阻断头颈鳞癌STAT3信号在抑制肿瘤干细胞及增强抗肿瘤免疫作用中的研究
发布时间：2018-07-23 11:05
【摘要】：头颈部鳞状细胞癌(head and neck squamous cell carcinoma, HNSCC,简称头颈鳞癌)占到全身恶性肿瘤的6%,每年全世界新增600,000例病人,位居全身恶性肿瘤第六位,且发病率呈现逐年上升趋势。尽管在过去的三十年中,整复外科、微创外科、精准放疗、化疗以及单抗治疗等治疗方式取得的巨大进展,头颈鳞癌的五年生存率依然只有约53%。因此,结合当今飞速发展的医学基础科学研究及交叉学科研究,在深刻认知头颈鳞癌发生、发展、复发及转移的机理上,寻找有效的治疗靶点以及探索有效的治疗方式,将大大提高我们对于头颈鳞癌的认识,同时对于改善头颈鳞癌的预后具有重要的意义。头颈鳞癌的一大特点就是肿瘤组织的异质性以及肿瘤组织构成的复杂的肿瘤微环境,肿瘤干细胞以及肿瘤浸润免疫细胞(髓源性抑制细胞)是其中非常重要的两种组成部分。肿瘤干细胞(cancer stem cells, CSCs)是肿瘤组织中一小部分具有无限增殖、自我更新及多分化潜能的细胞,肿瘤发生、发展、复发、转移以及化疗抵抗的根本原因,因此有效靶向肿瘤干细胞对于头颈鳞癌的治疗具有重要作用9-12。肿瘤免疫治疗作为Science杂志评选的2013年十大科技突破之一13-15,目前在头颈鳞癌的研究仍然方兴未艾。头颈鳞癌作为一种免疫抑制性肿瘤,其中的髓源性抑制细胞(Myeloid-derived suppressor cells,MDSCs)对于头颈鳞癌的的免疫抑制状态的形成及维持具有重要作用。信号转导与转录活化因子3(signal transducers and activators of transcription 3, STAT3)在不同的细胞及组织当中广泛存在,是一种具有信号转导以及转录调控能力的脱氧核糖核酸结合蛋白16-19。STAT3蛋白存在于细胞浆当中,被激活之后形成磷酸化二聚体,进入胞核,与核内DNA特定序列结合,调控特定的下游信号通路,从而达到调控细胞生命活动的目的。STAT3作为一种具有多种功能的转录调控因子,可以在肿瘤微环境中的肿瘤细胞以及免疫细胞当中被持续激活,通过这两个方面促进肿瘤的发生、发展。纳米技术(nanotechnology)作为21世纪战略技术的制高点。相对于传统化疗及靶向药物,利用纳米技术发展起来的纳米载药系统最大的优势就是：增加药物给药剂量、改善生物利用程度、增加靶向能力以及减轻毒副作用20-23。传统肿瘤研究领域,人们往往关注本学科的基础问题,从机制研究到转化研究比较欠缺。如何结合临床现状发现问题,根据问题探究问题所产生的原因,是比较传统的套路,以这种简单单一的思维方式去思考肿瘤这一具有进化能力的有智慧的存在,反而付出了很多努力却难以达到满意的效果。因此将最新的研究理念(多效应靶点)以及最新的纳米技术结合起来,从多学科多中心肿瘤治疗的角度出发,寻找可以同时靶向多种肿瘤特性的分子靶点应该是未来的趋势。第一部分探索STAT3信号对于头颈鳞癌干细胞的调控作用及其在头颈鳞癌化疗耐药中的作用目的：头颈鳞癌干细胞在头颈鳞癌发生发展、复发侵袭及转移等特性中都发挥着重要的作用。本部分内容研究目的在于探究多功能信号分子STAT3信号在头颈鳞癌中表达情况；其与肿瘤干细胞标志物(SOX2、OCT4、ALDH1、CD44)的相关性；其在头颈鳞癌化疗耐药中的作用以及阻断STAT3信号对头颈鳞癌肿瘤干细胞的抑制作用。方法：首先通过使用公共数据库Oncomine(癌症微阵列数据库)深入挖掘STAT3基因在已有癌症研究当中的表达情况；利用免疫组化检测本课题组建立的头颈鳞癌组织芯片当中STAT3以及肿瘤干细胞标志物SOX2、OCT4、ALDH1、CD44的表达情况；通过Aperio Scanscope数字化病理技术对其表达情况进行定量分析,同时利用Pearson correlation(皮尔森相关性)分析和Cluster(聚类分析)对其在不同组织中的表达进行聚类分析；通过采用克隆微球实验(sphere formation assay)、流式细胞术肿瘤干细胞表面标志物(CD44)、流式细胞术侧群细胞检测(side population assay)评估STAT3特异性小分子抑制剂对于头颈鳞癌干细胞的影响,使用头颈鳞癌裸鼠模型检测STAT3特异性小分子抑制剂联合传统化疗药物(顺铂、5-氟尿嘧啶、多西他赛)分析STAT3通路阻断对于头颈鳞癌化疗耐药的影响。结果：持续活化的STAT3信号在头颈鳞癌中的表达显著高于正常粘膜,关联性分析结果显示STAT3与肿瘤干细胞标志物(SOX2、OCT4、ALDH1、CD44)正相关。体外体内实验表明阻断STAT3信号可以有有效抑制头颈鳞癌肿瘤干细胞的增殖以及克服由于肿瘤干细胞引起的头颈鳞癌对于传统药物的化疗耐药。结论：头颈鳞癌组织中以及头颈鳞癌经术前诱导化疗组织中都可以发现STAT3信号的持续活化且与头颈鳞癌干细胞密切相关,阻断S TAT3信号可以有效抑制头颈肿瘤干细胞,提示STAT3信号在头颈鳞癌干细胞维持中的重要作用。第二部分探索STAT3通过调控髓源性抑制细胞在头颈鳞癌肿瘤免疫选逸中的作用目的：髓源性抑制细胞在肿瘤免疫逃逸中起重要作用,而STAT3作为一种多功能调节分子对于引起肿瘤免疫逃逸的一群同样发挥着重要作用。本部分研究目的是探索S TAT3信号在这群髓源性抑制细胞上的表达情况及作用,以及检测阻断ST/ T3信号在头颈鳞癌肿瘤免疫逃逸中的作用。方法：采用免疫组化检测STAT3信号以及髓源性抑制细胞信号(CD11b、Gr1, MDSC; CD68、CD163, TAM)在头颈鳞癌组织芯片的表达情况,通过Aperio Scanscope对其表达情况进行定量分析,利用Pearson correlation分析和Cluster对其在不同组织中的表达进行聚类分析；采用免疫荧光双染检测STAT3信号在Tgfbrl/Pten双基因条件性敲除小鼠髓源性抑制细胞上的表达情况；使用STAT3特异性小分子抑制剂作用于免疫健全时间可诱导组织特异性Tgfbrl/Pten双基因条件性敲除头颈鳞癌小鼠,采用免疫流式技术检测转基因小鼠中免疫抑制性细胞MDSC、TAM的变化以及效应性细胞CD4、CD8T细胞数量的改变,检测阻断STAT3信号对于头颈鳞癌抗肿瘤免疫增强的作用。结果：头颈鳞癌中STAT3信号与髓源性抑制细胞信号的表达高度相关,免疫荧光双染结果显示STAT3表达在髓源性抑制细胞上,阻断STAT3信号可以有效降低转基因小鼠中免疫抑制性细胞MDSC、T AM的数量,提高效应性免疫细胞CD4、CD8T细胞的数量,从而提高头颈鳞癌抗肿瘤免疫效应。结论：阻断STAT3信号对肿瘤免疫抑制性细胞群体具有明显的抑制效应,并增强免疫效应细胞的作用,说明阻断STAT3信号可以有效提高头颈鳞癌抗肿瘤免疫效应。第三部分探索明胶纳米颗粒包裹的STAT3抑制剂及多西他赛对头颈鳞癌的作用目的：以上两部分的研究结果证明STAT3信号在头颈鳞癌肿瘤干细胞以及肿瘤免疫细胞两方面都发挥着重要的作用,STAT3作为头颈鳞癌治疗靶点显示出巨大的可能性。本部分实验的目的是通过采用生物相容性良好的明胶纳米材料对STAT3小分子抑制剂及传统化疗药物多西他赛(Docetaxel,DTX)进行纳米载药体系荷载,探索新型纳米载药技术在头颈鳞癌基础研究中的应用前景。方法：采用免疫组化以及公共数据库TCGA检测明胶酶(MMP-2、MMP-9)在头颈鳞癌中的表达情况；将生物相容性良好的明胶材料制备成纳米颗粒,使用透射电镜等手段检测纳米颗粒的特性；使用CCK-8检测明胶颗粒的生物毒性；检测STAT3小分子抑制剂及多西他赛荷载于明胶纳米颗粒效率：使用头颈鳞癌细胞系体外实验通过比较STAT3小分子抑制剂、多西他赛单独给药以及明胶荷载的药物之间的治疗效果,检测纳米颗粒荷载药物相对于传统药物的优势。结果：两种明胶酶在头颈鳞癌中表达明显升高,制备的明胶纳米颗粒具备显著的纳米颗粒特性,单纯纳米颗粒对于正常细胞系及头颈鳞癌细胞系都不会产生任何杀伤作用,STAT3小分子抑制剂及多西他赛都可以有效的被荷载与纳米颗粒当中,相对于单纯小分子抑制剂,不降低其本身的生物学效应,展现了良好的治疗效果。结论：明胶纳米颗粒荷载S TAT3、分子抑制剂以及多西他赛不影响药物本身作用的情况下,同样展现良好的抗肿瘤作用。
