2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38737Oral submucous fibrosis (OSF) is a pre-malignant fibrotic lesion of the mouth in areca quid chewers. It is probably a consequence of disturbances in the hemeostatic equilibrium between synthesis and degradation of extracellular matrix molecules (ECM). Thus, matrix metalloproteinases (MMPs) and plasminogen activator (PA)/plasmin system play an important role in the pathogenesis of OSF. In our present studying, we emphasize the proteinase and stromal-cell-derived factor 1 (SDF-1) and CXCR4 expression in OSF and oral cancer. Interaction between the SDF-1 chemokine and its receptor CXCR4 has been found to play an important role in tumorigenicity, proliferation, metastasis and angiogenesis in many cancers, such as lung cancer, breast cancer, melanoma, glioblastoma, pancreatic cancer, cholangiocarcinoma and basal cell carcinoma cells. Over the past decades, extensive efforts has been made to find the role of SDF-1 and CXCR4 axis in tumor progression, either as frequently investigated in variety of tumor cells and cell lines, but it has been difficult to find a clear-cut relationship between the expression of SDF-1 and CXCR4 in normal and malignant cell. However, little evidence of a direct relationship between OSF, oral cancer and SDF-1 and CXCR4 axis expression. Thus, in this study, we propose to study the expression of SDF-1 and CXCR4 axis in human normal buccal mucosal and oral submucous fibrosis and oral cancer. With a few established techniques, including zymography, western blotting, RT-PCR and EMSA for MMPs, PAI-1 and SDF-1 and CXCR4 , our Lab is very capable of performing studies on genes relevant to ECM-degradation, such as MMPs, TIMPs, and PAI-1 to clarify their involvements in the pathogenesis of OSF and oral cancer. Therefore, in the first year of this study, we propose to (1) analyze the expressions of SDF-1 and CXCR4 from cell extracts from OSF and normal buccal mucosa fibroblast via western blotting assay. (2) analyze the mRNA levels of SDF-1 and CXCR4 from OSF and normal buccal mucosa fibroblast via RT-PCR assay. (3) Using immunohistochemistry to investigate the expression of SDF-1 and CXCR4 from OSF and normal buccal mucosa. (4) To verify whether arecoline or areca nut extracts could affect SDF-1 and CXCR4 production by human buccal mucosa fibroblasts used western blots and RT-PCR. (5)further investigate the signal pathways for regulated arecoline and areca nut extracts affect SDF-1 and CXCR4 production by human buccal mucosa fibroblasts, including MAPK family, PI3K/Akt pathway, and finially (6) further investigate