HSF1在子宫内膜癌细胞株中拮抗氧化损伤作用的体外实验研究
发布时间：2018-09-18 16:30
【摘要】：【背景】 子宫内膜癌是对女性健康产生极大威胁的恶性肿瘤，近年来其发病率和病死率都呈现上升趋势，俨然已经成为一个严重的公共卫生难题，它作为严重危害女性健康的疾病已受到全世界的广泛关注。美国2012年有47130名女性被诊断患有子宫内膜癌并且有8010例因其死亡。在2004-2005年卫生部恶性肿瘤回顾调查中，显示子宫内膜癌死亡率以约2.71/10万高居女性恶性肿瘤死亡率第十位（2012中国卫生统计年鉴）。尽管多数子宫内膜癌病例是早期，但由于晚期以及低分化子宫内膜癌的预后极差，其5年生存率并不令人满意，因此子宫内膜癌的早期发现和及时治疗显得至关重要。 热休克因子1（Heat shock factor1，HSF1）是调节细胞热休克蛋白（HSPs）表达的主要转录因子，热休克反应被激活后HSF1能迅速改变HSP基因位点并增加它们的表达，，热休克蛋白可以帮助错误折叠的多肽重新折叠并抑制蛋白聚合。这有利于细胞抑制蛋白应激毒性和保持蛋白质体内平衡，使生物具有可感应应力的分子机制来检测和中和蛋白损伤。HSF1已经通过细胞检测、器官模型检测、进而包括酵母、线虫、啮齿目动物等生物水平的分析全面描述了它的功能。 尽管HSF1帮助细胞和有机体对抗严重有害刺激的作用已被广泛研究并取得了一些研究结果，但仍然有许多问题亟待探讨：HSF1对下游因子调控的功能是否参与肿瘤发生发展？HSF1对细胞的保护作用在其参与子宫内膜癌发生发展、氧化应激、细胞周期调控及凋亡等多种关键的细胞反应中发挥着怎样的作用？干预HSF1后对这些细胞反应又有何影响？ 为了验证HSF1在子宫内膜癌中的表达及其作用，本实验以不同浓度的过氧化氢分别作用于子宫内膜癌细胞株，观察细胞的生存情况、HSF1的表达变化情况以及细胞受损伤状况、抗氧化系统和线粒体功能改变情况，探讨HSF1对子宫内膜癌细胞株抗凋亡能力的影响及其作用机制，从而为以HSF1为子宫内膜癌的治疗靶点提供新的思路。 【研究目的】 1.探讨HSF1在子宫内膜癌细胞株的表达及其作用 2.探讨HSF1对子宫内膜癌细胞株抗凋亡能力的影响及其作用机制 【研究方法】 1.应用实时荧光定量PCR、Western blot技术检测子宫内膜癌细胞中HSF1的表达水平。 2.应用实时荧光定量PCR的方法检测子宫内膜癌细胞受到不同浓度过氧化氢刺激后HSF1的表达变化。 3. MTT法检测细胞受到不同浓度过氧化氢刺激后的受抑制情况。 4.应用RNAi技术下调HSF1在子宫内膜癌细胞中的表达，实时荧光定量PCR、Western blot技术检测下调后HSF1表达水平的变化。 5. MTT法检测下调HSF1后，各组细胞受到过氧化氢刺激时的受抑制情况。 6. Annexin V-PI双染色法检测下调HSF1前后，各组细胞的凋亡情况。 7.使用流式细胞仪检测下调HSF1前后，各组细胞的周期变化。 8.使用酶标仪检测下调HSF1前后，各组细胞丙二醛含量、过氧化氢酶活性、谷胱甘肽含量、总抗氧化能力以及ATP生成情况。 9.使用流式细胞仪检测下调HSF1前后，各组细胞内活性氧含量。 【实验结果】 1.在Ishikawa细胞中HSF1的mRNA表达水平最高，HEC-1-B其次，RL95-2最低。 2.在Ishikawa细胞中HSF1的蛋白表达水平最高，HEC-1-B其次，RL95-2最低。Ishikawa细胞当受到300μmol/L过氧化氢溶液刺激时HSF1表达出现了明显的上调，刺激浓度为500μmol/L时HSF1表达继续增高，而在浓度到达700μmol/L时细胞内HSF1的表达却较500μmol/L略有降低，组间差异具有统计学意义；HEC-1B细胞在受到300μmol/L过氧化氢刺激时HSF1表达出现明显增高，而当刺激浓度为500μmol/L时HSF1的表达出现降低，以700μmol/L浓度刺激时甚至低于基水平，组间差异具有统计学意义；RL95-2细胞在50μmol/L低浓度过氧化氢刺激时HSF1表达即出现明显上升，当浓度300μmol/L时HSF1表达与低浓度相比已有所降低，当大于等于500μmol/L过氧化氢刺激时HSF1则出现了低于基水平的表达，组间差异具有统计学意义。 3. Ishikawa细胞和HEC-1-B细胞分别在受到浓度为700μmol/L和500μmol/L的H2O2刺激后，细胞抑制率显著增高，具有统计学意义；而RL95-2细胞在受到300μmol/L的H2O2刺激后，细胞抑制率即出现具有统计学意义的明显改变。 