發(fā)布時(shí)間：2018-03-14 23:04

  本文選題：口腔黏膜鱗癌　切入點(diǎn)：HSF1　出處：《南京醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：口腔黏膜鱗癌(oral squamous cell carcinoma,OSCC)是口腔頜面部常見(jiàn)的惡性腫瘤,局部浸潤(rùn)和遠(yuǎn)處轉(zhuǎn)移是患者致死的最主要原因。研究表明腫瘤的發(fā)展除了癌細(xì)胞自身的惡性增殖以外,更是依賴于癌細(xì)胞與腫瘤微環(huán)境的相互作用,微環(huán)境與口腔癌侵襲轉(zhuǎn)移及預(yù)后密切相關(guān)。癌相關(guān)成纖維細(xì)胞(Cancer associated fibroblasts,CAFs)是口腔癌微環(huán)境中最主要的細(xì)胞,可通過(guò)改建腫瘤細(xì)胞外基質(zhì)(Extracellular matrix,ECM)、誘導(dǎo)血管生成、上皮-間質(zhì)轉(zhuǎn)化(Epithelial-mesenchymaltransitio,EMT)、免疫抑制等直接或間接促進(jìn)口腔癌侵襲轉(zhuǎn)移。研究認(rèn)為熱休克因子1(Heat shock factor 1,HSF1)在腫瘤侵襲轉(zhuǎn)移過(guò)程中發(fā)揮了核心調(diào)控作用。目的:探討HSF1調(diào)控CAFs與口腔癌侵襲轉(zhuǎn)移的關(guān)系方法:本研究首先通過(guò)收集臨床上121例口腔癌臨床標(biāo)本,以正常黏膜為對(duì)照,對(duì)其進(jìn)行HSF1和CAFs特異性標(biāo)記物α-SMA的免疫組織化學(xué)染色,統(tǒng)計(jì)分析其與口腔癌臨床病理特征及預(yù)后的關(guān)系;其次我們通過(guò)體外組織塊培養(yǎng)法分離培養(yǎng)CAFs和正常成纖維細(xì)胞(Normal fibroblasts,NFs),采用Real-time RT-PCR、免疫蛋白印跡技術(shù)和免疫熒光技術(shù)對(duì)其進(jìn)行鑒定,同時(shí)采用間接共培養(yǎng)、細(xì)胞劃痕和侵襲實(shí)驗(yàn)檢測(cè)其對(duì)口腔癌細(xì)胞侵襲轉(zhuǎn)移的影響;在此基礎(chǔ)上構(gòu)建HSF1干擾載體病毒,轉(zhuǎn)染CAFs,檢測(cè)其表型的變化以及其對(duì)口腔癌侵襲轉(zhuǎn)移的影響。結(jié)果:研究結(jié)果表明在121例口腔癌組織標(biāo)本中,腫瘤細(xì)胞中HSF1高表達(dá)68例(56.20%),癌間質(zhì)成纖維細(xì)胞中HSF1高表達(dá)40例(33.06%),α-SMA高表達(dá)80例(66.12%)。HSF1在口腔癌細(xì)胞中的表達(dá)水平與病理分級(jí)、復(fù)發(fā)和死亡正相關(guān);基質(zhì)成纖維細(xì)胞中HSF1的高表達(dá)與腫瘤復(fù)發(fā)、死亡正相關(guān);α-SMA的表達(dá)與T、N、臨床分期、病理分級(jí)、復(fù)發(fā)和死亡顯著相關(guān)。相關(guān)性分析顯示腫瘤細(xì)胞和癌間質(zhì)成纖維細(xì)胞中的HSF1和α-SMA的表達(dá)之間存在統(tǒng)計(jì)學(xué)相關(guān)性,同時(shí)腫瘤細(xì)胞與癌間質(zhì)成纖維細(xì)胞中的HSF1之間也存在統(tǒng)計(jì)學(xué)相關(guān)性。生存分析結(jié)果顯示腫瘤細(xì)胞及間質(zhì)成纖維細(xì)胞中HSF1、α-SMA在口腔癌中的高表達(dá)與患者的總生存期具有顯著相關(guān)性。單因素及多因素分析結(jié)果顯示癌間質(zhì)成纖維細(xì)胞中HSF1的表達(dá)與預(yù)后差有顯著相關(guān),提示其可作為口腔癌患者預(yù)后的獨(dú)立預(yù)測(cè)指標(biāo)。體外成功獲得口腔癌原代CAFs和NFs,兩者在基因和蛋白水平都存在明顯差異;相對(duì)于NFs,CAFs改變了口腔癌細(xì)胞Cal27的形態(tài)、遷移及侵襲能力,CAFs明顯促進(jìn)了口腔癌細(xì)胞的侵襲轉(zhuǎn)移。組織學(xué)及分子生物學(xué)實(shí)驗(yàn)均顯示HSF1高表達(dá)于CAFs,通過(guò)慢病毒干擾載體轉(zhuǎn)染細(xì)胞,下調(diào)CAFs中HSF1的表達(dá)后,CAFs標(biāo)記物FAP、FSP-1表達(dá)下降,但α-SMA表達(dá)未見(jiàn)明顯變化。同時(shí)下調(diào)HSF1后CAFs對(duì)Cal27的遷移及侵襲能力均減弱,HSF1通過(guò)調(diào)控CAFs促進(jìn)了口腔癌細(xì)胞的侵襲轉(zhuǎn)移能力。結(jié)論:HSF1調(diào)控CAFs在口腔癌侵襲和轉(zhuǎn)移中發(fā)揮重要作用,有可能成為口腔癌患者預(yù)后預(yù)測(cè)指標(biāo)。
[Abstract]:Oral squamous cell carcinoma (OSCC) is a common malignant tumor in oral and maxillofacial region. Local invasion and distant metastasis are the main causes of death. It is also dependent on the interaction between cancer cells and tumor microenvironment, which is closely related to the invasion, metastasis and prognosis of oral cancer. Cancer associated fibroblast cells (Cafs) are the most important cells in the microenvironment of oral cancer. Angiogenesis can be induced by transforming extracellular matrix extracellular matrix (ECM). Epithelial-mesenchymal transitiotor (EMT), immunosuppressive and so on, can directly or indirectly promote the invasion and metastasis of oral cancer. It is believed that heat shock factor 1 heat shock factor 1 (HSF1) plays a central role in the process of tumor invasion and metastasis. Objective: to investigate the regulation of CAFs by HSF1. Methods: in this study, 121 clinical specimens of oral carcinoma were collected. The HSF1 and CAFs specific markers 偽 -SMA were stained with immunohistochemical staining in normal mucosa as control. The relationship between 偽 -SMA and clinicopathological features and prognosis of oral carcinoma was analyzed statistically. Secondly, CAFs and normal fibroblasts were isolated and cultured by tissue mass culture method in vitro. Real-time RT-PCR, Western blot and immunofluorescence techniques were used to identify them, and indirect co-culture was used. The effect of scratch and invasion assay on the invasion and metastasis of oral cancer cells was detected, and the HSF1 interference vector virus was constructed. After transfection of CAFs, the phenotype of CAFsand its effect on the invasion and metastasis of oral carcinoma were detected. The overexpression of HSF1 was found in 68 cases (56.20%), the high expression of HSF1 in stromal fibroblasts (40 cases) and the expression of 偽 -SMA in 80 cases (66.12%) were positively correlated with pathological grade, recurrence and death. The high expression of HSF1 in stromal fibroblasts was positively correlated with tumor recurrence and death. The correlation analysis showed that the expression of HSF1 and 偽 -SMA in tumor cells and stromal fibroblasts were significantly correlated. The survival analysis showed that the high expression of HSF1 and 偽 -SMA in oral carcinoma was associated with the total survival time of the patients. Univariate and multivariate analysis showed that the expression of HSF1 in the stromal fibroblasts was significantly correlated with the poor prognosis. The results suggest that it can be used as an independent predictor of the prognosis of oral cancer patients. There are significant differences in gene and protein levels between primary CAFs and NFS obtained from oral carcinoma in vitro, and the morphology of Cal27 in oral cancer cells has been changed compared with NFS. The migration and invasion ability of CAFs significantly promoted the invasion and metastasis of oral cancer cells. Histopathological and molecular biological experiments showed that HSF1 was highly expressed in CAFs.The expression of FAPF FSP-1 was decreased after the expression of HSF1 in CAFs was down-regulated by lentivirus interference vector transfection. But the expression of 偽 -SMA did not change significantly. At the same time, the migration and invasion ability of CAFs to Cal27 decreased after down-regulation of HSF1. HSF1 promoted the ability of invasion and metastasis of oral cancer cells by regulating CAFs. Conclusion the regulation of CAFs by CAFs plays an important role in the invasion and metastasis of oral carcinoma. It may be a predictor of prognosis in patients with oral cancer.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R739.8
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本文編號(hào)：1613319