阻斷頭頸鱗癌STAT3信號在抑制腫瘤干細(xì)胞及增強(qiáng)抗腫瘤免疫作用中的研究
發(fā)布時(shí)間：2018-07-23 11:05
【摘要】：頭頸部鱗狀細(xì)胞癌(head and neck squamous cell carcinoma, HNSCC,簡稱頭頸鱗癌)占到全身惡性腫瘤的6%,每年全世界新增600,000例病人,位居全身惡性腫瘤第六位,且發(fā)病率呈現(xiàn)逐年上升趨勢。盡管在過去的三十年中,整復(fù)外科、微創(chuàng)外科、精準(zhǔn)放療、化療以及單抗治療等治療方式取得的巨大進(jìn)展,頭頸鱗癌的五年生存率依然只有約53%。因此,結(jié)合當(dāng)今飛速發(fā)展的醫(yī)學(xué)基礎(chǔ)科學(xué)研究及交叉學(xué)科研究,在深刻認(rèn)知頭頸鱗癌發(fā)生、發(fā)展、復(fù)發(fā)及轉(zhuǎn)移的機(jī)理上,尋找有效的治療靶點(diǎn)以及探索有效的治療方式,將大大提高我們對于頭頸鱗癌的認(rèn)識,同時(shí)對于改善頭頸鱗癌的預(yù)后具有重要的意義。頭頸鱗癌的一大特點(diǎn)就是腫瘤組織的異質(zhì)性以及腫瘤組織構(gòu)成的復(fù)雜的腫瘤微環(huán)境,腫瘤干細(xì)胞以及腫瘤浸潤免疫細(xì)胞(髓源性抑制細(xì)胞)是其中非常重要的兩種組成部分。腫瘤干細(xì)胞(cancer stem cells, CSCs)是腫瘤組織中一小部分具有無限增殖、自我更新及多分化潛能的細(xì)胞,腫瘤發(fā)生、發(fā)展、復(fù)發(fā)、轉(zhuǎn)移以及化療抵抗的根本原因,因此有效靶向腫瘤干細(xì)胞對于頭頸鱗癌的治療具有重要作用9-12。腫瘤免疫治療作為Science雜志評選的2013年十大科技突破之一13-15,目前在頭頸鱗癌的研究仍然方興未艾。頭頸鱗癌作為一種免疫抑制性腫瘤,其中的髓源性抑制細(xì)胞(Myeloid-derived suppressor cells,MDSCs)對于頭頸鱗癌的的免疫抑制狀態(tài)的形成及維持具有重要作用。信號轉(zhuǎn)導(dǎo)與轉(zhuǎn)錄活化因子3(signal transducers and activators of transcription 3, STAT3)在不同的細(xì)胞及組織當(dāng)中廣泛存在,是一種具有信號轉(zhuǎn)導(dǎo)以及轉(zhuǎn)錄調(diào)控能力的脫氧核糖核酸結(jié)合蛋白16-19。STAT3蛋白存在于細(xì)胞漿當(dāng)中,被激活之后形成磷酸化二聚體,進(jìn)入胞核,與核內(nèi)DNA特定序列結(jié)合,調(diào)控特定的下游信號通路,從而達(dá)到調(diào)控細(xì)胞生命活動的目的。STAT3作為一種具有多種功能的轉(zhuǎn)錄調(diào)控因子,可以在腫瘤微環(huán)境中的腫瘤細(xì)胞以及免疫細(xì)胞當(dāng)中被持續(xù)激活,通過這兩個(gè)方面促進(jìn)腫瘤的發(fā)生、發(fā)展。納米技術(shù)(nanotechnology)作為21世紀(jì)戰(zhàn)略技術(shù)的制高點(diǎn)。相對于傳統(tǒng)化療及靶向藥物,利用納米技術(shù)發(fā)展起來的納米載藥系統(tǒng)最大的優(yōu)勢就是：增加藥物給藥劑量、改善生物利用程度、增加靶向能力以及減輕毒副作用20-23。傳統(tǒng)腫瘤研究領(lǐng)域,人們往往關(guān)注本學(xué)科的基礎(chǔ)問題,從機(jī)制研究到轉(zhuǎn)化研究比較欠缺。如何結(jié)合臨床現(xiàn)狀發(fā)現(xiàn)問題,根據(jù)問題探究問題所產(chǎn)生的原因,是比較傳統(tǒng)的套路,以這種簡單單一的思維方式去思考腫瘤這一具有進(jìn)化能力的有智慧的存在,反而付出了很多努力卻難以達(dá)到滿意的效果。