LGK974合并放療對MLH1表達完整和MLH1表達缺失的細胞株的放療增敏作用。2.通過克隆形成實驗分別觀察LGK974合并放療對瞬時導入MLH1結(jié)腸癌細胞株HCT116(MLH1表達是缺失的)的放療增敏作用。3.通過比較結(jié)腸癌HCT116細胞株(MLH1表達缺失)分別經(jīng)對照組DMSO和實驗組LGK974處理后合并放療,用免疫熒光檢測細胞中γH2AX的變化來反映LGK974通過抑制DNA損傷修復增強MLH1缺失腫瘤細胞的放療敏感性。4.通過蛋白激酶組學Kinomic Profiling實驗檢測LGK974放療增敏的可能磷酸化靶點。結(jié)果:1.LGK974在MLH1表達完整的宮頸癌HeLa細胞株中無放療增敏作用,而在MLH1表達缺失的結(jié)直腸癌HCT116細胞株、子宮內(nèi)膜癌AN3CA細胞株、卵巢癌順鉑耐藥A2789/cp70細胞株中則有放療增敏作用。2.野生型MLH1重新導入HCT116細胞株后LGK974的放療增敏作用明顯減弱。3.LGK974相對對照組DMSO而言,合并放療后HCT116中檢測到的γH2AX數(shù)目明顯減少,細胞DNA損傷修復能力降低,說明LGK974有放療增敏作用;而把MLH1導入HCT116后發(fā)現(xiàn)LGK974相對對照組DMSO而言,合并放療后HCT116中檢測到的γH2AX數(shù)目明顯增多,細胞DNA損傷修復能力增強,LGK974放療增敏作用減弱。4.蛋白激酶組學實驗(Kinomic Profiling)發(fā)現(xiàn)LGK974合并放療后PRKACα和PRKACβ磷酸化作用減弱,提示它們可能為LGK974放療增敏的潛在靶點。結(jié)論:作為一種新型Wnt信號通路抑制劑,LGK974在MLH-1表達缺失的腫瘤中通過抑制DNA損傷修復和作用于PRKACα和PRKACβ磷酸化靶點產(chǎn)生放療增敏作用。這些發(fā)現(xiàn)說明LGK974可能會成為一種新型的針對MLH1表達缺失腫瘤的放療增敏劑,PRKACα和PRKACβ磷酸化靶點有可能是潛在的放療增敏靶點。第二部分:預測頭頸部鱗癌放療敏感性MiRNA的篩選與驗證目的:頭頸部鱗癌中預測放療敏感性MiRNA的篩選與驗證方法:1.通過選取一對分別取自共濟失調(diào)-毛細血管擴張癥ATM患者(患者)及其直系親屬(正常)的人淋巴母細胞樣細胞系來分析miRNA差異表達與電離放射的關(guān)系,兩組分別經(jīng)放療和未放療處理后對比進行完整的miRNA芯片實驗,得到28個表達有差異的miRNA。2.在目前最大的癌癥數(shù)據(jù)庫TCGA數(shù)據(jù)庫中通過找尋病理類型為鱗狀細胞癌病例來評估這28個miRNA的臨床相關(guān)性。3.利用MDA反向階段蛋白質(zhì)芯片技術(shù)檢測TCGA數(shù)據(jù)庫中頭頸鱗癌樣本中的蛋白表達水平,分別計算ATM在放射敏感和放射抗拒的患者中的平均表達水平。4.通過檢測頭頸鱗癌細胞系Cal27,驗證放射治療前后在抑制或不抑制ATM情況下的miRNA表達譜與最初的miRNA篩選結(jié)果及TCGA臨床數(shù)據(jù)一致。5.利用STRING數(shù)據(jù)庫(一個搜尋已知蛋白質(zhì)之間和預測蛋白質(zhì)之間相互作用的系統(tǒng))(STRING9.1),對這5個可預測頭頸部鱗癌放射敏感性的miRNA分子的調(diào)控目標進行通路分析(pathway analysis)。結(jié)果:共有28個miRNA在放射治療后出現(xiàn)不同的差異表達,但都依賴于共濟失調(diào)-毛細血管擴張癥突變(Ataxia-TelangiectasiaMutated,ATM)激酶。通過驗證這些miRNA最終確認有5個miRNA分子標志物可預測頭頸部鱗癌放射敏感性,且這些患者中的ATM表達水平與放射敏感性相關(guān)。結(jié)論:miRNA分子標志物可用于臨床預測頭頸鱗癌放射治療的敏感性。第三部分:RAS蛋白促進人腦膜瘤細胞增殖且抑制其凋亡目的:探討RAS蛋白對人腦膜瘤細胞生長的影響。方法:將人腦膜瘤IOMM-LEE細胞分為空白對照組(細胞未進行任何藥物處理)、陰性對照組(細胞經(jīng)等體積生理鹽水代替藥物)和FTS處理組(細胞經(jīng)FTS處理),采用四甲基偶氮唑鹽微量酶反應比色法(MTT法),FTS(75μmol/L)處理48h后采用流式細胞技術(shù)檢測降低RAS活性后IOMM-LEE細胞的增殖和凋亡情況,用Western Blot檢測ERK和AKT信號通路;按腎包膜下移植法建立人腦膜瘤動物模型,小鼠隨機分為實驗組(50mg/kg組、75mg/kg組和100mg/kg,皮下注射FTS)和對照組,免疫組化增殖細胞檢測核抗原(PCNA),用Western Blot檢測ERK和AKT信號通路。結(jié)果:實驗發(fā)現(xiàn)在75 μmol/LFTS濃度下,IOMM-LEE細胞的存活率隨時間的推移而明顯下降(P0.05)。75 μmol/LFTS濃度處理48h后,處理組細胞凋亡較空白組及陰性對照組均明顯增加(均P0.05);且細胞周期檢測結(jié)果顯示:與空白組及陰性對照組相比,處理組細胞生長阻滯在G1期,差異有統(tǒng)計學意義(P0.05)。75 μmol/LFTS濃度處理48h后,相對于空白組和陰性對照組,處理組ERK和AKT的磷酸化水平顯著降低(P0.05)。體內(nèi)實驗結(jié)果顯示:FTS處理后,與對照組和50mg/kg組相比,75mg/kg組和100mg/kg組腫瘤體積均明顯減小,PCNALI均明顯降低,p-ERK和p-AKT水平明顯降低(均P0.05)。結(jié)論:RAS蛋白在腦膜瘤細胞中高表達,抑制其活性可能通過下調(diào)ERK和AKT信號通路,進而調(diào)控細胞的生長。放療療效的本質(zhì)是射線導致細胞凋亡,RAS在腫瘤放射治療中可能是一個可調(diào)控的重要靶點。
[Abstract]:Part one: a new WNT pathway inhibitor LGK974 radiosensitizing effect and Mechanism Study: To explore the effect and mechanism of radiation sensitization of WNT signal transduction pathway inhibitor LGK974 in tumor cells. Method: 1. through cloning and forming experiment, the radiation enhancement of LGK974 combined with radiotherapy to complete and MLH1 deletion of MLH1 surface was observed. The sensitivity of.2. through the cloning and formation test to observe the radiation sensitization effect of LGK974 combined with radiotherapy on the transient introduction of MLH1 colon cancer cell line HCT116 (MLH1 expression is missing).3. by comparing the colon cancer HCT116 cell line (MLH1 expression deletion) by the control group DMSO and the experimental group LGK974 after the combined radiotherapy, using immunofluorescence detection finely. The changes in the intracellular gamma H2AX reflect the radiation sensitivity of LGK974 by inhibiting DNA damage and repairing the radiation sensitivity of MLH1 missing tumor cells. The possible phosphorylation targets of LGK974 radiation sensitization by the protein kinase group Kinomic Profiling test were detected by the protein kinase group Kinomic Profiling experiment. Colorectal cancer HCT116 cell lines with LH1 deletion, endometrial carcinoma AN3CA cell lines, ovarian cancer cisplatin resistant A2789/cp70 cell lines, the radiation sensitization effect of.2. wild type MLH1 re introduced to HCT116 cell lines after.2. was significantly weakened by.3.LGK974 relative to the control group DMSO, and the combined radiotherapy was detected in HCT116 after radiotherapy. The number of gamma H2AX decreased significantly and the ability of DNA injury and repair decreased, indicating that LGK974 had radiation sensitization. After MLH1 was introduced into HCT116, LGK974 relative to the control group DMSO, the number of gamma H2AX detected in HCT116 was significantly increased after the combined radiotherapy, and the ability to repair the DNA injury was enhanced and the LGK974 radiotherapy sensitization weakened the.4. protein kinase group. The study (Kinomic Profiling) found that PRKAC alpha and PRKAC beta phosphorylation in LGK974 combined with radiotherapy weakened, suggesting that they may be potential targets for sensitizing LGK974 radiation. Conclusion: as a new type of Wnt signal pathway inhibitor, LGK974 can inhibit DNA damage repair and act on PRKAC alpha and PRKAC beta phosphoric acid in the tumor of MLH-1 expression deletion. These findings