發(fā)布時(shí)間：2018-03-11 03:14

  本文選題：醋酸棉　切入點(diǎn)：酚腺樣囊性癌　出處：《昆明醫(yī)科大學(xué)》2014年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的 1.觀察體外應(yīng)用不同濃度醋酸棉酚(GAA)對(duì)人腺樣囊性癌肺高轉(zhuǎn)移細(xì)胞ACC-M生長(zhǎng)及凋亡的影響； 2.檢測(cè)不同濃度GAA對(duì)人腺樣囊性癌肺高轉(zhuǎn)移細(xì)胞ACC-M E-cadherin、p16 基因的蛋白表達(dá)的影響；3.檢測(cè)不同濃度GAA對(duì)人腺樣囊性癌肺高轉(zhuǎn)移細(xì)胞ACC-M甲基轉(zhuǎn)移酶1(DNMT1)的蛋白及mRNA表達(dá)影響。初步探討棉酚的抗腫瘤機(jī)制。 方法 1.常規(guī)培養(yǎng)人腺樣囊性癌肺高轉(zhuǎn)移細(xì)胞ACC-M、人肺成纖維細(xì)胞HFL1,用不同濃度GAA處理,倒置顯微鏡下觀察兩種細(xì)胞的生長(zhǎng)變化及形態(tài)學(xué)改變； 2.通過(guò)CCK-8實(shí)驗(yàn)檢測(cè)不同濃度GAA對(duì)人腺樣囊性癌肺高轉(zhuǎn)移細(xì)胞ACC-M.人肺成纖維細(xì)胞HFL1生長(zhǎng)的影響,并計(jì)算GAA處理后24,48,72h的ICso值； 3.應(yīng)用流式細(xì)胞術(shù)(FCM)檢測(cè)不同濃度GAA處理人腺樣囊性癌肺高轉(zhuǎn)移細(xì)胞ACC-M后24,48,72h的凋亡變化情況； 4.應(yīng)用Western-blot檢測(cè)不同濃度GAA處理人腺樣囊性癌肺高轉(zhuǎn)移細(xì)胞ACC-M后E-cadherin、p16、甲基轉(zhuǎn)移酶1(DNMT1)基因的蛋白表達(dá)變化; 5.應(yīng)用實(shí)時(shí)熒光定量PCR (RT-PCR)檢測(cè)不同濃度GAA處理人腺樣囊性癌肺高轉(zhuǎn)移細(xì)胞ACC-M甲基轉(zhuǎn)移酶1(DNMT1)的mRNA表達(dá)情況。 結(jié)果 1.CCK-8法檢測(cè)顯示,不同濃度GAA對(duì)人腺樣囊性癌肺高轉(zhuǎn)移細(xì)胞ACC-M.人肺成纖維細(xì)胞HFL1均有抑制作用,在一定劑量范圍內(nèi)具有濃度和時(shí)間依賴(lài)性。測(cè)得ACC-M的IC5o值：24h IC50=33.20μnol/L,48h IC50=15.50μmol/L,72h IC50=8.60μmol/L。 HFL1的IC5o值：24h IC50=35.13μmol/L,48hIC50=28.00μmol/L,72h IC50=26.80μmol/L; 2.FCM檢測(cè)顯示,經(jīng)終濃度為10μmol/L、20μmol/L、30μmol/L及40μmol/L的GAA處理后,人腺樣囊性癌肺高轉(zhuǎn)移細(xì)胞ACC-M的凋亡率與濃度呈正相關(guān); 3. Western-blot檢測(cè)蛋白表達(dá)結(jié)果顯示,24h：E-cadherin和p16蛋白表達(dá)增高,DNMT1降低；48h：E-cadherin和p16蛋白表達(dá)增高,DNMT1無(wú)明顯變化；72h：E-cadherin蛋白表達(dá)增高,p16, DNMT1無(wú)明顯變化； 4.實(shí)時(shí)熒光定量PCR檢測(cè)顯示,經(jīng)GAA處理后,ACC-M細(xì)胞實(shí)驗(yàn)組DNMT1的nRNA相對(duì)表達(dá)量與未加GAA的對(duì)照組相比表達(dá)降低(P0.05),且隨GAA濃度增加呈劑量依賴(lài)性降低。 結(jié)論 1.GAA能夠抑制人腺樣囊性癌肺高轉(zhuǎn)移細(xì)胞ACC-M生長(zhǎng)并誘導(dǎo)細(xì)胞凋亡,在一定劑量范圍內(nèi)具有濃度和時(shí)間依賴(lài)性。 2.一定濃度的GAA能使ACC-M細(xì)胞E-cadherin、p16蛋白表達(dá)上升,可能是GAA抗腫瘤的作用靶點(diǎn)。 3.一定濃度的GAA能降低ACC-M細(xì)胞DNMT1的蛋白表達(dá)并使mRNA表達(dá)水平下調(diào)。提示GAA能使DNMT1活性下調(diào)從而發(fā)揮抑癌作用,這可能是GAA的抗腫瘤機(jī)制之一。
[Abstract]:objective
1. the effects of different concentrations of gossypol (GAA) on the growth and apoptosis of lung high metastatic cells of human adenoid cystic carcinoma (ACC-M) were observed in vitro.
2. ACC-M E-cadherin, p16, and p16 in human adenoid cystic carcinoma of human adenoid cystic carcinoma cells were detected by GAA
The effect of gene protein expression; 3., detect the effect of different concentrations of GAA on the expression of ACC-M methyltransferase 1 (DNMT1) protein and mRNA in human lung adenocarcinoma cells with high metastatic adenoid cystic carcinoma, and preliminarily explore the anti-tumor mechanism of gossypol.
Method
1., the ACC-M and HFL1 of human lung adenocarcinoma cell line were cultured routinely. The growth and morphological changes of two kinds of cells were observed by inverted microscope under different concentrations of GAA.