4. Ishikawa细胞转染siRNA后HSF1的mRNA表达水平明显降低，与空白组和阴性对照组存在显著性差异（P0.05），而阴性对照组与空白组无明显组间差异（P0.05）。 5. Ishikawa细胞转染siRNA后HSF1的蛋白表达水平明显降低，与空白组和阴性对照组存在显著性差异（P0.05），而阴性对照组与空白组无明显组间差异（P0.05）。 6. MTT结果显示转染组Ishikawa细胞在受到H2O2溶液刺激后细胞抑制率显著增高且具有统计学意义。 7.转染siRNA后的Ishikawa细胞与空白对照组相比，在受到H2O2溶液刺激时G2+S期比例减少且差异具有统计学意义（P0.05）。 8.转染siRNA后的Ishikawa细胞在受到H2O2溶液刺激时，与对照组相比细胞内丙二醛含量明显上升且差异具有统计学意义（P0.05）。 9.正常状态与应激状态下的Ishikawa细胞与对照组相比过氧化氢酶活性均有所减弱且差异具有统计学意义（P0.05）。 10. Ishikawa细胞与对照组相比总谷胱甘肽含量减少且差异具有统计学意义（P0.05）。 11.转染组Ishikawa细胞在受到H2O2溶液刺激时，与对照组相比总抗氧化能力减弱且差异具有统计学意义（P0.05）。 12.转染组Ishikawa细胞在受到H2O2溶液刺激时，与对照组相比ATP水平降低且差异具有统计学意义（P0.05）。 13.转染siRNA后的Ishikawa细胞与对照组相比，在受到H2O2溶液刺激时活性氧含量明显增高且差异具有统计学意义（P0.05）。 【结论】 1. MTT结果与三株细胞内HSF1基水平测定的结果呈现相关性，HSF1基水平最高的Ishikawa细胞抑制率50%时的刺激浓度最高，为700μmol/L；HSF1基水平最低的RL95-2细胞出现抑制率50%时的刺激浓度最低，为300μmol/L；而HSF1基水平介于两者之间的HEC-1-B细胞出现抑制率50%时也处于500μmol/L的中间浓度。这提示HSF1的表达可能与肿瘤应激能力相关，促进子宫内膜癌的发生。 2.下调HSF1在子宫内膜癌细胞中的表达后，一系列的检测显示HSF1与子宫内膜癌细胞的抗凋亡能力相关，HSF1可能是一种子宫内膜肿瘤促进因子，它被激活时增加了子宫内膜癌细胞抗性，从而促进了子宫内膜癌发生发展。HSF1也许是一个较好的候选标志物，有可能作为可利用的肿瘤病程干预潜在靶点。
[Abstract]:[background]
Endometrial cancer is a malignant tumor that poses a great threat to women's health. In recent years, the incidence and mortality of endometrial cancer have shown an upward trend. It has become a serious public health problem. As a serious disease endangering women's health, it has attracted worldwide attention. Endometrial cancer is the leading cause of death in women. In the 2004-2005 retrospective survey of malignant tumors conducted by the Ministry of Health, the mortality rate of endometrial cancer was about 271/100,000, ranking tenth among female malignant tumors (China Health Statistics Yearbook 2012). Although most cases of endometrial cancer are early, advanced and poorly differentiated endometrial cancer is due to poor differentiation. The prognosis of endometrial carcinoma is very poor, and its 5-year survival rate is not satisfactory. Therefore, early detection and timely treatment of endometrial carcinoma is very important.
