他莫昔芬处理子宫内膜癌细胞后ARID1A、ERα、ERβ、PR、P53变化的研究

发布时间：2019-06-20 17:34

【摘要】：目的：子宫内膜癌是女性生殖系统最常见的恶性肿瘤，子宫内膜癌的发生、发展与很多因素有关，如长期雌激素刺激同时无孕激素拮抗等机制。同时，他莫昔芬（tamoxifen TAM）作用后，子宫内膜癌的发生率明显升高。但是，目前对于TAM引起子宫内膜癌的发病机制仍不清楚。关于染色体重组复合物的研究日益增多，具有抑制DNA合成的作用，故其突变低表达能够促进细胞无限分裂，进而发展成癌。ARID1A（AT-richinteracting domain containing protein，又称脑蛋白120、SMARCF1、p270等）是染色质重组复合物之一，同时作为抑癌基因，在多种肿瘤组织中存在突变。目前对于ARID1A的结构研究较多，对其在子宫内膜癌中的表达及生物学功能的研究较少。本研究通过检测ARID1A、ERα、ERβ、PR、P53在子宫内膜癌细胞（高分化Ishikawa、中分化HEC-1A及低分化KLE）中的基因和蛋白的表达水平，并进一步分析TAM及雌激素（estrogen E2）处理后子宫内膜癌细胞中ARID1A、ERα、ERβ、PR、P53基因和蛋白的表达水平变化，探讨ARID1A在子宫内膜癌的发生、发展中的可能作用机制。方法： 1应用实时定量qRT-PCR法检测正常子宫内膜组织、子宫内膜癌细胞株（Ishikawa、HEC-1A、KLE）中ARID1A、ERα、ERβ、PR、P53mRNA的表达水平。 2应用实时定量qRT-PCR法检测TAM及E2依据不同浓度及不同时间处理细胞后ARID1A、ERα、ERβ、PR、P53mRNA的表达水平。 3应用Western-blot法检测正常子宫内膜组织、子宫内膜癌细胞株（Ishikawa、HEC-1A、KLE）中BAF250a、ERα、ERβ、PR、P53蛋白的表达水平。 4应用Western-blot法检测TAM及E2依据不同浓度及不同时间处理细胞后BAF250a、ERα、ERβ、PR、P53蛋白的表达水平。 5应用流式细胞学测定TAM及E2依据不同浓度及不同时间处理细胞后细胞周期变化情况。结果： 1ARID1A、ERα、ERβ mRNA及蛋白在三种子宫内膜癌细胞中的表达均显著低于其在正常子宫内膜组织中的表达，经过TAM或E2处理后，ARID1A、ERα、ERβ mRNA及蛋白表达量随着药物浓度的增加而降低，随着药物处理时间的延长而降低(P 0.05)，而PR、P53mRNA及蛋白在三种子宫内膜癌细胞中的表达低于其在正常子宫内膜组织中的表达，经过TAM或E2处理后，PR、P53mRNA及蛋白表达量随着药物浓度的增加而增加，随着药物处理时间的延长而增加，但均低于其在正常子宫内膜组织中的表达(P 0.05)，呈时间-剂量依赖性。 2经过不同浓度TAM或E2在不同时间处理三种子宫内膜癌细胞（Ishikawa、HEC-1A、KLE）后，三种细胞均表现为G0/G1期缩短，S期延长，且随着药物浓度的增高及处理时间的延长，G0/G1期逐渐缩短，S期逐渐延长（P0.05）。结论： 1TAM或E2处理子宫内膜癌细胞后ARID1A、ERα、ERβmRNA及蛋白的表达明显低于其在未经药物处理子宫内膜癌中的表达，而PR、P53mRNA及蛋白的表达明显高于其在未经药物处理子宫内膜癌中的表达。 2TAM与E2可能通过下调ARID1A的表达而引起子宫内膜癌的发生，，而且ARID1A的下调可能与ER、P53存在一定相互作用关系。 3经TAM与E2处理后子宫内膜癌细胞的细胞周期均表现为G0/G1期缩短，S期延长。
[Abstract]:Objective: Endometrial carcinoma is the most common malignant tumor of female reproductive system. The occurrence and development of endometrial carcinoma are related to many factors, such as long-term estrogen stimulation and no such mechanism as progestogen antagonism. At the same time, tamoxifen (tamoxifen) was used to increase the incidence of endometrial carcinoma. However, the pathogenesis of endometrial cancer is still unclear for TAM. The research on the chromosomal recombination complex has been increasing, and has the effect of inhibiting the DNA synthesis, so that the mutant low expression can promote the infinite division of the cell, and further develop the cancer. ARID1A (AT-ricinting domain containment protein, also known as brain protein 120, SMARCF1, p270, etc.) is one of the recombinant complexes of chromatin, and as a tumor suppressor gene, there are mutations in various tumor tissues. At present, there are many studies on the structure of ARID1A, and the study of its expression and biological function in endometrial carcinoma is less. The expression of ARID1A, ER, ER, PR, P53 in endometrial cancer cells (HEC-1A and low-differentiated KLE) and the expression levels of genes and proteins in endometrial cancer cells (HEC-1A and low-differentiated KLE) were detected and the ARID, ER, ER and ER levels in the endometrial cancer cells were further analyzed. The expression level of PR, P53 gene and protein was changed, and the possible mechanism of ARID1A in the development and development of endometrial carcinoma was discussed. square Method:1 A real-time quantitative qRT-PCR was used to detect the expression of ARID1A, ER, ER, PR, P53mRNA in normal endometrium and endometrial carcinoma (Ishikawa, HEC-1A, KLE). The levels of AID1A, ER, ER, PR, P53mRNA were detected by real-time quantitative qRT-PCR. The level of expression of BAF250a, ER, ER, PR, P53 in normal endometrial tissue, endometrial carcinoma cell (Ishikawa, HEC-1A, KLE) was detected by Western-blot. The expression level of the protein was detected by Western-blot, and then the cells were treated with different concentrations and different time. The BAF250a, ER antigen, ER antigen, PR, P were treated with different concentrations and time. The expression level of 53 protein was measured by flow cytometry. After the cell Results: The expression of ARID1A, ER, ER, ER-mRNA and protein in three endometrial cancer cells was significantly lower than that in normal endometrial tissues. The expression of PR, P53mRNA and protein in the three endometrial cancer cells decreased with the increase of the drug concentration (P 0.05). The expression of PR, P53mRNA and protein in the three endometrial cancer cells was lower than that in the normal endometrial tissues. The expression of PR, P53mRNA and protein after treatment with TAM or E2 The amount of expression increases with the increase of the drug concentration, which increases with the increase of the drug treatment time, but is lower than the expression of the drug in the normal endometrium (P 0.05) Time-dose-dependent. After treatment of three endometrial cancer cells (Ishikawa, HEC-1A, KLE) at different times through different concentrations of TAM or E2, the three cells showed a decrease in G0/ G1 phase and an extension of S phase, and the G0/ G1 phase was decreased with the increase of drug concentration and the time of treatment. gradual shortening Conclusion: The expression of ARID1A, ER, ER, ER-mRNA and protein in the endometrial carcinoma cells after treatment with 1TAM or E2 was significantly lower than that of the non-drug-treated endometrial carcinoma, while the expression of PR, P53mRNA and protein was significantly lower. significantly higher than that in endometrial cancer without drug treatment. TAM and E2 may cause endometrial cancer by down-regulation of the expression of ARID1A, and ARID1 A's down-regulation may have a certain interaction with ER and P53. After treatment with TAM and E2, the endometrium
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33

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