【摘要】：【背景】 子宮內(nèi)膜癌是對(duì)女性健康產(chǎn)生極大威脅的惡性腫瘤，近年來(lái)其發(fā)病率和病死率都呈現(xiàn)上升趨勢(shì)，儼然已經(jīng)成為一個(gè)嚴(yán)重的公共衛(wèi)生難題，它作為嚴(yán)重危害女性健康的疾病已受到全世界的廣泛關(guān)注。美國(guó)2012年有47130名女性被診斷患有子宮內(nèi)膜癌并且有8010例因其死亡。在2004-2005年衛(wèi)生部惡性腫瘤回顧調(diào)查中，顯示子宮內(nèi)膜癌死亡率以約2.71/10萬(wàn)高居女性惡性腫瘤死亡率第十位（2012中國(guó)衛(wèi)生統(tǒng)計(jì)年鑒）。盡管多數(shù)子宮內(nèi)膜癌病例是早期，但由于晚期以及低分化子宮內(nèi)膜癌的預(yù)后極差，其5年生存率并不令人滿意，因此子宮內(nèi)膜癌的早期發(fā)現(xiàn)和及時(shí)治療顯得至關(guān)重要。 熱休克因子1（Heat shock factor1，HSF1）是調(diào)節(jié)細(xì)胞熱休克蛋白（HSPs）表達(dá)的主要轉(zhuǎn)錄因子，熱休克反應(yīng)被激活后HSF1能迅速改變HSP基因位點(diǎn)并增加它們的表達(dá)，，熱休克蛋白可以幫助錯(cuò)誤折疊的多肽重新折疊并抑制蛋白聚合。這有利于細(xì)胞抑制蛋白應(yīng)激毒性和保持蛋白質(zhì)體內(nèi)平衡，使生物具有可感應(yīng)應(yīng)力的分子機(jī)制來(lái)檢測(cè)和中和蛋白損傷。HSF1已經(jīng)通過(guò)細(xì)胞檢測(cè)、器官模型檢測(cè)、進(jìn)而包括酵母、線蟲(chóng)、嚙齒目動(dòng)物等生物水平的分析全面描述了它的功能。 盡管HSF1幫助細(xì)胞和有機(jī)體對(duì)抗嚴(yán)重有害刺激的作用已被廣泛研究并取得了一些研究結(jié)果，但仍然有許多問(wèn)題亟待探討：HSF1對(duì)下游因子調(diào)控的功能是否參與腫瘤發(fā)生發(fā)展？HSF1對(duì)細(xì)胞的保護(hù)作用在其參與子宮內(nèi)膜癌發(fā)生發(fā)展、氧化應(yīng)激、細(xì)胞周期調(diào)控及凋亡等多種關(guān)鍵的細(xì)胞反應(yīng)中發(fā)揮著怎樣的作用？干預(yù)HSF1后對(duì)這些細(xì)胞反應(yīng)又有何影響？ 為了驗(yàn)證HSF1在子宮內(nèi)膜癌中的表達(dá)及其作用，本實(shí)驗(yàn)以不同濃度的過(guò)氧化氫分別作用于子宮內(nèi)膜癌細(xì)胞株，觀察細(xì)胞的生存情況、HSF1的表達(dá)變化情況以及細(xì)胞受損傷狀況、抗氧化系統(tǒng)和線粒體功能改變情況，探討HSF1對(duì)子宮內(nèi)膜癌細(xì)胞株抗凋亡能力的影響及其作用機(jī)制，從而為以HSF1為子宮內(nèi)膜癌的治療靶點(diǎn)提供新的思路。 【研究目的】 1.探討HSF1在子宮內(nèi)膜癌細(xì)胞株的表達(dá)及其作用 2.探討HSF1對(duì)子宮內(nèi)膜癌細(xì)胞株抗凋亡能力的影響及其作用機(jī)制 【研究方法】 1.應(yīng)用實(shí)時(shí)熒光定量PCR、Western blot技術(shù)檢測(cè)子宮內(nèi)膜癌細(xì)胞中HSF1的表達(dá)水平。 2.應(yīng)用實(shí)時(shí)熒光定量PCR的方法檢測(cè)子宮內(nèi)膜癌細(xì)胞受到不同濃度過(guò)氧化氫刺激后HSF1的表達(dá)變化。 3. MTT法檢測(cè)細(xì)胞受到不同濃度過(guò)氧化氫刺激后的受抑制情況。 