[Abstract]:The head and neck squamous cell carcinoma (head and neck squamous cell carcinoma, HNSCC, referred to as head and neck squamous cell carcinoma) accounts for 6% of the malignant tumor of the whole body. Every year, 600000 patients are added to the whole world. The number of malignant tumors in the whole body is sixth, and the incidence is increasing year by year. Although in the past thirty years, reconstructive surgery, minimally invasive surgery, precision radiotherapy, Great progress has been made in the treatment of chemotherapy and McAb therapy. The five year survival rate of head and neck squamous cell carcinoma is still only about 53%.. Therefore, combined with the rapid development of basic medical science research and cross disciplinary research, we can find effective therapeutic targets and explore the mechanism of the occurrence, development, recurrence and transfer of head and neck squamous cell carcinoma. Effective treatment will greatly improve our understanding of head and neck squamous cell carcinoma and improve the prognosis of head and neck squamous cell carcinoma. One of the major features of head and neck squamous cell carcinoma is the heterogeneity of tumor tissue and the complex tumor microenvironment made up of tumor tissue, tumor stem cells and tumor infiltrating immune cells (myelogenous inhibition) Cell making) is a very important two component. Cancer stem cells (CSCs) is a small part of tumor tissue with unlimited proliferation, self renewal and multiple differentiation potential, the root cause of tumor occurrence, development, recurrence, metastasis and chemotherapy resistance, so the tumor stem cells are effectively targeted to the head and neck scales. Cancer therapy plays an important role in 9-12. tumor immunotherapy as one of the ten major scientific and technological breakthroughs selected by Science magazine in 2013. The study of head and neck squamous cell carcinoma is still in the ascendant. Head and neck squamous cell carcinoma is a kind of immunosuppressive tumor, the Myeloid-derived suppressor cells (MDSCs) in the head and neck scales The formation and maintenance of the immunosuppressive state of cancer is important. Signal transduction and transcription activator 3 (signal transducers and activators of transcription 3, STAT3) exist widely in different cells and tissues. It is a deoxyribonucleic acid 16-19.STAT with signal transduction and transcription control ability. 3 protein exists in the cytoplasm, and is activated after the formation of phosphorylated two polymer, entering the nucleus, combining with the specific sequence of DNA in the nucleus, regulating the specific downstream signal pathway, so as to achieve the purpose of regulating cell life activity as a transcription regulator with multiple functions, which can be used in tumor cells in the tumor microenvironment. And immune cells are continuously activated in these two aspects to promote the development of cancer, development. Nanotechnology (nanotechnology) as the commanding point of strategic technology in twenty-first Century. Compared with traditional chemotherapy and targeted drugs, the most important advantage of nanotechnology developed with nanotechnology is to increase the dosage of drugs and improve the drug delivery system. People often pay attention to the basic problems of this subject, from the mechanism research to the transformation research. How to combine the clinical status to find out the problems and the causes of the problems in 20-23. is a comparative traditional way, with this simple way. A single way of thinking to think about the evolutionary ability of cancer, the intelligent existence, has paid a lot of effort but difficult to achieve satisfactory results. Therefore, combining the latest research ideas (multi effect targets) and the latest nanotechnology, we can find the target from the perspective of multidisciplinary and multi center cancer treatment. The molecular targets for various tumor characteristics