DEPTOR调节食管鳞状细胞癌化疗敏感性的不同功能的研究
发布时间：2019-06-20 04:53
【摘要】：一、研究背景与目的DEPTOR作为mTORCl及mTORC2内源性抑制因子,在调控mTOR信号通路活性起重要作用。研究发现在多种肿瘤中DEPTOR的表达被下调,如乳腺癌、前列腺癌、肺癌、慢性髓系白血病,这使得mTOR信号通路处于过度激活状态而促进肿瘤生长。然而在部分多发性骨髓瘤中DEPTOR却意外的处于过表达状态,DEPTOR的高表达可抑制mTOR通路的活性,通过负反馈机制最终导致Akt的活化,促进多发性骨髓瘤细胞的存活。目前,已有两篇文章报道了DEPTOR与食管癌预后的关系。其中一研究结果显示:DEPTOR在食管鳞癌患者中的表达上调,并提示患者较短的生存期。然而,另一研究结果表明:DEPTOR的表达在食管鳞癌中下调,并提示较差的预后。根据这两组研究的结论提示我们,DEPTOR在食管癌中的表达量及其在预测食管鳞状细胞癌(ESCC)预后中的作用,仍未定论,存在一定的争议。我们本次实验的目的在于:1、确定DEPTOR在食管癌中的表达情况,及其对食管鳞状细胞癌预后的影响。2、DEPTOR对食管鳞状细胞癌化疗反应的影响。二、研究方法1、在南方医院胸外科收集23例食管癌标本,将标本分为两部分,一部分进行组织固定,包埋、切片及免疫组化染色,观察DEPTOR蛋白在组织中的表达部位以及表达水平;另一部分组织标本研磨并提取蛋白,采用免疫印迹的方法检测方法检测DEPTOR蛋白在组织中的表达水平。2、于上海芯超公司购买组织芯片2套,共含180例食管癌标本。进行免疫组化染色,对DEPTOR的染色结果进行评分,并做生存期分析。3、选取4种食管癌细胞系,1种永生化正常食管上皮细胞系,采用免疫印迹的方式,检测该5种细胞中DEPTOR的表达水平,并根据DEPTOR的表达水平,以永生化正常食管上皮为参考,进行分类。4、对4种食管癌细胞,培养过程中加入化疗药物,采用CCK-8的检测方式,观察4种食管癌细胞的存活情况。对4种食管癌细胞进行干扰和过表达,降低和升高DEPTOR在细胞中的表达水平,并加入化疗药物,探究DEPTOR表达水平的变化,对细胞化疗敏感性的影响。5、利用免疫印迹方式,探究DEPTOR影响食管癌细胞化疗敏感性的具体分子机制。三、研究结果我们的研究结果显示DEPTOR在肿瘤组织中的表达显著增加,并且在早期ESCC患者中提示较高的生存率。体外研究显示,可根据正常食管鳞状上皮细胞系HET-1A中的DEPTOR表达量,将ESCC细胞系分为相对高和低DEPTOR表达的亚群。在我们的研究中,不同水平的DEPTOR表达影响着食管癌细胞系对化疗药物的反应。在相对低表达的细胞系中,DEPTOR通过降低Akt活性增加化疗敏感性。在相对高表达的细胞系中,DEPTOR通过在下调核糖体蛋白S6激酶(S6K)后负反馈激活IRS1-P13K-Akt-Survivin通路,从而增加细胞的化疗耐药。
[Abstract]:Background and objective DEPTOR, as an intrinsic inhibitor of mTORCl and mTORC2, plays an important role in regulating the activity of mTOR signaling pathway. It has been found that the expression of DEPTOR is down-regulated in a variety of tumors, such as breast cancer, prostate cancer, lung cancer and chronic myeloid leukemia, which makes the mTOR signaling pathway overactivated and promotes tumor growth. However, DEPTOR is unexpectedly overexpressed in some multiple myeloma. The high expression of DEPTOR can inhibit the activity of mTOR pathway, and eventually lead to the activation of Akt through negative feedback mechanism, and promote the survival of multiple myeloma cells. At present, two articles have reported the relationship between DEPTOR and prognosis of esophageal carcinoma. One of the results showed that the expression of DEPTOR was up-regulated in patients with esophageal squamous cell carcinoma and suggested a shorter survival time. However, another study showed that the expression of DEPTOR was down-regulated in esophageal squamous cell carcinoma and suggested poor prognosis. According to the conclusions of these two groups, the expression of DEPTOR in esophageal carcinoma and its role in predicting the prognosis of esophageal squamous cell carcinoma (ESCC) are still uncertain and controversial. The purpose of our experiment was to determine the expression of DEPTOR in esophageal carcinoma and its effect on the prognosis of esophageal squamous cell carcinoma. 2, the effect of DEPTOR on chemotherapy response of esophageal squamous cell carcinoma. 2. Methods 1. 23 specimens of esophageal carcinoma were collected in thoracic surgery of Southern Hospital. The specimens were divided into two parts, one of which was fixed, embedded, sectioned and stained by immunohistochemistry, and the expression site and expression level of DEPTOR protein in the tissue were observed. The other part of the tissue specimens were ground and extracted, and the expression of DEPTOR protein in the tissues was detected by immunoblotting. 2. Two sets of tissue chips were purchased in Shanghai Xinchao Company, including a total of 180 esophageal cancer specimens. The results of DEPTOR staining were evaluated by immunohistochemical staining, and the survival time was analyzed. 3. Four kinds of esophageal cancer cell lines and one immortalized normal esophageal epithelial cell line were selected to detect the expression of DEPTOR in the five kinds of cells by immunoblotting. According to the expression level of DEPTOR, 4 kinds of esophageal cancer cells were classified with reference to immortalized normal esophageal epithelial cells. Chemotherapeutic drugs were added in the culture process and CCK-8 was used to observe the survival of four kinds of esophageal cancer cells. Four kinds of esophageal cancer cells were interfered with and overexpressed, the expression level of DEPTOR in cells was decreased and increased, and chemotherapeutic drugs were added to explore the change of DEPTOR expression level and the effect on the chemosensitivity of esophageal cancer cells. 5. The specific molecular mechanism of DEPTOR affecting the chemosensitivity of esophageal cancer cells was explored by immunoblotting. Third, our results showed a significant increase in the expression of DEPTOR in tumor tissues, and suggested a higher survival rate in patients with early ESCC. In vitro studies showed that ESCC cell lines could be divided into relatively high and low DEPTOR subpopulations according to the expression of DEPTOR in normal esophageal scaly epithelial cell line HET-1A. In our study, different levels of DEPTOR expression affected the response of esophageal cancer cell lines to chemotherapeutic drugs. In relatively low expression cell lines, DEPTOR increased chemotherapy sensitivity by reducing Akt activity. In the cell lines with relatively high expression, DEPTOR activated the IRS1-P13K-Akt-Survivin pathway by negative feedback after down-regulating ribosomal protein S6 kinase (S6K), thus increasing the chemoresistance of the cells.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.1