因此將最新的研究理念(多效應(yīng)靶點(diǎn))以及最新的納米技術(shù)結(jié)合起來,從多學(xué)科多中心腫瘤治療的角度出發(fā),尋找可以同時(shí)靶向多種腫瘤特性的分子靶點(diǎn)應(yīng)該是未來的趨勢。第一部分探索STAT3信號對于頭頸鱗癌干細(xì)胞的調(diào)控作用及其在頭頸鱗癌化療耐藥中的作用目的：頭頸鱗癌干細(xì)胞在頭頸鱗癌發(fā)生發(fā)展、復(fù)發(fā)侵襲及轉(zhuǎn)移等特性中都發(fā)揮著重要的作用。本部分內(nèi)容研究目的在于探究多功能信號分子STAT3信號在頭頸鱗癌中表達(dá)情況；其與腫瘤干細(xì)胞標(biāo)志物(SOX2、OCT4、ALDH1、CD44)的相關(guān)性；其在頭頸鱗癌化療耐藥中的作用以及阻斷STAT3信號對頭頸鱗癌腫瘤干細(xì)胞的抑制作用。方法：首先通過使用公共數(shù)據(jù)庫Oncomine(癌癥微陣列數(shù)據(jù)庫)深入挖掘STAT3基因在已有癌癥研究當(dāng)中的表達(dá)情況；利用免疫組化檢測本課題組建立的頭頸鱗癌組織芯片當(dāng)中STAT3以及腫瘤干細(xì)胞標(biāo)志物SOX2、OCT4、ALDH1、CD44的表達(dá)情況；通過Aperio Scanscope數(shù)字化病理技術(shù)對其表達(dá)情況進(jìn)行定量分析,同時(shí)利用Pearson correlation(皮爾森相關(guān)性)分析和Cluster(聚類分析)對其在不同組織中的表達(dá)進(jìn)行聚類分析；通過采用克隆微球?qū)嶒?yàn)(sphere formation assay)、流式細(xì)胞術(shù)腫瘤干細(xì)胞表面標(biāo)志物(CD44)、流式細(xì)胞術(shù)側(cè)群細(xì)胞檢測(side population assay)評估STAT3特異性小分子抑制劑對于頭頸鱗癌干細(xì)胞的影響,使用頭頸鱗癌裸鼠模型檢測STAT3特異性小分子抑制劑聯(lián)合傳統(tǒng)化療藥物(順鉑、5-氟尿嘧啶、多西他賽)分析STAT3通路阻斷對于頭頸鱗癌化療耐藥的影響。結(jié)果：持續(xù)活化的STAT3信號在頭頸鱗癌中的表達(dá)顯著高于正常粘膜,關(guān)聯(lián)性分析結(jié)果顯示STAT3與腫瘤干細(xì)胞標(biāo)志物(SOX2、OCT4、ALDH1、CD44)正相關(guān)。體外體內(nèi)實(shí)驗(yàn)表明阻斷STAT3信號可以有有效抑制頭頸鱗癌腫瘤干細(xì)胞的增殖以及克服由于腫瘤干細(xì)胞引起的頭頸鱗癌對于傳統(tǒng)藥物的化療耐藥。結(jié)論：頭頸鱗癌組織中以及頭頸鱗癌經(jīng)術(shù)前誘導(dǎo)化療組織中都可以發(fā)現(xiàn)STAT3信號的持續(xù)活化且與頭頸鱗癌干細(xì)胞密切相關(guān),阻斷S TAT3信號可以有效抑制頭頸腫瘤干細(xì)胞,提示STAT3信號在頭頸鱗癌干細(xì)胞維持中的重要作用。第二部分探索STAT3通過調(diào)控髓源性抑制細(xì)胞在頭頸鱗癌腫瘤免疫選逸中的作用目的：髓源性抑制細(xì)胞在腫瘤免疫逃逸中起重要作用,而STAT3作為一種多功能調(diào)節(jié)分子對于引起腫瘤免疫逃逸的一群同樣發(fā)揮著重要作用。本部分研究目的是探索S TAT3信號在這群髓源性抑制細(xì)胞上的表達(dá)情況及作用,以及檢測阻斷ST/ T3信號在頭頸鱗癌腫瘤免疫逃逸中的作用。