should be the future trend. Part 1 explore the role of STAT3 signal in the control of head and neck squamous carcinoma stem cells and its role in the chemotherapy resistance of head and neck squamous cell carcinoma. The purpose of this study is to investigate the expression of multifunction signal molecule STAT3 signal in head and neck squamous cell carcinoma, its correlation with tumor stem cell markers (SOX2, OCT4, ALDH1, CD44), its role in chemotherapy resistance of head and neck squamous cell carcinoma and the inhibition effect of STAT3 signal on head and neck squamous carcinoma stem cells. Use the public database Oncomine (cancer microarray database) to dig into the expression of STAT3 gene in the existing cancer research; use immunohistochemistry to detect the expression of STAT3 and tumor stem cell markers SOX2, OCT4, ALDH1, CD44 in the head and neck squamous cell carcinoma tissue chip established by this group; and through Aperio Scanscope number. Pearson correlation (Pearson correlation) analysis and Cluster (cluster analysis) were used to cluster analysis of its expression in different tissues; by using cloned microsphere test (sphere formation assay), the surface marker of tumor stem cells (CD44) in flow cytometry was used. Flow cytometry (side population assay) evaluation of the effect of STAT3 specific small molecule inhibitors on head and neck cancer stem cells. Using nude mice model of head and neck squamous cell carcinoma to detect STAT3 specific small molecule inhibitors combined with traditional chemotherapeutic drugs (cisplatin, 5- fluorouracil, docetaxel) analysis of STAT3 pathway blocking the scalp scale Results: the expression of continuous activated STAT3 signal in the head and neck squamous cell carcinoma was significantly higher than that of the normal mucosa, and the correlation analysis showed that STAT3 was positively related to the tumor stem cell markers (SOX2, OCT4, ALDH1, CD44). In vitro experiments showed that blocking the STAT3 signal could effectively inhibit the increase of the head and neck cancer stem cells. Conclusion: the continuous activation of STAT3 signal in head and neck squamous carcinoma and head and neck squamous carcinoma can be found to be closely related to the head and neck cancer stem cells in the head and neck squamous carcinoma tissue and the squamous cell carcinoma of head and neck. The obstruction of S TAT3 signal can effectively inhibit the head and neck tumor. Stem cells, suggesting the important role of STAT3 signal in the maintenance of head and neck squamous cell cancer stem cells. The second part is to explore the role of STAT3 by regulating the immunization of medullary suppressor cells in the immunization of head and neck squamous cell carcinoma: the myelinated suppressor cells play an important role in the tumor immune escape, and STAT3 is used as a multifunctional regulator. A group of tumor immune escape also plays an important role. The purpose of this part is to explore the expression and role of S TAT3 signal on this group of myelinated suppressor cells, and to detect the role of blocking ST/ T3 signal in the immune escape of head and neck squamous cell carcinoma. Methods: using immunization to detect STAT3 signal and myelinated inhibition. The expression of cell signal (CD11b, Gr1, MDSC; CD68, CD163, TAM) in the squamous cell carcinoma of the head and neck was quantified by Aperio Scanscope, and the expression of its expression in different tissues was analyzed by Pearson correlation analysis and Cluster, and the immunofluorescence double staining was used to detect the STAT3 signals. The expression of the medullary suppressor cells in the double gene conditionality knockout mice; the use of STAT3 specific small molecule inhibitors in the