Heat shock factor 1 (HSF1) is a major transcription factor that regulates the expression of heat shock proteins (HSPs). HSF1 can rapidly alter and increase the expression of HSP genes after heat shock response is activated. HSF1 can help misfolded polypeptides to fold and inhibit protein polymerization. This is conducive to cell inhibition. Protein stress toxicity and the maintenance of protein homeostasis enable organisms to detect and neutralize protein damage by molecular mechanisms that induce stress. HSF1 has been fully described by cell detection, organ model testing, and further analysis of biological levels including yeast, nematode, rodent and so on.
Although the role of HSF1 in helping cells and organisms resist severe and harmful stimuli has been extensively studied and some results have been obtained, there are still many questions to be discussed urgently: whether the function of HSF1 in regulating downstream factors is involved in tumorigenesis and development, and whether the protective effect of HSF1 on cells is involved in the development and oxidation of endometrial carcinoma. What are the roles of stimulation, cell cycle regulation, apoptosis and other key cell responses? What are the effects of HSF1 intervention on these cell responses?
In order to verify the expression and role of HSF1 in endometrial carcinoma, different concentrations of hydrogen peroxide were used to treat endometrial carcinoma cell lines. The survival, expression and damage of HSF1, antioxidant system and mitochondrial function were observed. The effects of HSF1 on endometrial carcinoma cells were investigated. The effect of HSF1 on the anti-apoptosis ability and its mechanism provide a new idea for the treatment of endometrial carcinoma.
[research purposes]
1. to investigate the expression and role of HSF1 in endometrial carcinoma cell lines.
2. to investigate the effect of HSF1 on the anti apoptotic ability of endometrial carcinoma cell lines and its mechanism.
[research methods]
1. real-time fluorescence quantitative PCR and Western blot were used to detect the expression level of HSF1 in endometrial carcinoma cells.
2. Real-time fluorescence quantitative PCR was used to detect the expression of HSF1 in endometrial carcinoma cells stimulated by different concentrations of hydrogen peroxide.
3. MTT method was used to detect the inhibition of cells after stimulation with different concentrations of hydrogen peroxide.
4. The expression of HSF1 in endometrial carcinoma cells was down-regulated by RNAi, real-time fluorescence quantitative PCR and Western blot.
5. MTT method was used to detect the inhibition of cells stimulated by hydrogen peroxide after down-regulation of HSF1.
6. Annexin V-PI double staining was used to detect the apoptosis of cells in each group before and after down-regulation of HSF1.
7. flow cytometry was used to detect cell cycle changes before and after down-regulation of HSF1.
8. Malondialdehyde content, catalase activity, glutathione content, total antioxidant capacity and ATP production were detected by enzyme labeling.
9. flow cytometry was used to detect reactive oxygen species in each group before and after HSF1 downregulation.
[experimental results]
1. in Ishikawa cells, the expression level of HSF1 mRNA was the highest, followed by HEC-1-B, and RL95-2 was the lowest.