方法：采用免疫組化檢測STAT3信號以及髓源性抑制細(xì)胞信號(CD11b、Gr1, MDSC; CD68、CD163, TAM)在頭頸鱗癌組織芯片的表達(dá)情況,通過Aperio Scanscope對其表達(dá)情況進(jìn)行定量分析,利用Pearson correlation分析和Cluster對其在不同組織中的表達(dá)進(jìn)行聚類分析；采用免疫熒光雙染檢測STAT3信號在Tgfbrl/Pten雙基因條件性敲除小鼠髓源性抑制細(xì)胞上的表達(dá)情況；使用STAT3特異性小分子抑制劑作用于免疫健全時(shí)間可誘導(dǎo)組織特異性Tgfbrl/Pten雙基因條件性敲除頭頸鱗癌小鼠,采用免疫流式技術(shù)檢測轉(zhuǎn)基因小鼠中免疫抑制性細(xì)胞MDSC、TAM的變化以及效應(yīng)性細(xì)胞CD4、CD8T細(xì)胞數(shù)量的改變,檢測阻斷STAT3信號對于頭頸鱗癌抗腫瘤免疫增強(qiáng)的作用。結(jié)果：頭頸鱗癌中STAT3信號與髓源性抑制細(xì)胞信號的表達(dá)高度相關(guān),免疫熒光雙染結(jié)果顯示STAT3表達(dá)在髓源性抑制細(xì)胞上,阻斷STAT3信號可以有效降低轉(zhuǎn)基因小鼠中免疫抑制性細(xì)胞MDSC、T AM的數(shù)量,提高效應(yīng)性免疫細(xì)胞CD4、CD8T細(xì)胞的數(shù)量,從而提高頭頸鱗癌抗腫瘤免疫效應(yīng)。結(jié)論：阻斷STAT3信號對腫瘤免疫抑制性細(xì)胞群體具有明顯的抑制效應(yīng),并增強(qiáng)免疫效應(yīng)細(xì)胞的作用,說明阻斷STAT3信號可以有效提高頭頸鱗癌抗腫瘤免疫效應(yīng)。第三部分探索明膠納米顆粒包裹的STAT3抑制劑及多西他賽對頭頸鱗癌的作用目的：以上兩部分的研究結(jié)果證明STAT3信號在頭頸鱗癌腫瘤干細(xì)胞以及腫瘤免疫細(xì)胞兩方面都發(fā)揮著重要的作用,STAT3作為頭頸鱗癌治療靶點(diǎn)顯示出巨大的可能性。本部分實(shí)驗(yàn)的目的是通過采用生物相容性良好的明膠納米材料對STAT3小分子抑制劑及傳統(tǒng)化療藥物多西他賽(Docetaxel,DTX)進(jìn)行納米載藥體系荷載,探索新型納米載藥技術(shù)在頭頸鱗癌基礎(chǔ)研究中的應(yīng)用前景。方法：采用免疫組化以及公共數(shù)據(jù)庫TCGA檢測明膠酶(MMP-2、MMP-9)在頭頸鱗癌中的表達(dá)情況；將生物相容性良好的明膠材料制備成納米顆粒,使用透射電鏡等手段檢測納米顆粒的特性；使用CCK-8檢測明膠顆粒的生物毒性；檢測STAT3小分子抑制劑及多西他賽荷載于明膠納米顆粒效率：使用頭頸鱗癌細(xì)胞系體外實(shí)驗(yàn)通過比較STAT3小分子抑制劑、多西他賽單獨(dú)給藥以及明膠荷載的藥物之間的治療效果,檢測納米顆粒荷載藥物相對于傳統(tǒng)藥物的優(yōu)勢。結(jié)果：兩種明膠酶在頭頸鱗癌中表達(dá)明顯升高,制備的明膠納米顆粒具備顯著的納米顆粒特性,單純納米顆粒對于正常細(xì)胞系及頭頸鱗癌細(xì)胞系都不會產(chǎn)生任何殺傷作用,STAT3小分子抑制劑及多西他賽都可以有效的被荷載與納米顆粒當(dāng)中,相對于單純小分子抑制劑,不降低其本身的生物學(xué)效應(yīng),展現(xiàn)了良好的治療效果。結(jié)論：明膠納米顆粒荷載S TAT3、分子抑制劑以及多西他賽不影響藥物本身作用的情況下,同樣展現(xiàn)良好的抗腫瘤作用。
[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.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R739.91
本文編號：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.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R739.91
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