immune sound time can induce tissue specific Tgfbrl/Pten double gene knockout in the head and neck squamous cell carcinoma mice, and the immunofluorescence technique is used to detect the changes of MDSC and TAM in the immunosuppressive cells of the transgenic mice. The changes in the number of CD4 and CD8T cells in the effector cells were used to detect the anti tumor immunity enhancement of the head and neck squamous cell carcinoma by blocking the STAT3 signal. Results: the STAT3 signal in the squamous cell carcinoma of the head and neck is highly correlated with the expression of the myelinated suppressor cell signal. The results of immunofluorescence double staining show that the expression of STAT3 is on the myeloamedullary suppressor cells, and the STAT3 signal is blocked. It can effectively reduce the number of MDSC, T AM, increase the number of CD4 and CD8T cells in the effector cells, thus improve the anti-tumor immunity effect of the squamous cell carcinoma of the head and neck. Conclusion: blocking the STAT3 signal has obvious inhibitory effect on the tumor immune suppressor cell population and enhancing the effect of immune effect cells. The results showed that blocking STAT3 signal could effectively improve the anti-tumor immunity effect of head and neck squamous cell carcinoma. Third the purpose of exploring the effect of STAT3 inhibitor and docetaxel on head and neck squamous cell carcinoma was explored. The results of the above two parts showed that the STAT3 signal was found in two aspects of the head and neck cancer stem cells and the tumor immune cells. STAT3 as a target for the treatment of head and neck squamous cell carcinoma shows great possibility. The purpose of this part of the experiment is to explore new nano drug loading techniques by using biocompatible gelatin nanomaterials to load STAT3 small molecule inhibitors and traditional chemotherapeutic drug Docetaxel (DTX) in nano drug loading system. The application prospect in the basic study of head and neck squamous cell carcinoma. Methods: the expression of MMP-2 (MMP-9) in the squamous cell carcinoma of head and neck was detected by immunohistochemistry and public database TCGA; the biocompatible gelatin materials were prepared into nanoparticles, and the properties of the nanoparticles were detected by transmission electron microscopy. CCK-8 examination was used to detect the properties of the nanoparticles. Biotoxicity of gelatin particles; detection of STAT3 small molecule inhibitors and docetaxel load on gelatin nanoparticles efficiency: in vitro experiments using head and neck squamous cell carcinoma cell lines by comparing the treatment effects of STAT3 small molecule inhibitors, docetaxel alone and gelatin loaded drugs, detection of nanoparticle load drug relative Results: the advantages of traditional medicine. Results: the expression of two kinds of Gelatinase in head and neck squamous cell carcinoma is obviously increased. The prepared gelatin nanoparticles have significant nanoparticle characteristics. Pure nanoparticles can not produce any killing effect on normal cell lines and head and neck squamous cell carcinoma cell lines. STAT3 small molecule inhibitors and docetaxel can be effective. The load and nano particles, relative to the simple small molecule inhibitors, do not reduce their biological effects and show good therapeutic effects. Conclusion: gelatin nanoparticles load S TAT3, molecular inhibitors and docetaxel do not affect the effect of the drug itself, and also exhibit good antitumor effect.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R739.91
本文编号：2139199
