2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38671Cervical intraepithelial neoplasia (CIN) is a pre-malignant lesion of the cervical cancer. Invasive squamous cell carcinoma (SCC) of the cervix has been found to be the end stage of progressive intraepithelial dysplastic atypia occurring within the metaplastic epithelium of the cervical transformation zone. 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 CIN and cervical cancer. In our present studying, we emphasize the proteinase and stromal-cell-derived factor 1 (SDF-1) and CXCR4 expression in CIN and cervical 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 CIN, cervical 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 CIN and cervical 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 CIN and cervical cancer. Therefore, in the first year of this study, we propose to (1) analyze the expressions of SDF-1 and CXCR4 from plasma on CIN and cervical cancer patients via ELISA assay. (2) analyze the mRNA levels of SDF-1 and CXCR4 from CIN and cervical cancer patients via RT-PCR assay. (3) Using immunohistochemistry to investigate the expression of SDF-1 and CXCR4 from CIN and cervical cancer patients. (4) To verify whether recombinant SDF-1 could affect SDF-1 and CXCR4 production by human cervical epithelial cell used western blots and RT-PCR. (5)further investigate the signal pathways for regulated recombinant SDF-1 affect SDF-1 and CXCR4 production by cervical epithelial cell, 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 paired human surgical tumor/adjacent normal specimens of cervical cancer; (2) examine 100 paired cervical 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 paired human surgical tumor/adjacent normal specimens of cervical cancer via RT-PCR assay. (4) Using PCR-RFLP to investigate the genotype of SDF-1 gene of 150 CIN1-3 patients and 150 cervical cancer. In the third year of this study, we propose to (1) analyze the effect of CXCR4 inhibitors (AMD3100) on viability and invasion of cervical cancer cell SiHa, HeLa and CaSki cell line. (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 cervical cancer cell. (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 cervical cancer cell inoculated mice.子宮頸上皮內贅瘤 (Cervical intraepithelial neoplasia ; CIN)是一種癌前病變，此種病變有可能進展成侵入性的子宮頸癌。而造成癌症轉移的主因主要是因為matrix metalloproteinase的大量表現，促使惡性腫瘤細胞分解基底膜造成局部的侵襲和遠處的轉移。因此，本實驗室一直致力於膠原蛋白相關的分解基因如基質金屬蛋白水解酵素 (matrix metalloproteinases; MMPs)及轉移抑制基因的(non-metastasis gene)與子宮頸上皮內贅瘤及子宮頸癌的研究。 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MMP-2及MMP-9在由子宮頸上皮內贅瘤癌化成子宮頸癌的過程中扮演著重要的角色。而又有文獻發現SDF-1及CXCR4會調控著MMP其內生性抑制劑TIMPs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)在子宮頸上皮內贅瘤及子宮頸癌的表現。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 ELISA、Western blotting 技術以及 RT-PCR、EMSA 等技術，以從事相關子宮頸上皮內贅瘤及子宮頸癌相關基因的探討。因此，本計畫擬於第一年進行【1】利用ELISA呈色方式分析正常人及不同時期的子宮頸上皮內贅瘤(CIN1-3)患者其血漿中基質細胞衍生因子(SDF-1)及其受體(CXCR4)的蛋白表現。【2】利用RT-PCR及real time RT-PCR分析正常人及不同時期的子宮頸上皮內贅瘤(CIN1-3)患者其子宮頸組織中其SDF-1及CXCR4的mRNA基因層次表現。【3】利用immunohistochemistry分析正常人及不同時期的子宮頸上皮內贅瘤(CIN1-3)患者組織切片上SDF-1及CXCR4的差異性變化。【4】在人類正常子宮頸細胞中，加入recombinant SDF-1，利用Western blotting, MTT assay及RT-PCR的技術分析recombinant SDF-1對人類正常子宮頸細胞其增生能力及其相關基因的表現為何?【5】進一步利用訊息傳遞路徑的抑制劑探討recombinant SDF-1對誘導人類正常子宮頸細胞中其增生能力及其相關基因的相關訊息傳遞路徑及其蛋白變化，如MAPK family、PI3K/Akt pathway。本計畫擬於第二年進行【1】收集100位子宮頸癌病人的手術切除組織。以Western blotting方式分析子宮頸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，對子宮頸癌病人的發生原因、轉移與否及一些臨床症狀做進一步的分析。【2】擬收集100 位子宮頸癌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，對子宮頸癌病人的發生原因、轉移與否及一些臨床症狀做進一步的分析。【3】擬收集100位子宮頸癌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)分析子宮頸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，對子宮頸癌病人的發生原因、轉移與否及一些臨床症狀做進一步的分析。【4】擬收集150位子宮頸癌、150位不同時期的子宮頸上皮內贅瘤(CIN1-3)病人及300位健康者的血液，萃取其genomic DNA，利用PCR-RFLP的方式，探討子宮頸癌及不同時期的子宮頸上皮內贅瘤病人的SDF-1的基因多型性。並進一步以病患同意授權之資料，對子宮頸癌及不同時期的子宮頸上皮內贅瘤病人的發生原因、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處理不同的人類子宮頸癌細胞株，如SiHa, HeLa及 CaSki cell line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探討子宮頸癌細胞株在處理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處理不同的人類子宮頸癌細胞株，如SiHa, HeLa及 CaSki cell line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，探討子宮頸癌細胞株在處理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殖入子宮頸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， 其子宮頸癌細胞移動、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系統改變子宮頸癌細胞分泌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子宮頸癌細胞株，如SiHa, HeLa及 CaSki cell line的裸鼠其腫瘤大小、重量及轉移至肺部的能力，以評估應用的可行性。Project