the regulation of transcription factors via western blotting and EMSA assay to determine the transactivity of these transcription factors. In the second year of this study, we propose to (1) examine the SDF-1 and CXCR4 in 100 OSF patients and 100 paired human surgical tumor/adjacent normal specimens of oral cancer; (2) examine each 100 OSF patients and 100 paired oral cancer specimens with the tumor progression or tumor stages related to SDF-1 and CXCR4 expression. (3) analyze the mRNA levels of SDF-1 and CXCR4 from 100 OSF patients and 100 paired human surgical tumor/adjacent normal specimens of oral cancer via RT-PCR assay. (4) Using PCR-RFLP to investigate the genotype of SDF-1 gene of 150 OSF patients and 150 oral cancer. In the third year of this study, we propose to (1) analyze the effect of CXCR4 inhibitors (AMD3100) on viability and invasion of oral cancer cell SCC-4, SAS, SCC-25 and OC2. (2) investigate the mechanism involved in reduced invasiveness by CXCR4 inhibitors (AMD3100), including changed adhesion capability and invasion ability and ECM degraded proteinase (MMPs, u-PA) activities. (3) analyze the effect of CXCR4 RNAi on viability, invasion and ECM degraded proteinase (MMPs, u-PA) activities of oral cancer cell SCC-4. (4) further investigate the signal pathways (MAPK family ERK 1/2、p38 and JNK 1/2、phosphatidylinositol 3-kinase （PI3K）/Akt pathway) involved in reduced invasiveness by CXCR4 inhibitors. (5) investigate the regulation of transcription factors(CREB, AP-2, SP-1, NF-κB, c-Jun, c-Fos, SP-1 and Ets-1) via western blotting and EMSA assay to determine the transactivity of these transcription factors. (6) via an animal model, we propose to determine the effect of CXCR4 inhibitors (AMD3100) on metastasis of oral cancer cell inoculated mice.口腔黏膜下纖維化症(oral submucous fibrosis；OSF)為一種口腔癌前期病變，主要 與檳榔的嚼食習慣有關，而口腔黏膜下纖維化症後大約在3到16年間就有可能會發展成 口腔癌。而造成口腔黏膜下纖維化症及口腔癌的主要原因之ㄧ是細胞外基質中膠原蛋白 異常的堆積及分解所導致的。基質金屬蛋白水解酶 (matrix metalloproteinases；MMPs) 及纖維蛋白溶解系統(plasminogen activator (PA)/plasmin system)在細胞外基質的組成及 分解過程中扮演很重要的角色。而本實驗室一直致力於結締組織內膠原蛋白相關的分解 基因如MMPs 及其抑制劑 TIMPs 及胞漿素原活化劑的抑制劑(PAIs)與口腔相關疾病的 研究，尤其是在口腔黏膜下纖維化症與口腔癌的致病機轉。 基質細胞衍生因子(stromal-derived factor-1;SDF-1)又稱為CXCL12，為一趨化激素 (chemokines)，可與其受體蛋白CXCR4結合，進而進行其訊息傳遞。而趨化激素除了擁 有能夠引導白血球趨化性的能力外，也與感染與發炎現象有關。而近年來，SDF-1及其 受體蛋白CXCR4也被發現在不同癌細胞中有高度表現，並與其腫瘤的生長、遷移、吸附 與血管新生的癌化發生過程有關。而本實驗室之前發現口腔黏膜下纖維化症病人的纖維 母細胞較人類正常頰黏膜組織有著大量PAI-1、HIF-1α 及TIMP的表現。而又有文獻發現 SDF-1及CXCR4會調控著TIMPs、PAI-1與HIF-1α的表現，並且與 MMP-2及-9有密切的 相關性。因此，本計劃擬探討基質細胞衍生因子(SDF-1)及其受體(CXCR4)在口腔黏膜下 纖維化及口腔癌的表現。直至目前，本實驗室已經建立了MMPs 之 zymography 技術， TIMPs、PAI-1、SDF-1及CXCR4之 Western blotting 技術以及 RT-PCR、EMSA 等技術， 以從事相關口腔黏膜下纖維化及口腔癌相關基因的探討。