[Abstract]:The head and neck squamous cell carcinoma (head and neck squamous cell carcinoma, HNSCC, referred to as head and neck squamous cell carcinoma) accounts for 6% of the malignant tumor of the whole body. Every year, 600000 patients are added to the whole world. The number of malignant tumors in the whole body is sixth, and the incidence is increasing year by year. Although in the past thirty years, reconstructive surgery, minimally invasive surgery, precision radiotherapy, Great progress has been made in the treatment of chemotherapy and McAb therapy. The five year survival rate of head and neck squamous cell carcinoma is still only about 53%.. Therefore, combined with the rapid development of basic medical science research and cross disciplinary research, we can find effective therapeutic targets and explore the mechanism of the occurrence, development, recurrence and transfer of head and neck squamous cell carcinoma. Effective treatment will greatly improve our understanding of head and neck squamous cell carcinoma and improve the prognosis of head and neck squamous cell carcinoma. One of the major features of head and neck squamous cell carcinoma is the heterogeneity of tumor tissue and the complex tumor microenvironment made up of tumor tissue, tumor stem cells and tumor infiltrating immune cells (myelogenous inhibition) Cell making) is a very important two component. Cancer stem cells (CSCs) is a small part of tumor tissue with unlimited proliferation, self renewal and multiple differentiation potential, the root cause of tumor occurrence, development, recurrence, metastasis and chemotherapy resistance, so the tumor stem cells are effectively targeted to the head and neck scales. Cancer therapy plays an important role in 9-12. tumor immunotherapy as one of the ten major scientific and technological breakthroughs selected by Science magazine in 2013. The study of head and neck squamous cell carcinoma is still in the ascendant. Head and neck squamous cell carcinoma is a kind of immunosuppressive tumor, the Myeloid-derived suppressor cells (MDSCs) in the head and neck scales The formation and maintenance of the immunosuppressive state of cancer is important. Signal transduction and transcription activator 3 (signal transducers and activators of transcription 3, STAT3) exist widely in different cells and tissues. It is a deoxyribonucleic acid 16-19.STAT with signal transduction and transcription control ability. 3 protein exists in the cytoplasm, and is activated after the formation of phosphorylated two polymer, entering the nucleus, combining with the specific sequence of DNA in the nucleus, regulating the specific downstream signal pathway, so as to achieve the purpose of regulating cell life activity as a transcription regulator with multiple functions, which can be used in tumor cells in the tumor microenvironment. And immune cells are continuously activated in these two aspects to promote the development of cancer, development. Nanotechnology (nanotechnology) as the commanding point of strategic technology in twenty-first Century. Compared with traditional chemotherapy and targeted drugs, the most important advantage of nanotechnology developed with nanotechnology is to increase the dosage of drugs and improve the drug delivery system. People often pay attention to the basic problems of this subject, from the mechanism research to the transformation research. How to combine the clinical status to find out the problems and the causes of the problems in 20-23. is a comparative traditional way, with this simple way. A single way of thinking to think about the evolutionary ability of cancer, the intelligent existence, has paid a lot of effort but difficult to achieve satisfactory results. Therefore, combining the latest research ideas (multi effect targets) and the latest nanotechnology, we can find the target from the perspective of