热休克因子1调控癌相关成纤维细胞促口腔癌侵袭转移机制研究

发布时间：2018-03-14 23:04

  本文选题：口腔黏膜鳞癌　切入点：HSF1　出处：《南京医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：口腔黏膜鳞癌(oral squamous cell carcinoma,OSCC)是口腔颌面部常见的恶性肿瘤,局部浸润和远处转移是患者致死的最主要原因。研究表明肿瘤的发展除了癌细胞自身的恶性增殖以外,更是依赖于癌细胞与肿瘤微环境的相互作用,微环境与口腔癌侵袭转移及预后密切相关。癌相关成纤维细胞(Cancer associated fibroblasts,CAFs)是口腔癌微环境中最主要的细胞,可通过改建肿瘤细胞外基质(Extracellular matrix,ECM)、诱导血管生成、上皮-间质转化(Epithelial-mesenchymaltransitio,EMT)、免疫抑制等直接或间接促进口腔癌侵袭转移。研究认为热休克因子1(Heat shock factor 1,HSF1)在肿瘤侵袭转移过程中发挥了核心调控作用。目的:探讨HSF1调控CAFs与口腔癌侵袭转移的关系方法:本研究首先通过收集临床上121例口腔癌临床标本,以正常黏膜为对照,对其进行HSF1和CAFs特异性标记物α-SMA的免疫组织化学染色,统计分析其与口腔癌临床病理特征及预后的关系;其次我们通过体外组织块培养法分离培养CAFs和正常成纤维细胞(Normal fibroblasts,NFs),采用Real-time RT-PCR、免疫蛋白印迹技术和免疫荧光技术对其进行鉴定,同时采用间接共培养、细胞划痕和侵袭实验检测其对口腔癌细胞侵袭转移的影响;在此基础上构建HSF1干扰载体病毒,转染CAFs,检测其表型的变化以及其对口腔癌侵袭转移的影响。结果:研究结果表明在121例口腔癌组织标本中,肿瘤细胞中HSF1高表达68例(56.20%),癌间质成纤维细胞中HSF1高表达40例(33.06%),α-SMA高表达80例(66.12%)。HSF1在口腔癌细胞中的表达水平与病理分级、复发和死亡正相关;基质成纤维细胞中HSF1的高表达与肿瘤复发、死亡正相关;α-SMA的表达与T、N、临床分期、病理分级、复发和死亡显著相关。相关性分析显示肿瘤细胞和癌间质成纤维细胞中的HSF1和α-SMA的表达之间存在统计学相关性,同时肿瘤细胞与癌间质成纤维细胞中的HSF1之间也存在统计学相关性。生存分析结果显示肿瘤细胞及间质成纤维细胞中HSF1、α-SMA在口腔癌中的高表达与患者的总生存期具有显著相关性。单因素及多因素分析结果显示癌间质成纤维细胞中HSF1的表达与预后差有显着相关,提示其可作为口腔癌患者预后的独立预测指标。体外成功获得口腔癌原代CAFs和NFs,两者在基因和蛋白水平都存在明显差异;相对于NFs,CAFs改变了口腔癌细胞Cal27的形态、迁移及侵袭能力,CAFs明显促进了口腔癌细胞的侵袭转移。组织学及分子生物学实验均显示HSF1高表达于CAFs,通过慢病毒干扰载体转染细胞,下调CAFs中HSF1的表达后,CAFs标记物FAP、FSP-1表达下降,但α-SMA表达未见明显变化。同时下调HSF1后CAFs对Cal27的迁移及侵袭能力均减弱,HSF1通过调控CAFs促进了口腔癌细胞的侵袭转移能力。结论:HSF1调控CAFs在口腔癌侵袭和转移中发挥重要作用,有可能成为口腔癌患者预后预测指标。
[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 伪 -SMA were stained with immunohistochemical staining in normal mucosa as control. The relationship between 伪 -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 伪 -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 伪 -SMA in tumor cells and stromal fibroblasts were significantly correlated. The survival analysis showed that the high expression of HSF1 and 伪 -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 伪 -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.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.8
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本文编号：1613319