4.應(yīng)用RNAi技術(shù)下調(diào)HSF1在子宮內(nèi)膜癌細(xì)胞中的表達(dá)，實(shí)時(shí)熒光定量PCR、Western blot技術(shù)檢測(cè)下調(diào)后HSF1表達(dá)水平的變化。 5. MTT法檢測(cè)下調(diào)HSF1后，各組細(xì)胞受到過(guò)氧化氫刺激時(shí)的受抑制情況。 6. Annexin V-PI雙染色法檢測(cè)下調(diào)HSF1前后，各組細(xì)胞的凋亡情況。 7.使用流式細(xì)胞儀檢測(cè)下調(diào)HSF1前后，各組細(xì)胞的周期變化。 8.使用酶標(biāo)儀檢測(cè)下調(diào)HSF1前后，各組細(xì)胞丙二醛含量、過(guò)氧化氫酶活性、谷胱甘肽含量、總抗氧化能力以及ATP生成情況。 9.使用流式細(xì)胞儀檢測(cè)下調(diào)HSF1前后，各組細(xì)胞內(nèi)活性氧含量。 【實(shí)驗(yàn)結(jié)果】 1.在Ishikawa細(xì)胞中HSF1的mRNA表達(dá)水平最高，HEC-1-B其次，RL95-2最低。 2.在Ishikawa細(xì)胞中HSF1的蛋白表達(dá)水平最高，HEC-1-B其次，RL95-2最低。Ishikawa細(xì)胞當(dāng)受到300μmol/L過(guò)氧化氫溶液刺激時(shí)HSF1表達(dá)出現(xiàn)了明顯的上調(diào)，刺激濃度為500μmol/L時(shí)HSF1表達(dá)繼續(xù)增高，而在濃度到達(dá)700μmol/L時(shí)細(xì)胞內(nèi)HSF1的表達(dá)卻較500μmol/L略有降低，組間差異具有統(tǒng)計(jì)學(xué)意義；HEC-1B細(xì)胞在受到300μmol/L過(guò)氧化氫刺激時(shí)HSF1表達(dá)出現(xiàn)明顯增高，而當(dāng)刺激濃度為500μmol/L時(shí)HSF1的表達(dá)出現(xiàn)降低，以700μmol/L濃度刺激時(shí)甚至低于基水平，組間差異具有統(tǒng)計(jì)學(xué)意義；RL95-2細(xì)胞在50μmol/L低濃度過(guò)氧化氫刺激時(shí)HSF1表達(dá)即出現(xiàn)明顯上升，當(dāng)濃度300μmol/L時(shí)HSF1表達(dá)與低濃度相比已有所降低，當(dāng)大于等于500μmol/L過(guò)氧化氫刺激時(shí)HSF1則出現(xiàn)了低于基水平的表達(dá)，組間差異具有統(tǒng)計(jì)學(xué)意義。 3. Ishikawa細(xì)胞和HEC-1-B細(xì)胞分別在受到濃度為700μmol/L和500μmol/L的H2O2刺激后，細(xì)胞抑制率顯著增高，具有統(tǒng)計(jì)學(xué)意義；而RL95-2細(xì)胞在受到300μmol/L的H2O2刺激后，細(xì)胞抑制率即出現(xiàn)具有統(tǒng)計(jì)學(xué)意義的明顯改變。 4. Ishikawa細(xì)胞轉(zhuǎn)染siRNA后HSF1的mRNA表達(dá)水平明顯降低，與空白組和陰性對(duì)照組存在顯著性差異（P0.05），而陰性對(duì)照組與空白組無(wú)明顯組間差異（P0.05）。 5. Ishikawa細(xì)胞轉(zhuǎn)染siRNA后HSF1的蛋白表達(dá)水平明顯降低，與空白組和陰性對(duì)照組存在顯著性差異（P0.05），而陰性對(duì)照組與空白組無(wú)明顯組間差異（P0.05）。 