本文選題：三氧化二砷 + 前列腺癌��； 參考：《鄭州大學》2014年碩士論文

【摘要】：背景和目的 前列腺癌是在男性泌尿生殖系統(tǒng)中最常見的惡性腫瘤之一。目前治療前列腺癌的主要方法有手術，激素拮抗治療，化療和放療。雖然大多數(shù)前列腺癌患者最初的雄激素去勢有效，但他們通常會在數(shù)月至數(shù)年后出現(xiàn)在雄激素非依賴性。雄激素非依賴性的前列腺癌的預后比較差，且多對化療和放射治療不敏感。目前激素非依賴型前列腺癌對放、化療抵抗的機制目前還不明確。因此了解腫瘤細胞如何產(chǎn)生耐藥及如何才能逆轉耐藥是急需解決的重要問題。 近幾年來已有多項試驗證實KEAP1-NRF2-ARE信號通路與多重耐藥機制相關聯(lián)[1,2]，目前已經(jīng)在NSCL、乳腺癌、結腸癌等腫瘤組織中發(fā)現(xiàn)KEAP1基因的突變，使表達相關蛋白下降且NRF2相關蛋白高表達[3,4]，已有實驗證實在非小細胞肺癌，結腸癌，前列腺癌DU145細胞中KEAP1基因啟動子區(qū)CpG島高甲基化而低表達[5,6]，前列腺癌PC3細胞中KEAP1基因高表達，所以本實驗選擇PC3細胞做為陽性對照[7]。我國學者將三氧化二砷用于治療急性早幼粒細胞白血病取得顯著療效，引起了普遍關注；隨后證實其對實體瘤也能獲得同樣效果，同時三氧化二砷的去甲基化作用也有報道。本研究通過用不同濃度As2O3溶液對前列腺癌DU145細胞進行處理，檢測藥物對細胞增殖抑制影響及對細胞中KEAP1基因表達的影響，初步探索As2O3對KEAP1基因可能的作用機制，從而為激素非依賴性前列腺癌的治療提供新的治療藥物。 材料和方法 1．雄激素非依賴性前列腺癌DU145細胞，用10%滅活胎牛血清含雙抗的新鮮DMEM-H培養(yǎng)液在細胞培養(yǎng)箱中（T：37℃,5%CO2）常規(guī)培養(yǎng)；PC-3細胞用含10%滅活胎牛血清的新鮮RPMI-1640培養(yǎng)液培養(yǎng)。 2．應用MTT法檢測不同濃度(0、0.5、1.0、2.0、4.0、6.0、8.0μmol/L)的As2O3不同作用時間(24、48、72h)對DU145細胞的生長抑制作用。 3.應用RT-PCR方法檢測不同濃度（0、0.5、1.0、2.0、4.0μmol/L）As2O3作用于各組DU145細胞72h后KEAP1基因mRNA表達情況。 4.應用Western blot方法檢測不同濃度組(0.0、0.5、1.0、2.0、4.0μmol/L）As2O3的作用激素非依賴性前列腺癌DU145細胞株72h后KEAP1蛋白表達的情況，PC3細胞組作為對照組。 5．統(tǒng)計學處理：實驗數(shù)據(jù)用SPSS17.0統(tǒng)計軟件進行處理，各組數(shù)據(jù)間采用非參數(shù)檢驗、t檢驗及方差分析進行差異比較，檢驗水準α=0.05。 結果 1．MTT結果顯示：隨著As203濃度的升高對DU145細胞抑制作用越明顯，在8．0μmol/L時抑制率最高；根據(jù)不同時間點對抑制率的測定在72h時抑制率最高。因此As203對DU145細胞的抑制作用具有劑量依賴性和時間依賴性。 2．RT-PCR結果顯示：在不含As2O3的處理組中，前列腺癌DU145細胞的KEAP1基因mRNA表達不明顯，經(jīng)As2O3處理72h后，KEAP1基因表達逐漸增強，在2.0μmol/L時條帶亮度最大，表達最強。 3．Western blot結果顯示：陰性對照組keap1蛋白未表達；不同濃度As2O3處理人前列腺癌DU145細胞72h后，各觀察組KEAP1蛋白均出現(xiàn)表達；2.0μmol/L As2O3處理組與陽性對照PC-3組顯示的KEAP1蛋白條帶最深。 結論 1.As2O3對前列腺癌DU145細胞的增殖具有抑制作用，，并且隨著劑量的增大或時間的增長抑制作用而逐漸增強，呈劑量依賴性和時間依賴性。 2.As2O3可以誘導KEAP1基因的mRNA和KEAP蛋白表達增加。
[Abstract]:Background and purpose
Prostate cancer is one of the most common malignant tumors in the male genitourinary system. The main methods for treating prostate cancer are surgery, hormone antagonism, chemotherapy and radiotherapy. Although the initial androgen deprivation of most prostate cancer patients is effective, they usually appear in androgens for months to years. The prognosis of hormone non dependent prostate cancer is poor, and it is not sensitive to chemotherapy and radiation therapy. The mechanism of hormone non dependent prostate cancer therapy is not clear at present. Therefore, it is an important problem to understand how cancer cells produce resistance and how to reverse the drug resistance.