multidisciplinary and multi center cancer treatment. The molecular targets for various tumor characteristics should be the future trend. Part 1 explore the role of STAT3 signal in the control of head and neck squamous carcinoma stem cells and its role in the chemotherapy resistance of head and neck squamous cell carcinoma. The purpose of this study is to investigate the expression of multifunction signal molecule STAT3 signal in head and neck squamous cell carcinoma, its correlation with tumor stem cell markers (SOX2, OCT4, ALDH1, CD44), its role in chemotherapy resistance of head and neck squamous cell carcinoma and the inhibition effect of STAT3 signal on head and neck squamous carcinoma stem cells. Use the public database Oncomine (cancer microarray database) to dig into the expression of STAT3 gene in the existing cancer research; use immunohistochemistry to detect the expression of STAT3 and tumor stem cell markers SOX2, OCT4, ALDH1, CD44 in the head and neck squamous cell carcinoma tissue chip established by this group; and through Aperio Scanscope number. Pearson correlation (Pearson correlation) analysis and Cluster (cluster analysis) were used to cluster analysis of its expression in different tissues; by using cloned microsphere test (sphere formation assay), the surface marker of tumor stem cells (CD44) in flow cytometry was used. Flow cytometry (side population assay) evaluation of the effect of STAT3 specific small molecule inhibitors on head and neck cancer stem cells. Using nude mice model of head and neck squamous cell carcinoma to detect STAT3 specific small molecule inhibitors combined with traditional chemotherapeutic drugs (cisplatin, 5- fluorouracil, docetaxel) analysis of STAT3 pathway blocking the scalp scale Results: the expression of continuous activated STAT3 signal in the head and neck squamous cell carcinoma was significantly higher than that of the normal mucosa, and the correlation analysis showed that STAT3 was positively related to the tumor stem cell markers (SOX2, OCT4, ALDH1, CD44). In vitro experiments showed that blocking the STAT3 signal could effectively inhibit the increase of the head and neck cancer stem cells. Conclusion: the continuous activation of STAT3 signal in head and neck squamous carcinoma and head and neck squamous carcinoma can be found to be closely related to the head and neck cancer stem cells in the head and neck squamous carcinoma tissue and the squamous cell carcinoma of head and neck. The obstruction of S TAT3 signal can effectively inhibit the head and neck tumor. Stem cells, suggesting the important role of STAT3 signal in the maintenance of head and neck squamous cell cancer stem cells. The second part is to explore the role of STAT3 by regulating the immunization of medullary suppressor cells in the immunization of head and neck squamous cell carcinoma: the myelinated suppressor cells play an important role in the tumor immune escape, and STAT3 is used as a multifunctional regulator. A group of tumor immune escape also plays an important role. The purpose of this part is to explore the expression and role of S TAT3 signal on this group of myelinated suppressor cells, and to detect the role of blocking ST/ T3 signal in the immune escape of head and neck squamous cell carcinoma. Methods: using immunization to detect STAT3 signal and myelinated inhibition. The expression of cell signal (CD11b, Gr1, MDSC; CD68, CD163, TAM) in the squamous cell carcinoma of the head and neck was quantified by Aperio Scanscope, and the expression of its expression in different tissues was analyzed by Pearson correlation analysis and Cluster, and the immunofluorescence double staining was used to detect the STAT3 signals. The expression of the medullary suppressor cells in