suggest that LGK974 may become a new type of radiation sensitizer for MLH1 expression deletion tumors. PRKAC alpha and PRKAC beta phosphorylation targets may be potential targets for radiation sensitization. Second: the screening and validation of MiRNA for the prediction of radiation sensitivity of head and neck squamous cell carcinoma: head and neck scales Screening and validation methods for predicting radiation sensitivity MiRNA in cancer: 1. the relationship between differential expression of miRNA and ionizing radiation was analyzed by selecting a pair of ATM patients and their direct relative (normal) lymphoblastoid cell lines from patients with ataxia - telangiectasia (patients) and their direct relatives (normal). The two groups were compared after radiotherapy and no radiotherapy respectively. A complete miRNA chip experiment was carried out to obtain 28 differentially expressed miRNA.2. in the current largest cancer database TCGA database to evaluate the clinical relevance of the 28 miRNA by finding the pathological type of squamous cell carcinoma..3. was used to detect the head and neck squamous cell carcinoma in the TCGA database by the MDA reverse phase protein chip technology. The level of protein expression, the average expression level of ATM in patients with radiosensitivity and radiological resistance,.4. was detected by the detection of Cal27 in the head and neck cancer cell line, which verified that the miRNA expression profiles under the condition of inhibition or non inhibition of ATM before and after radiotherapy were consistent with the initial miRNA screening results and TCGA clinical data and.5. using the STRING database. A system for searching for the interaction between known proteins and predicting proteins (STRING9.1), pathway analysis (pathway analysis) for these 5 miRNA molecules that predict radiosensitivity of the head and neck squamous cell carcinoma (pathway analysis). Results: a total of 28 miRNA had different differential expressions after radiation therapy, but they all depended on ataxia - Ataxia-TelangiectasiaMutated (ATM) kinase. By verifying these miRNA, 5 miRNA markers can be confirmed to predict radiosensitivity in the head and neck squamous cell carcinoma, and the level of ATM expression in these patients is associated with radiosensitivity. Conclusion: the miRNA molecular marker can be used to predict the radiation of head and neck squamous cell carcinoma The sensitivity of treatment. The third part: RAS protein promotes the proliferation of meningioma cells and inhibits its apoptosis: the effect of RAS protein on the growth of human meningioma cells. Methods: the human meningioma IOMM-LEE cells are divided into blank control group (the cells are not treated with any drug treatment), and the negative control group (cell via equal volume of physiological saline instead of the drug) ) and FTS treatment group (cells treated by FTS), the proliferation and apoptosis of IOMM-LEE cells were detected by flow cytometry with four methylazazolazolide Microenzyme reaction colorimetric assay (MTT) and FTS (75 mol/L), and ERK and AKT signal pathways were detected by Western Blot, and human meningioma was established by subcapsular transplantation. The mice were randomly divided into experimental group (group 50mg/kg, group 75mg/kg and 100mg/kg, subcutaneous injection of FTS) and control group. Immuno proliferating cells were used to detect nuclear antigen (PCNA), and ERK and AKT signal pathways were detected by Western Blot. Results: the experimental results showed that the survival rate of IOMM-LEE cells decreased significantly at the concentration of 75 micron mol/LFTS (P0.05) (P0.05) significantly decreased with time (P0.05). When the concentration of.75 mu mol/LFTS was treated with 48h, the cell apoptosis in the treatment group was significantly higher than that in the blank group and the negative control group (all P0.05). And the cell cycle detection results showed that the cell growth block in the treatment group was compared with the blank group and the negative control group, and the difference was statistically significant (P0.05).75 mu mol/LFTS concentration treatment 48h, compared with the blank space of the blank group and the negative control group. In the group and the negative control group, the phosphorylation level of ERK and AKT in the treatment group was significantly lower (P0.05). In vivo experiment results showed that after FTS treatment, the tumor volume of 75mg/kg and 100mg/kg groups decreased significantly compared with the control group and 50mg/kg group, PCNALI decreased obviously, p-ERK and p-AKT water level obviously decreased (P0.05). Conclusion: RAS protein is in meningioma cells. The expression of middle height, which inhibits its activity, may regulate the growth of cells by downregulating the ERK and AKT signaling pathways. The essence of the radiotherapy effect is that radiation leads to cell apoptosis. RAS may be an important regulatory target in tumor radiotherapy.
【學位授予單位】：南昌大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R730.55