In recent years, many experiments have proved that KEAP1-NRF2-ARE signaling pathway is associated with multidrug resistance mechanism [1,2]. The mutation of KEAP1 gene has been found in NSCL, breast cancer, colon cancer and other tumor tissues. The expression related protein is decreased and the NRF2 related protein is highly expressed [3,4]. It has been proved to be in the non small cell lung cancer, colon cancer, and the front row. The CpG island of KEAP1 gene promoter region of adenocarcinoma DU145 cells is highly methylation and low expression of [5,6], and the KEAP1 gene in PC3 cells of prostate cancer is highly expressed. Therefore, this experiment selected PC3 cells as a positive control [7]. in China to use arsenic trioxide to treat acute promyelocytic leukemia. It is confirmed that it can also achieve the same effect on solid tumor, and the demethylation of arsenic trioxide is also reported. This study was conducted by using different concentrations of As2O3 solution to treat DU145 cells in prostate cancer, to detect the effect of drugs on cell proliferation inhibition and to the expression of KEAP1 gene in cells, and to explore the possibility of As2O3 to the KEAP1 gene. Therefore, it provides a new treatment for hormone independent prostate cancer.
Materials and methods
1. the androgen independent prostate cancer DU145 cells were cultured in the cell culture box (T:37, 5%CO2) with 10% inactivated fetal bovine serum containing fresh DMEM-H culture in the cell culture box (T:37, 5%CO2), and the PC-3 cells were cultured with fresh RPMI-1640 culture containing 10% inactivated fetal bovine serum.
2. MTT method was used to detect the growth inhibition of DU145 cells with different concentrations (0,0.5,1.0,2.0,4.0,6.0,8.0 mu mol/L) of As2O3 at different time of action (24,48,72h).
3. RT-PCR method was used to detect mRNA expression of KEAP1 gene after different concentrations (0,0.5,1.0,2.0,4.0 mol/L) As2O3 acting on 72h cells of each group.
4. the Western blot method was used to detect the expression of KEAP1 protein in the non dependent prostate cancer DU145 cell line 72h, and the PC3 cell group was used as the control group. The Western blot method was used to detect the different concentration group (0.0,0.5,1.0,2.0,4.0 mu mol/L).
5. statistical processing: the experimental data were processed by SPSS17.0 statistical software. The data between each group were tested by nonparametric test, t test and variance analysis were compared, and the level of alpha =0.05. was tested.
Result
1.MTT results showed that the inhibition of DU145 cells was more obvious with the increase of As203 concentration and the highest inhibition rate at 8 mol/L; the inhibition rate was highest at 72h at different time points. Therefore, the inhibitory effect of As203 on DU145 cells was dose-dependent and time dependent.
2.RT-PCR results showed that in the treatment group without As2O3, the expression of KEAP1 gene mRNA in DU145 cells of prostate cancer was not obvious. After As2O3 treatment 72h, the expression of KEAP1 gene was increased gradually, and the band brightness was the greatest and the expression was the strongest at 2 u.
The results of 3.Western blot showed that the Keap1 protein in the negative control group was not expressed, and the KEAP1 protein was expressed in all the observation groups after 72h of As2O3 treated DU145 cells of human prostate cancer, and the KEAP1 protein bands in the 2 u mol/L As2O3 treatment group and the positive control PC-3 group were the deepest.
conclusion
1.As2O3 has a inhibitory effect on the proliferation of DU145 cells in prostate cancer and gradually increases with the dose increase or time growth inhibition, which is dose-dependent and time dependent.
2.As2O3 can induce the expression of mRNA and KEAP protein in KEAP1 gene.
【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R737.25

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