因此，本計畫擬於第一年進行 【1】利用Western blotting分析人類正常頰黏膜及口腔黏膜下纖維化所培養的纖維母細胞 其基質細胞衍生因子(SDF-1)及其受體(CXCR4)的蛋白表現，並利用ELISA呈色方式分析 其分泌SDF-1的差異性表現。【2】利用RT-PCR及real time RT-PCR分析人類正常頰黏膜 及口腔黏膜下纖維化纖維母細胞其SDF-1及CXCR4的mRNA基因層次表現。【3】利用 immunohistochemistry分析人類正常頰黏膜及口腔黏膜下纖維化組織切片上SDF-1及 CXCR4的差異性變化。【4】在人類正常頰黏膜纖維母細胞中，分別加入arecoline 及 areca nut extracts，利用Western blotting, ELISA及RT-PCR的技術分析 arecoline 及areca nut extracts 對人類正常頰黏膜纖維母細胞中SDF-1及CXCR4的表現為何? 【5】進一步利用 訊息傳遞路徑的抑制劑探討arecoline及areca nut extracts對誘導人類正常頰黏膜纖維母細 胞中SDF-1及CXCR4的相關訊息傳遞路徑及其蛋白變化，如MAPK family、PI3K/Akt pathway。【6】以EMSA的分析方式探討arecoline及areca nut extracts是否影響SDF-1及 CXCR4的相關轉錄因子表現或與其target binding sequence結合能力。本計畫擬於第二年 進行【1】擬收集100位口腔癌及100位口腔黏膜下纖維化病人的手術切除組織。以Western blotting方式分析口腔癌病人其手術切除癌組織及其週遭較正常組織中SDF-1及CXCR4 的蛋白表現。並進一步以病患同意授權之資料，對口腔癌病人的發生原因、轉移與否及 一些臨床症狀做進一步的分析。【2】擬收集100位口腔癌及100位口腔黏膜下纖維化病人 的手術切除組織。以其手術切除癌組織及其週遭較正常組織中，取出組織並以RT-PCR 及即時RT-PCR (Real time RT-PCR)分析其組織中SDF-1及CXCR4的mRNA level的表現。 並進一步以病患同意授權之資料，對口腔癌病人的發生原因、轉移與否及一些臨床症狀 做進一步的分析。【3】擬收集100位口腔癌及100位口腔黏膜下纖維化病人的手術切除組 織檢體，製作成病理切片。利用以immunohistochemistry (免疫組織染色方式)分析口腔癌 病人其切片組織中SDF-1及CXCR4的蛋白表現。並進一步以病患同意授權之資料，對口 腔癌病人的發生原因、轉移與否及一些臨床症狀做進一步的分析。【4】擬收集150位口 腔癌及150位口腔黏膜下纖維化病人的血液，萃取其genomic DNA，利用PCR-RFLP的方 式，探討口腔癌與口腔黏膜下纖維化病人的SDF-1的基因多型性。並進一步以病患同意 授權之資料，對口腔癌病人的發生原因、轉移與否及一些臨床症狀做進一步的分析。本 計畫擬於第三年進行【1】以CXCR4 inhibitor (AMD3100)及 CXCR4-neutralizing antibody 處理不同的人類口腔癌細胞株，如SCC-4, SAS, SCC-25, OC2等，以改變細胞株CXCR4 的表現量，並觀察SDF-1與CXCR4的表現情形，更進一步利用migration/invasion assay、 adhesion assay探討口腔癌細胞株在處理CXCR4 inhibitors的細胞移動、侵襲和轉移能力之 變化情形。【2】以CXCR4 inhibitor (AMD3100)及 CXCR4-neutralizing antibody處理不同 的人類口腔癌細胞株，如SCC-4, SAS, SCC-25, OC2等，以改變細胞株中CXCR4的表現 量，進一步利用zymography、western blotting及 RT-PCR 等技術，探討口腔癌細胞株在 處理CXCR4 inhibitors的情況下，其細胞分泌基質分解相關蛋白酶，如 MMP-2、MMP-9、 u-PA、u-PAR和基質分解相關蛋白酶抑制劑，如TIMP-1、TIMP-2、PAI-1及PAI-2之變化 情形。【3】構築CXCR4的RNAi vectors以liposome或adenovirus將RNAi殖入口腔癌細胞中 以改變細胞株中CXCR4活性，進一步觀察在處理CXCR4的RNAi vectors的情況下或 CXCR4的RNAi stable colnes， 其口腔癌細胞移動、侵襲和轉移能力之變化情形，及細 胞分泌基質分解相關蛋白酶，如 MMP-2、MMP-9、u-PA、t-PA和基質分解相關蛋白酶 抑制劑，如TIMP-1、TIMP-2、PAI-1及PAI-2之變化情形。【4】進一步觀察CXCR4 inhibitor 及RNAi系統改變口腔癌細胞分泌ECM分解酵素和ECM分解酵素抑制劑的機制分析，如 影響侵襲能力的基質金屬蛋白水解酶活性有關的訊息傳遞MAPK family ERK 1/2、p38及 JNK 1/2、phosphatidylinositol 3-kinase (PI3K)/Akt pathway。【5】以electrophoretic mobility shift assay (EMSA)、Western blotting 探討CXCR4 inhibitor 及RNAi系統是否抑制上述基 因的相關轉錄因子表現，或與其target binding sequence的結合能力，如：MMP-2的轉錄 因子CREB、AP-2、SP-1，MMP-9的轉錄因子AP-1、NF-κB、c-Jun、c-Fos、SP-1及u-PA 的轉錄因子Ets-1、NF-κB等。【6】選用CXCR4 inhibitor (AMD3100)或建立CXCR4的RNAi stable colnes，以動物模式，分析其對於接種不同的人類口腔癌細胞株，如SCC-4, SAS, SCC-25, OC2的老鼠其腫瘤大小、重量及轉移至肺部的能力，以評估應用的可行性。Project