6. MTT結(jié)果顯示轉(zhuǎn)染組Ishikawa細(xì)胞在受到H2O2溶液刺激后細(xì)胞抑制率顯著增高且具有統(tǒng)計(jì)學(xué)意義。 7.轉(zhuǎn)染siRNA后的Ishikawa細(xì)胞與空白對(duì)照組相比，在受到H2O2溶液刺激時(shí)G2+S期比例減少且差異具有統(tǒng)計(jì)學(xué)意義（P0.05）。 8.轉(zhuǎn)染siRNA后的Ishikawa細(xì)胞在受到H2O2溶液刺激時(shí)，與對(duì)照組相比細(xì)胞內(nèi)丙二醛含量明顯上升且差異具有統(tǒng)計(jì)學(xué)意義（P0.05）。 9.正常狀態(tài)與應(yīng)激狀態(tài)下的Ishikawa細(xì)胞與對(duì)照組相比過(guò)氧化氫酶活性均有所減弱且差異具有統(tǒng)計(jì)學(xué)意義（P0.05）。 10. Ishikawa細(xì)胞與對(duì)照組相比總谷胱甘肽含量減少且差異具有統(tǒng)計(jì)學(xué)意義（P0.05）。 11.轉(zhuǎn)染組Ishikawa細(xì)胞在受到H2O2溶液刺激時(shí)，與對(duì)照組相比總抗氧化能力減弱且差異具有統(tǒng)計(jì)學(xué)意義（P0.05）。 12.轉(zhuǎn)染組Ishikawa細(xì)胞在受到H2O2溶液刺激時(shí)，與對(duì)照組相比ATP水平降低且差異具有統(tǒng)計(jì)學(xué)意義（P0.05）。 13.轉(zhuǎn)染siRNA后的Ishikawa細(xì)胞與對(duì)照組相比，在受到H2O2溶液刺激時(shí)活性氧含量明顯增高且差異具有統(tǒng)計(jì)學(xué)意義（P0.05）。 【結(jié)論】 1. MTT結(jié)果與三株細(xì)胞內(nèi)HSF1基水平測(cè)定的結(jié)果呈現(xiàn)相關(guān)性，HSF1基水平最高的Ishikawa細(xì)胞抑制率50%時(shí)的刺激濃度最高，為700μmol/L；HSF1基水平最低的RL95-2細(xì)胞出現(xiàn)抑制率50%時(shí)的刺激濃度最低，為300μmol/L；而HSF1基水平介于兩者之間的HEC-1-B細(xì)胞出現(xiàn)抑制率50%時(shí)也處于500μmol/L的中間濃度。這提示HSF1的表達(dá)可能與腫瘤應(yīng)激能力相關(guān)，促進(jìn)子宮內(nèi)膜癌的發(fā)生。 2.下調(diào)HSF1在子宮內(nèi)膜癌細(xì)胞中的表達(dá)后，一系列的檢測(cè)顯示HSF1與子宮內(nèi)膜癌細(xì)胞的抗凋亡能力相關(guān)，HSF1可能是一種子宮內(nèi)膜腫瘤促進(jìn)因子，它被激活時(shí)增加了子宮內(nèi)膜癌細(xì)胞抗性，從而促進(jìn)了子宮內(nèi)膜癌發(fā)生發(fā)展。HSF1也許是一個(gè)較好的候選標(biāo)志物，有可能作為可利用的腫瘤病程干預(yù)潛在靶點(diǎn)。
[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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R737.33

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相關(guān)期刊論文 前1條

1 閆惠琴;馬力;周清華;;Nrf2在腫瘤化學(xué)防御方面的研究進(jìn)展[J];中國(guó)肺癌雜志;2011年07期

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