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2 姚亦帆;長鏈非編碼RNA-X53654對肺上皮增殖、遷移和放射敏感性的影響及機制的研究[D];蘇州大學;2015年

3 郭銀;尼妥珠單抗對食管鱗癌ECA-109細胞放射敏感性的影響及機制研究[D];河北醫(yī)科大學;2015年

4 袁小鵬;抑制STAT3通路對人腦膠質(zhì)瘤細胞放射敏感性的影響及其作用機制研究[D];蘇州大學;2015年

5 蘇玉飛;自噬誘導對大腸癌細胞放射敏感性的影響[D];蘇州大學;2015年

6 鄭安平;COX-2、XRCC1及RASSF1在食管鱗癌中的表達及其與放射敏感性的研究[D];河南科技大學;2015年

7 邱雋;SiRNA、Apigenin、LY294002、Wortmannin 抑制GLUT-1及PI3K/Akt信號通路影響喉癌放射敏感性體內(nèi)研究[D];浙江大學;2015年

8 楊文影;抑制MUC1胞內(nèi)段對人肺腺癌A549細胞放射敏感性的影響及機制研究[D];重慶醫(yī)科大學;2015年

9 李陽;人肺腺癌放射抗拒細胞株H1299R的建立及miR-183調(diào)控HIF-1α影響H1299R放射敏感性的初步研究[D];遵義醫(yī)學院;2016年

10 劉偉;長鏈非編碼RNA-UCA1對大腸癌細胞放射敏感性的影響及其機制初探[D];蘇州大學;2016年

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