2. The expression of HSF1 in Ishikawa cells was the highest, followed by HEC-1-B and RL95-2. The expression of HSF1 in Ishikawa cells was significantly up-regulated when stimulated by 300 micromol/L hydrogen peroxide solution. The expression of HSF1 in Ishikawa cells continued to increase when stimulated by 500 micromol/L, but slightly higher than that in 500 micromol/L when stimulated by 700 micromol/L. The expression of HSF1 in HEC-1B cells increased significantly when stimulated by 300 micromol/L hydrogen peroxide, but decreased when stimulated by 500 micromol/L, even lower than the basal level when stimulated by 700 micromol/L. The difference was statistically significant between groups. The expression of HSF1 increased significantly when the concentration of hydrogen peroxide was 300 micromol/L, and decreased when the concentration of hydrogen peroxide was more than 500 micromol/L. The expression of HSF1 was lower than the basal level when the concentration of hydrogen peroxide was more than 500 micromol/L.
3. Ishikawa cells and HEC-1-B cells were stimulated by H2O2 at concentrations of 700 micromol/L and 500 micromol/L, respectively, and the cell inhibition rate increased significantly, which was statistically significant; RL95-2 cells were stimulated by H2O2 at a concentration of 300 micromol/L, and the cell inhibition rate changed significantly.
4. The expression of HSF1 mRNA in Ishikawa cells transfected with siRNA was significantly lower than that in blank group and negative control group (P 0.05), but there was no significant difference between negative control group and blank group (P 0.05).
5. The expression level of HSF1 protein in Ishikawa cells transfected with siRNA was significantly lower than that in the blank and negative control groups (P 0.05), but there was no significant difference between the negative control group and the blank group (P 0.05).
6. MTT results showed that the inhibitory rate of Ishikawa cells in transfection group was significantly increased after being stimulated by H2O2 solution.
7. Compared with the blank control group, the proportion of G2+S phase in Ishikawa cells transfected with siRNA decreased and the difference was statistically significant (P 0.05).
8. When Ishikawa cells transfected with siRNA were stimulated by H2O2 solution, the content of malondialdehyde in cells increased significantly compared with the control group (P 0.05).
9. The catalase activity of Ishikawa cells in normal state and stress state was lower than that of control group, and the difference was statistically significant (P 0.05).
10. The total glutathione content of Ishikawa cells was significantly lower than that of the control group (P 0.05).
11. The total antioxidant capacity of Ishikawa cells in the transfection group was weaker than that in the control group when stimulated by H2O2 solution (P 0.05).
12. The ATP level of Ishikawa cells in transfection group was significantly lower than that of control group when stimulated by H2O2 solution (P 0.05).
13. Compared with the control group, the content of reactive oxygen species in Ishikawa cells transfected with siRNA was significantly higher when stimulated by H2O2 solution (P 0.05).
[Conclusion]
1. MTT results were correlated with the results of HSF1 level determination in three cell lines. The stimulation concentration of Ishikawa cells with the highest HSF1 level was the highest at 50% inhibition rate, which was 700 micromol/L; the lowest at 50% inhibition rate of HL95-2 cells with the lowest HSF1 level, which was 300 micromol/L; and the level of HSF1 between them was HEC-1-1-2. The expression of HSF1 may be related to tumor stress and promote the development of endometrial carcinoma.
