2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38795Pelvic inflammatory disease (PID) is caused by micro-organisms which colonize in the endocervix and ascend to the endometrium and fallopian tubes. The clinical diagnosis includes the presence of an upper genital tract infection and inflammation in the patient. PID comprises a spectrum of inflammatory disorders of any combination of endometritis, salpingitis, tubo-ovarian abscess (TOA), and pelvic peritonitis. PID is difficult to diagnose because of a wide variation in the symptoms and signs. Early diagnosis with the help of successful laboratory testing is one way to avoid invasive laparoscopy or laparotomy and is beneficial for the patients, therefore our laboratory made efforts in the investigation of the mechanism of pelvic inflammatory disease and the relationship of different plasma protein concentrations and PID. Plasminogen activator/ plasma system is the inhibitor of the production of fibrin and acts on the degradation of fibrin. As well, is associated with the degradation of tissue basement, fibrination, tissue regeneration, and vascular angiogenesis, as well as the activation of macrophage and metastasis of tumor. However, it has been found that plasminogen activator is associated with the development of inflammation. In this study, we will investigate the association between plasminogen activator and pelvic inflammatory disease. Up to now, our laboratory has established some of the assays, such as zymography, Western blotting, RT-PCR, and EMSA for our investigation of PID. Using casein zymography assay, we found that the plasma concentrations of u-PA of PID patients were elevated before they received treatment compared with after receiving treatment in our preliminary data. During the first year of this proposed project, we will 【1】examine the plasma concentration of u-PA, soluble u-PAR, t-PA, PAI-1, and PAI-2 of one hundred PID patients compare with those of one hundred healthy individuals using ELISA assay. Additionally, we will estimate the interaction effect between those proteins and clinical information such as risk factors of PID, infection types of bacteria, white blood counts, neutrophils, and CRP, after obtaining the permissions from subjects. 【2】examine the plasma concentration of u-PA, soluble u-PAR, t-PA, PAI-1, and PAI-2 of one hundred PID patients before and after receiving treatment using ELISA assay. Additionally, we will estimate the interaction effect between those proteins and clinical information such as risk factors of PID, infection types of bacteria, white blood counts, neutrophils, and CRP, after obtaining the permissions from subjects, and estimate the relationship between PID prognosis and those proteins. 【3】 estimate mRNA expression levels of u-PA, u-PAR, t-PA, PAI-1, and PAI-2 of PBMC obtained from peripheral blood of one hundred PID patients before and after receiving treatment using RT-PCR and real time RT-PCR, particular for the estimation of u-PAR mRNA expression. As well, we will further estimate the different expression level and ratio between soluble u-PAR and membrane u-PAR. In addition, we will estimate the interaction effect between those proteins and clinical information such as risk factors of PID, infection types of bacteria, white blood counts, neutrophils, and CRP, after obtaining the permissions from subjects. 【4】estimate activity of plasma t-PA and u-PA of one hundred PID patients before and after receiving treatment using casein zymography. In addition, we will estimate the interaction effect between those proteins and clinical information such as risk factors of PID, infection types of bacteria, white blood counts, neutrophils, and CRP, after obtaining the permissions from subjects. From year two through year three, we will 【1】estimate the contributions of gene polymorphisms of u-PA, u-PAR, t-PA, PAI-1, and PAI-2 on the development of PID using PCR-RFLP after extration of genomic DNA from one hundred PID patients and one hundred healthy individuals. Additionally, we will estimate the interaction effect between those gene polymorphisms and clinical information such as risk factors of PID, infection types of bacteria, white blood counts, neutrophils, and CRP, after obtaining the permissions from subjects, and estimate the relationship between PID prognosis and those gene polymorphisms as well as the relationship between protein expression and gene polymorphism, and these effects on PID prognosis. 