the double gene conditionality knockout mice; the use of STAT3 specific small molecule inhibitors in the immune sound time can induce tissue specific Tgfbrl/Pten double gene knockout in the head and neck squamous cell carcinoma mice, and the immunofluorescence technique is used to detect the changes of MDSC and TAM in the immunosuppressive cells of the transgenic mice. The changes in the number of CD4 and CD8T cells in the effector cells were used to detect the anti tumor immunity enhancement of the head and neck squamous cell carcinoma by blocking the STAT3 signal. Results: the STAT3 signal in the squamous cell carcinoma of the head and neck is highly correlated with the expression of the myelinated suppressor cell signal. The results of immunofluorescence double staining show that the expression of STAT3 is on the myeloamedullary suppressor cells, and the STAT3 signal is blocked. It can effectively reduce the number of MDSC, T AM, increase the number of CD4 and CD8T cells in the effector cells, thus improve the anti-tumor immunity effect of the squamous cell carcinoma of the head and neck. Conclusion: blocking the STAT3 signal has obvious inhibitory effect on the tumor immune suppressor cell population and enhancing the effect of immune effect cells. The results showed that blocking STAT3 signal could effectively improve the anti-tumor immunity effect of head and neck squamous cell carcinoma. Third the purpose of exploring the effect of STAT3 inhibitor and docetaxel on head and neck squamous cell carcinoma was explored. The results of the above two parts showed that the STAT3 signal was found in two aspects of the head and neck cancer stem cells and the tumor immune cells. STAT3 as a target for the treatment of head and neck squamous cell carcinoma shows great possibility. The purpose of this part of the experiment is to explore new nano drug loading techniques by using biocompatible gelatin nanomaterials to load STAT3 small molecule inhibitors and traditional chemotherapeutic drug Docetaxel (DTX) in nano drug loading system. The application prospect in the basic study of head and neck squamous cell carcinoma. Methods: the expression of MMP-2 (MMP-9) in the squamous cell carcinoma of head and neck was detected by immunohistochemistry and public database TCGA; the biocompatible gelatin materials were prepared into nanoparticles, and the properties of the nanoparticles were detected by transmission electron microscopy. CCK-8 examination was used to detect the properties of the nanoparticles. Biotoxicity of gelatin particles; detection of STAT3 small molecule inhibitors and docetaxel load on gelatin nanoparticles efficiency: in vitro experiments using head and neck squamous cell carcinoma cell lines by comparing the treatment effects of STAT3 small molecule inhibitors, docetaxel alone and gelatin loaded drugs, detection of nanoparticle load drug relative Results: the advantages of traditional medicine. Results: the expression of two kinds of Gelatinase in head and neck squamous cell carcinoma is obviously increased. The prepared gelatin nanoparticles have significant nanoparticle characteristics. Pure nanoparticles can not produce any killing effect on normal cell lines and head and neck squamous cell carcinoma cell lines. STAT3 small molecule inhibitors and docetaxel can be effective. The load and nano particles, relative to the simple small molecule inhibitors, do not reduce their biological effects and show good therapeutic effects. Conclusion: gelatin nanoparticles load S TAT3, molecular inhibitors and docetaxel do not affect the effect of the drug itself, and also exhibit good antitumor effect.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R739.91
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