2. After down-regulating the expression of HSF1 in endometrial cancer cells, a series of tests showed that HSF1 was related to the anti-apoptosis ability of endometrial cancer cells. HSF1 may be an endometrial tumor-promoting factor, which increases the resistance of endometrial cancer cells when activated, thus promoting the development of endometrial cancer. Candidate biomarkers may be used as potential targets for tumor progression.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33
本文编号：2248479
[Abstract]:[background]
Endometrial cancer is a malignant tumor that poses a great threat to women's health. In recent years, the incidence and mortality of endometrial cancer have shown an upward trend. It has become a serious public health problem. As a serious disease endangering women's health, it has attracted worldwide attention. Endometrial cancer is the leading cause of death in women. In the 2004-2005 retrospective survey of malignant tumors conducted by the Ministry of Health, the mortality rate of endometrial cancer was about 271/100,000, ranking tenth among female malignant tumors (China Health Statistics Yearbook 2012). Although most cases of endometrial cancer are early, advanced and poorly differentiated endometrial cancer is due to poor differentiation. The prognosis of endometrial carcinoma is very poor, and its 5-year survival rate is not satisfactory. Therefore, early detection and timely treatment of endometrial carcinoma is very important.
Heat shock factor 1 (HSF1) is a major transcription factor that regulates the expression of heat shock proteins (HSPs). HSF1 can rapidly alter and increase the expression of HSP genes after heat shock response is activated. HSF1 can help misfolded polypeptides to fold and inhibit protein polymerization. This is conducive to cell inhibition. Protein stress toxicity and the maintenance of protein homeostasis enable organisms to detect and neutralize protein damage by molecular mechanisms that induce stress. HSF1 has been fully described by cell detection, organ model testing, and further analysis of biological levels including yeast, nematode, rodent and so on.
Although the role of HSF1 in helping cells and organisms resist severe and harmful stimuli has been extensively studied and some results have been obtained, there are still many questions to be discussed urgently: whether the function of HSF1 in regulating downstream factors is involved in tumorigenesis and development, and whether the protective effect of HSF1 on cells is involved in the development and oxidation of endometrial carcinoma. What are the roles of stimulation, cell cycle regulation, apoptosis and other key cell responses? What are the effects of HSF1 intervention on these cell responses?
In order to verify the expression and role of HSF1 in endometrial carcinoma, different concentrations of hydrogen peroxide were used to treat endometrial carcinoma cell lines. The survival, expression and damage of HSF1, antioxidant system and mitochondrial function were observed. The effects of HSF1 on endometrial carcinoma cells were investigated. The effect of HSF1 on the anti-apoptosis ability and its mechanism provide a new idea for the treatment of endometrial carcinoma.
[research purposes]
1. to investigate the expression and role of HSF1 in endometrial carcinoma cell lines.
2. to investigate the effect of HSF1 on the anti apoptotic ability of endometrial carcinoma cell lines and its mechanism.
[research methods]
1. real-time fluorescence quantitative PCR and Western blot were used to detect the expression level of HSF1 in endometrial carcinoma cells.
2. Real-time fluorescence quantitative PCR was used to detect the expression of HSF1 in endometrial carcinoma cells stimulated by different concentrations of hydrogen peroxide.
3. MTT method was used to detect the inhibition of cells after stimulation with different concentrations of hydrogen peroxide.
4. The expression of HSF1 in endometrial carcinoma cells was down-regulated by RNAi, real-time fluorescence quantitative PCR and Western blot.
5. MTT method was used to detect the inhibition of cells stimulated by hydrogen peroxide after down-regulation of HSF1.
6. Annexin V-PI double staining was used to detect the apoptosis of cells in each group before and after down-regulation of HSF1.
7. flow cytometry was used to detect cell cycle changes before and after down-regulation of HSF1.
8. Malondialdehyde content, catalase activity, glutathione content, total antioxidant capacity and ATP production were detected by enzyme labeling.
9. flow cytometry was used to detect reactive oxygen species in each group before and after HSF1 downregulation.
[experimental results]
1. in Ishikawa cells, the expression level of HSF1 mRNA was the highest, followed by HEC-1-B, and RL95-2 was the lowest.
