肿瘤相关巨噬细胞促口腔癌侵袭转移的机制研究

发布时间：2018-07-31 14:37

【摘要】：口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)简称口腔癌,是头颈部最常见的恶性肿瘤,可造成患者进食、语言、呼吸等功能障碍及容貌、心理改变,晚期患者生存率低。局部复发和颈部淋巴结及远处转移是其致死的主要原因。近年来,肿瘤微环境在口腔癌发生发展中的作用备受关注,肿瘤相关巨噬细胞(tumor-associated macrophages,TAMs)作为主要的基质细胞,能诱导上皮间质转化(epithelial to mesenchymal transition,EMT),与口腔癌进展及预后密切相关。目的:分析CD68,CD163在口腔癌组织中的表达与患者临床病理特征、预后及EMT的关系,探讨TAMs诱导口腔癌细胞发生EMT促其侵袭转移的可能机制。方法:本研究首先通过免疫组化检测127例口腔癌组织标本中TAMs标记物CD68、CD163及EMT相关标记物的表达水平,统计分析其与口腔癌临床病理特征及预后的关系;同时,通过体外诱导分化M1型和M2型巨噬细胞,采用实时定量PCR,免疫蛋白印迹技术和免疫荧光对其表型进行鉴定;在此基础上,构建不同的炎症微环境,分别刺激口腔癌细胞cal27,通过实时定量PCR,免疫蛋白印迹技术和免疫荧光、细胞侵袭和划痕实验等方法检测了不同炎症微环境对口腔癌细胞侵袭、增殖能力的影响以及EMT相关的标记物表达水平,进一步探讨了 TAMs诱导口腔癌细胞发生EMT的机制。结果:CD68阳性巨噬细胞浸润影响患者生存率,而CD163阳性巨噬细胞浸润和口腔癌细胞高表达CD163均与患者的生存密切相关,是重要的独立预后因子。我们发现CD68和CD163不仅在巨噬细胞中表达,在口腔癌细胞中亦有表达,并且口腔癌细胞表达CD163与EMT标记物E-cadherin和vimentin显著相关,提示TAMs与口腔癌EMT密切相关。通过体外实验经GM-CSF刺激后巨噬细胞CD68表达升高,M-CSF刺激后巨噬细胞CD163,CD206,IL10表达上升,成功诱导分化获得M1型和M2型巨噬细胞。分别收集其条件培养基刺激口腔癌细胞cal27发现,M2型条件培养基能诱导cal27发生EMT,侵袭和转移能力增强。而分别加入TGF-β细胞因子后,M1型+TGF-β具有诱导cal27发生EMT,促其侵袭转移的能力;但是M2型+TGF-β则使cal27发生EMT的能力减弱,侵袭能力下降。相反,两组条件培养基中加入TGF-β抑制剂后,cal27发生EMT和侵袭转移能力均下降。结论:本研究发现,TAMs与口腔癌发生、发展及预后密切相关,其可能的机制为TAMs可诱导口腔癌细胞发生EMT而促进其侵袭转移,TGF-β在其中可能发挥着关键作用。
[Abstract]:Oral squamous cell carcinoma (oral squamous cell carcinomas) is the most common malignant tumor in head and neck. Local recurrence and cervical lymph node and distant metastasis were the main causes of death. In recent years, the role of tumor microenvironment in the occurrence and development of oral cancer has attracted much attention. As the main stromal cells, tumor-associated macrophages can induce the transition of epithelial stroma into (epithelial to mesenchymal, which is closely related to the progression and prognosis of oral cancer. Objective: to investigate the relationship between the expression of CD68 and CD163 and clinicopathological features, prognosis and EMT in oral cancer cells, and to explore the possible mechanism of EMT induced invasion and metastasis of oral cancer cells induced by TAMs. Methods: in this study, the expression of TAMs markers CD68, CD163 and EMT related markers were detected by immunohistochemistry in 127 cases of oral carcinoma, and the relationship between CD68 and CD163 and clinicopathological features and prognosis of oral carcinoma was analyzed statistically. The phenotypes of M 1 and M 2 macrophages were identified by real-time quantitative PCR, Western blot and immunofluorescence, and different inflammatory microenvironments were constructed on the basis of in vitro induction and differentiation of M 1 and M 2 macrophages. Oral cancer cell cal27 was stimulated by real-time quantitative PCR, Western blot, immunofluorescence, cell invasion and scratch test. The invasion of oral cancer cells was detected by different inflammatory microenvironments. The effect of TAMs on proliferation and the expression level of EMT related markers further explored the mechanism of EMT induced by TAMs. Results the survival rate of patients was affected by the infiltration of macrophages positive to CD68, while that of CD163 positive macrophages and high expression of CD163 in oral cancer cells were closely related to the survival of the patients, which was an important independent prognostic factor. We found that CD68 and CD163 were expressed not only in macrophages, but also in oral cancer cells. The expression of CD163 in oral cancer cells was significantly correlated with EMT markers E-cadherin and vimentin, suggesting that TAMs was closely related to EMT. The expression of CD68 in macrophages was increased after GM-CSF stimulation in vitro. After stimulation with M-CSF, the expression of CD163, CD206 and IL-10 in macrophages was increased, and M1 and M2 macrophages were successfully induced to differentiate into M 1 and M 2 type macrophages. The conditioned medium was collected to stimulate oral cancer cell cal27. It was found that M 2 conditioned medium could induce cal27 to develop EMTs and enhance the ability of invasion and metastasis. TGF- 尾 can induce the invasion and metastasis of cal27, but M2 TGF- 尾 can weaken the ability of cal27 to produce EMT and decrease the ability of invasiveness. On the contrary, TGF- 尾 inhibitor decreased the ability of EMT and invasion and metastasis in both groups. Conclusion: Tams are closely related to the occurrence, development and prognosis of oral cancer. The possible mechanism of TGF- 尾 may be that TAMs can induce the occurrence of EMT and promote the invasion and metastasis of oral cancer cells. TGF- 尾 may play a key role in this study.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R739.8

【相似文献】