2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38810Cervical 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 t-PA、u-PA、u-PAR、PAI-1 and PAI-2 expressions in CIN and cervical cancer. plasminogen activator/ plasmin system has been found to play an important role in tumorigenicity, proliferation, metastasis and angiogenesis in many cancers, such as lung cancer, breast cancer, melanoma and basal cell carcinoma cells. Over the past decades, extensive efforts has been made to find the role of u-PA and u-PAR 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 u-PA and u-PAR axis in normal and malignant cell. However, little evidence of a direct relationship between CIN, cervical cancer and u-PA and u-PAR axis expression. Thus, in this study, we propose to study the expression of u-PA and u-PAR axis in human CIN and cervical cancer. With a few established techniques, including zymography, western blotting, RT-PCR and EMSA for u-PA, MMPs, u-PAR and PAI-1 ,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 t-PA、u-PA、u-PAR、PAI-1 and PAI-2 from plasma on CIN and cervical cancer patients via ELISA assay. (2) analyze the mRNA levels of t-PA、 u-PA、u-PAR、PAI-1 and PAI-2 from CIN and cervical cancer patients via RT-PCR assay. (3) Using immunohistochemistry to investigate the expression of t-PA、u-PA、u-PAR、PAI-1 and PAI-2 from CIN and cervical cancer patients. (4) To verify whether recombinant u-PA could affect t-PA、u-PA、u-PAR、PAI-1 and PAI-2 production by human cervical epithelial cell used western blots and RT-PCR. (5)further investigate the signal pathways for regulated recombinant u-PA affect t-PA、u-PA、u-PAR、PAI-1 and PAI-2 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 t-PA、u-PA、u-PAR、PAI-1 and PAI-2 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 t-PA、u-PA、u-PAR、PAI-1 and PAI-2 expression. (3) analyze the mRNA levels of t-PA、u-PA、u-PAR、PAI-1 and PAI-2 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 t-PA、u-PA、u-PAR、 PAI-1 and PAI-2 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 u-PAR inhibitors on viability and invasion of cervical cancer cell SiHa, HeLa and CaSki cell line. (2) investigate the mechanism involved in reduced invasiveness by u-PAR inhibitors, including changed adhesion capability and invasion ability and ECM degraded proteinase (MMPs, u-PA) activities. (3) analyze the effect of u-PAR 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 u-PAR 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 u-PAR inhibitors 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。 胞漿素原活化劑及胞漿素系統(plasminogen activator/ plasmin system)是哺乳動物 血液中用來限制過度纖維蛋白(fibrin)形成及分解血塊的酵素系統，另和基底膜的降解、 組織纖維化及組織重組非常有關係。而此系統的組成物，依其所扮演角色不同，主要有 胞漿素原活化劑(plasminogen activators，PAs)及胞漿素原活化劑的抑制因子(plasminogen activators inhibitors ， PAIs) 及尿激酶型接受器(urokinase-type plasminogen activator receptor，u-PAR)。而u-PA及其receptor u-PAR在許多癌症的致病過程有密切的相關性已 是不爭的事實，而且u-PA及u-PAR也被發現會調控基質金屬蛋白水解酵素(MMPs)及其 抑制劑(TIMPs)。但是t-PA、u-PA、u-PAR及其抑制劑PAI-1、PAI-2這一個plasminogen activator / plasmin system與子宮頸癌的致癌過程及其相關性卻仍不清楚。直至目前，本 實驗室已經建立了u-PA及MMPs 之zymography 技術，TIMPs、u-PAR、PAI-1及PAI-2 之ELISA、Western blotting 技術以及Real Time RP-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 t-PA、u-PA、soluble u-PAR及其抑制劑PAI-1、PAI-2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t-PA、 u-PA 、u-PAR 及其抑制劑PAI-1 、PAI-2 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t-PA、u-PA、u-PAR及其抑制劑PAI-1、PAI-2的差異性變化。【4】在人類正常子宮 頸細胞中，加入recombinant u-PA，利用Western blotting、MTT及RT-PCR的技術分析 recombinant u-PA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 u-PA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】擬收集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t-PA、 u-PA、u-PAR及其抑制劑PAI-1、PAI-2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Real time RT-PCR分析其組織中t-PA、u-PA、u-PAR及其抑制劑PAI-1、 PAI-2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immunohistochemistry分析子宮頸癌病人其切片 組織中t-PA、u-PA、u-PAR及其抑制劑PAI-1、PAI-2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t-PA、u-PA、u-PAR及其抑制劑PAI-1、PAI-2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u-PAR inhibitor及u-PAR-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u-PAR的表現量，並觀察t-PA、u-PA、 u-PAR及其抑制劑PAI-1、PAI-2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u-PAR inhibitors的細胞移動、侵襲和轉移能力 之變化情形。【2】以u-PAR inhibitor及u-PAR-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u-PAR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 u-PAR 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 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u-PAR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u-PAR活性，進一步觀察在處理u-PAR的RNAi vectors的情況下或u-PAR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u-PAR 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u-PAR 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u-PAR inhibitor或建立u-PAR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 lines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