本文编号：2502949
[Abstract]:Background and objective DEPTOR, as an intrinsic inhibitor of mTORCl and mTORC2, plays an important role in regulating the activity of mTOR signaling pathway. It has been found that the expression of DEPTOR is down-regulated in a variety of tumors, such as breast cancer, prostate cancer, lung cancer and chronic myeloid leukemia, which makes the mTOR signaling pathway overactivated and promotes tumor growth. However, DEPTOR is unexpectedly overexpressed in some multiple myeloma. The high expression of DEPTOR can inhibit the activity of mTOR pathway, and eventually lead to the activation of Akt through negative feedback mechanism, and promote the survival of multiple myeloma cells. At present, two articles have reported the relationship between DEPTOR and prognosis of esophageal carcinoma. One of the results showed that the expression of DEPTOR was up-regulated in patients with esophageal squamous cell carcinoma and suggested a shorter survival time. However, another study showed that the expression of DEPTOR was down-regulated in esophageal squamous cell carcinoma and suggested poor prognosis. According to the conclusions of these two groups, the expression of DEPTOR in esophageal carcinoma and its role in predicting the prognosis of esophageal squamous cell carcinoma (ESCC) are still uncertain and controversial. The purpose of our experiment was to determine the expression of DEPTOR in esophageal carcinoma and its effect on the prognosis of esophageal squamous cell carcinoma. 2, the effect of DEPTOR on chemotherapy response of esophageal squamous cell carcinoma. 2. Methods 1. 23 specimens of esophageal carcinoma were collected in thoracic surgery of Southern Hospital. The specimens were divided into two parts, one of which was fixed, embedded, sectioned and stained by immunohistochemistry, and the expression site and expression level of DEPTOR protein in the tissue were observed. The other part of the tissue specimens were ground and extracted, and the expression of DEPTOR protein in the tissues was detected by immunoblotting. 2. Two sets of tissue chips were purchased in Shanghai Xinchao Company, including a total of 180 esophageal cancer specimens. The results of DEPTOR staining were evaluated by immunohistochemical staining, and the survival time was analyzed. 3. Four kinds of esophageal cancer cell lines and one immortalized normal esophageal epithelial cell line were selected to detect the expression of DEPTOR in the five kinds of cells by immunoblotting. According to the expression level of DEPTOR, 4 kinds of esophageal cancer cells were classified with reference to immortalized normal esophageal epithelial cells. Chemotherapeutic drugs were added in the culture process and CCK-8 was used to observe the survival of four kinds of esophageal cancer cells. Four kinds of esophageal cancer cells were interfered with and overexpressed, the expression level of DEPTOR in cells was decreased and increased, and chemotherapeutic drugs were added to explore the change of DEPTOR expression level and the effect on the chemosensitivity of esophageal cancer cells. 5. The specific molecular mechanism of DEPTOR affecting the chemosensitivity of esophageal cancer cells was explored by immunoblotting. Third, our results showed a significant increase in the expression of DEPTOR in tumor tissues, and suggested a higher survival rate in patients with early ESCC. In vitro studies showed that ESCC cell lines could be divided into relatively high and low DEPTOR subpopulations according to the expression of DEPTOR in normal esophageal scaly epithelial cell line HET-1A. In our study, different levels of DEPTOR expression affected the response of esophageal cancer cell lines to chemotherapeutic drugs. In relatively low expression cell lines, DEPTOR increased chemotherapy sensitivity by reducing Akt activity. In the cell lines with relatively high expression, DEPTOR activated the IRS1-P13K-Akt-Survivin pathway by negative feedback after down-regulating ribosomal protein S6 kinase (S6K), thus increasing the chemoresistance of the cells.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.1
【参考文献】
相关期刊论文 前3条
1 María José Domper Arnal;ángel Ferrández Arenas;ángel Lanas Arbeloa;;Esophageal cancer: Risk factors,screening and endoscopic treatment in Western and Eastern countries[J];World Journal of Gastroenterology;2015年26期
2 Yuwei Zhang;;Epidemiology of esophageal cancer[J];World Journal of Gastroenterology;2013年34期
3 ;Epidemiologic differences in esophageal cancer between Asian and Western populations[J];癌症;2012年06期
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