2., we detected the effects of different concentrations of GAA on the growth of HFL1 in human lung adenocarcinoma cells and ACC-M. lung fibroblasts, and the ICso value of 24,48,72h after GAA treatment by CCK-8 test.
3. the flow cytometry (FCM) was used to detect the changes in the apoptosis of 24,48,72h after ACC-M with different concentrations of GAA in human adenoid cystic carcinoma of the lung.
4. Western-blot was used to detect the protein expression of E-cadherin, p16 and methyltransferase 1 (DNMT1) gene after treatment with different concentrations of GAA in human lung adenocarcinoma cell line ACC-M.
5. using real-time fluorescent quantitative PCR (RT-PCR) to detect the mRNA expression of ACC-M methyltransferase 1 (DNMT1) in human adenoid cystic carcinoma of human adenoid cystic carcinoma with different concentrations of GAA.
Result
1.CCK-8 assay showed that different concentrations of GAA on human adenoid cystic carcinoma with lung metastasis of ACC-M. cells into human lung fibroblast cells HFL1 were inhibited, in a certain dose range is dependent on concentration and time. The measured ACC-M IC5o values: 24h IC50=33.20 nol/L, 48h IC50=15.50 mol/L, 72h IC50=8.60 mol/L. HFL1 the value of IC5o: 24h IC50=35.13 mol/L, 48hIC50=28.00 mol/L, 72h IC50=26.80 mol/L;
2.FCM detection showed that the apoptosis rate of ACC-M cells was positively correlated with the concentration of ACC-M after treatment with the final concentration of 10 mol/L, 20 mol/L, 30 mol/L and 40 mol/L GAA.
3. Western-blot detection protein expression results showed that 24h:E-cadherin and p16 protein expression increased, DNMT1 decreased, 48h:E-cadherin and p16 protein expression increased, DNMT1 did not change significantly, 72h:E-cadherin protein expression increased, p16 and DNMT1 did not change significantly.
4. real-time fluorescence quantitative PCR detection showed that after GAA treatment, the relative nRNA expression of DNMT1 in ACC-M cell group decreased compared with that in control group without GAA (P0.05), and decreased in a dose-dependent manner with increasing GAA concentration.
conclusion
1.GAA can inhibit the growth of ACC-M and induce apoptosis in human adenoid cystic carcinoma, which is dependent on concentration and time in a certain dose range.
2. a certain concentration of GAA can increase the expression of E-cadherin and p16 protein in ACC-M cells, which may be the target of anti-tumor action of GAA.
3., a certain concentration of GAA can decrease the expression of DNMT1 and down regulate the expression of mRNA in ACC-M cells, suggesting that GAA can reduce DNMT1 activity and play a role in inhibiting cancer, which may be one of the anti-tumor mechanisms of GAA.

【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R739.8

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