醋酸棉酚对人腺样囊性癌ACC-M细胞生长及E-cadherin、p16基因蛋白表达的影响

发布时间：2018-03-11 03:14

本文选题：醋酸棉　切入点：酚腺样囊性癌　出处：《昆明医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的 1.观察体外应用不同浓度醋酸棉酚(GAA)对人腺样囊性癌肺高转移细胞ACC-M生长及凋亡的影响； 2.检测不同浓度GAA对人腺样囊性癌肺高转移细胞ACC-M E-cadherin、p16 基因的蛋白表达的影响；3.检测不同浓度GAA对人腺样囊性癌肺高转移细胞ACC-M甲基转移酶1(DNMT1)的蛋白及mRNA表达影响。初步探讨棉酚的抗肿瘤机制。方法 1.常规培养人腺样囊性癌肺高转移细胞ACC-M、人肺成纤维细胞HFL1,用不同浓度GAA处理,倒置显微镜下观察两种细胞的生长变化及形态学改变； 2.通过CCK-8实验检测不同浓度GAA对人腺样囊性癌肺高转移细胞ACC-M.人肺成纤维细胞HFL1生长的影响,并计算GAA处理后24,48,72h的ICso值； 3.应用流式细胞术(FCM)检测不同浓度GAA处理人腺样囊性癌肺高转移细胞ACC-M后24,48,72h的凋亡变化情况； 4.应用Western-blot检测不同浓度GAA处理人腺样囊性癌肺高转移细胞ACC-M后E-cadherin、p16、甲基转移酶1(DNMT1)基因的蛋白表达变化; 5.应用实时荧光定量PCR (RT-PCR)检测不同浓度GAA处理人腺样囊性癌肺高转移细胞ACC-M甲基转移酶1(DNMT1)的mRNA表达情况。结果 1.CCK-8法检测显示,不同浓度GAA对人腺样囊性癌肺高转移细胞ACC-M.人肺成纤维细胞HFL1均有抑制作用,在一定剂量范围内具有浓度和时间依赖性。测得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检测显示,经终浓度为10μmol/L、20μmol/L、30μmol/L及40μmol/L的GAA处理后,人腺样囊性癌肺高转移细胞ACC-M的凋亡率与浓度呈正相关; 3. Western-blot检测蛋白表达结果显示,24h：E-cadherin和p16蛋白表达增高,DNMT1降低；48h：E-cadherin和p16蛋白表达增高,DNMT1无明显变化；72h：E-cadherin蛋白表达增高,p16, DNMT1无明显变化； 4.实时荧光定量PCR检测显示,经GAA处理后,ACC-M细胞实验组DNMT1的nRNA相对表达量与未加GAA的对照组相比表达降低(P0.05),且随GAA浓度增加呈剂量依赖性降低。结论 1.GAA能够抑制人腺样囊性癌肺高转移细胞ACC-M生长并诱导细胞凋亡,在一定剂量范围内具有浓度和时间依赖性。 2.一定浓度的GAA能使ACC-M细胞E-cadherin、p16蛋白表达上升,可能是GAA抗肿瘤的作用靶点。 3.一定浓度的GAA能降低ACC-M细胞DNMT1的蛋白表达并使mRNA表达水平下调。提示GAA能使DNMT1活性下调从而发挥抑癌作用,这可能是GAA的抗肿瘤机制之一。
[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.

【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.8

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