2. The expression of HSF1 in Ishikawa cells was the highest, followed by HEC-1-B and RL95-2. The expression of HSF1 in Ishikawa cells was significantly up-regulated when stimulated by 300 micromol/L hydrogen peroxide solution. The expression of HSF1 in Ishikawa cells continued to increase when stimulated by 500 micromol/L, but slightly higher than that in 500 micromol/L when stimulated by 700 micromol/L. The expression of HSF1 in HEC-1B cells increased significantly when stimulated by 300 micromol/L hydrogen peroxide, but decreased when stimulated by 500 micromol/L, even lower than the basal level when stimulated by 700 micromol/L. The difference was statistically significant between groups. The expression of HSF1 increased significantly when the concentration of hydrogen peroxide was 300 micromol/L, and decreased when the concentration of hydrogen peroxide was more than 500 micromol/L. The expression of HSF1 was lower than the basal level when the concentration of hydrogen peroxide was more than 500 micromol/L.
3. Ishikawa cells and HEC-1-B cells were stimulated by H2O2 at concentrations of 700 micromol/L and 500 micromol/L, respectively, and the cell inhibition rate increased significantly, which was statistically significant; RL95-2 cells were stimulated by H2O2 at a concentration of 300 micromol/L, and the cell inhibition rate changed significantly.
4. The expression of HSF1 mRNA in Ishikawa cells transfected with siRNA was significantly lower than that in blank group and negative control group (P 0.05), but there was no significant difference between negative control group and blank group (P 0.05).
5. The expression level of HSF1 protein in Ishikawa cells transfected with siRNA was significantly lower than that in the blank and negative control groups (P 0.05), but there was no significant difference between the negative control group and the blank group (P 0.05).
6. MTT results showed that the inhibitory rate of Ishikawa cells in transfection group was significantly increased after being stimulated by H2O2 solution.
7. Compared with the blank control group, the proportion of G2+S phase in Ishikawa cells transfected with siRNA decreased and the difference was statistically significant (P 0.05).
8. When Ishikawa cells transfected with siRNA were stimulated by H2O2 solution, the content of malondialdehyde in cells increased significantly compared with the control group (P 0.05).
9. The catalase activity of Ishikawa cells in normal state and stress state was lower than that of control group, and the difference was statistically significant (P 0.05).
10. The total glutathione content of Ishikawa cells was significantly lower than that of the control group (P 0.05).
11. The total antioxidant capacity of Ishikawa cells in the transfection group was weaker than that in the control group when stimulated by H2O2 solution (P 0.05).
12. The ATP level of Ishikawa cells in transfection group was significantly lower than that of control group when stimulated by H2O2 solution (P 0.05).
13. Compared with the control group, the content of reactive oxygen species in Ishikawa cells transfected with siRNA was significantly higher when stimulated by H2O2 solution (P 0.05).
[Conclusion]
1. MTT results were correlated with the results of HSF1 level determination in three cell lines. The stimulation concentration of Ishikawa cells with the highest HSF1 level was the highest at 50% inhibition rate, which was 700 micromol/L; the lowest at 50% inhibition rate of HL95-2 cells with the lowest HSF1 level, which was 300 micromol/L; and the level of HSF1 between them was HEC-1-1-2. The expression of HSF1 may be related to tumor stress and promote the development of endometrial carcinoma.
2. After down-regulating the expression of HSF1 in endometrial cancer cells, a series of tests showed that HSF1 was related to the anti-apoptosis ability of endometrial cancer cells. HSF1 may be an endometrial tumor-promoting factor, which increases the resistance of endometrial cancer cells when activated, thus promoting the development of endometrial cancer. Candidate biomarkers may be used as potential targets for tumor progression.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33
【参考文献】
相关期刊论文 前1条
1 闫惠琴;马力;周清华;;Nrf2在肿瘤化学防御方面的研究进展[J];中国肺癌杂志;2011年07期
本文编号：2248479
本文链接：https://www.wllwen.com/yixuelunwen/fuchankeerkelunwen/2248479.html
最近更新
教材专著