【2】 estimate the relationship between gene polymorphism and plasma protein expression level of u-PA, soluble u-PAR, t-PA, PAI-1, and, PAI-2, respectively, in PID patients as well as healthy controls. 【3】establish an in vitro study to demonstrate whether different gene polymorphisms will induce different expression levels of mRNA and protein by establisnment of vector and tranfection of specifically significantly differenc gene polymorphisms into HeLa 229 epithelial cell and ECC-1 human uterine epithelial cell, and then estimate those effects using RT-PCR, real time RT-PCR, western blotting and ELISA 【4】estimate gene polymorphism effect on gene expression of inflammatory factors, such as IL-1, TNF-alpha and IL-6, and inflammatory transduction mediators, such as MAPK family ERK 1/2, p38, JNK 1/2, and phosphatidylinositol 3-kinase（PI3K）/Akt pathway in HeLa 229 epithelial cell and ECC-1 human uterine epithelial cell after tansfection of those gene polymorhisms into the both cells. During the third year of this proposed project, we will 【1】estimate mRNA and protein levels of u-PA, soluble u-PAR, t-PA, PAI-1, and PAI-2 of of HeLa 229 epithelial cell, HEC 1B cell, and ECC-1 human uterine epithelial cell using RT-PCR, real time RT-PCR, western blotting, and ELISA assay, after transfection of IL-1, TNF-α, and LPS (lipopolysaccharide) into those epithelial cells. 【2】estimate whether the altered secretion levels of u-PA, soluble u-PAR, t-PA, PAI-1, and PAI-2 of epithelial cells, which induce by IL-, TNF-α, and LPS, are mediated through signal transduction pathway, such as MAPK family ERK 1/2、p38及JNK 1/2, phosphatidylinositol 3-kinase（PI3K）/Akt pathways. 【3】estimate the expression levels of u-PA, soluble u-PAR, t-PA, PAI-1, PAI-2 as well as of inflammatory factors, such as IL-1, TNF-alpha, and IL-6, of epithelial cells, such as HeLa 229 epithelial cell, HEC 1B cell or ECC-1 human uterine epithelial cell, using RT-PCR, real time RT-PCR, western blotting, and ELISA, after neutralization of uPAR of those cells by uPAR-neutralizing antibody. 【4】 estimate whether the secretion levels of IL-1, TNF-alpha, and IL-6 of epithelial cells will be altered after transfection of uPAR RNAi vectors into those cells by liposome or adenovirus. 【5】estimate whether the altered secretion levels of inflammatory cytokines, such as IL-1, TNF-α, and IL-6 , which induced by uPAR inhibitor and RNAi system, are mediated through signal transduction pathway, such as MAPK family ERK 1/2 、p38 及JNK 1/2, phosphatidylinositol 3-kinase（PI3K）/Akt pathways. 【6】Using electrophoretic mobility shift assay（EMSA）and Western blotting to estimate whether the expression of transcription factor of those proteins, such as the transcription factors CREB, AP-2, SP-1 of IL-1, transcription factors AP-1, NF-κB, c-Jun, c-Fos, SP-1 of IL-6, and tracsription factors Ets-1, NF-κB of u-PA, or the interation of receptor-ligand will be inhibited by uPAR inhibitor and RNAi .骨盆腔炎症包括了子宮內膜炎、骨盆腔腹膜炎、輸卵管卵巢囊腫、輸卵管炎、輸卵 管卵巢炎等，在定義上泛指女性的急性骨盆腔逆行性感染。在現今社會中不孕症夫婦的 比例有逐年增加的趨勢，而骨盆腔炎症就是造成女性不孕症的最主要原因。骨盆腔炎症 的症狀與許多骨盆相關疾病相似，所以很難經由一般的實驗室檢查來診斷，因此針對骨 盆腔炎症較具專一性、特異性的檢查在臨床診斷上就顯得很重要。本實驗室一直致力於 子宮頸癌的相關研究與骨盆腔炎症的相關致病機轉及探討骨盆腔炎症其血漿中的不同 蛋白表現。 纖維蛋白溶解系統(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。而此系統亦參與組織修補、血管新生、巨噬細胞活化及腫瘤轉移 等其它生理功能，並在這些過程中擔任蛋白水解的工作。然而，近年來亦有越來越多的 研究發現纖維蛋白溶解系統與不同的發炎反應有密切的相關性。因此，本計畫擬探討纖 維蛋白溶解系統在骨盆腔炎症的相關性表現。直至目前，本實驗室已經建立了u-PA 之 zymography 技術，PAI-1、t-PA及u-PAR之 Western blotting 技術以及 RT-PCR、EMSA 等技術，以從事骨盆腔炎症的相關致病機轉的探討。本實驗室初步利用casein zymography 方式測量骨盆腔炎症病人血漿中u-PA的表現，發現骨盆腔炎症病人治療前血漿中u-PA 的表現量比治療後血漿中u-PA的表現量高。因此，我們初步發現u-PA可能參與骨盆腔炎 症的致病過程。因此，本計畫擬於第一年進行【1】收集100位正常人及100位骨盆腔炎 症患者治療前的血漿，以ELISA的分析方式探討正常人及骨盆腔炎症血漿中u-PA、soluble u-PAR、t-PA、PAI-1及PAI-2的蛋白表現及其差異。並進一步以病患同意授權之資料，對 骨盆腔炎症的發生原因、細菌感染與否及一些臨床診斷如WBC數目、Neutrophils數目、 及CRP數值等做進一步的分析。【2】擬收集100位骨盆腔炎症患者治療前及治療後的血 漿，以ELISA的分析方式探討骨盆腔炎症患者治療前及治療後血漿中u-PA、soluble u-PAR、t-PA、PAI-1及PAI-2等的蛋白表現及其差異。並進一步以病患同意授權之資料， 對骨盆腔炎症的發生原因、細菌感染與否及一些臨床診斷如WBC數目、Neutrophils數目 及CRP數值等做進一步的分析。並進一步探討這些酵素的表現量與其預後的相關性。【3】 利用RT-PCR及real time RT-PCR分析100位骨盆腔炎症患者治療前及治療後的周邊血液 單核球(PBMC)中u-PA、u-PAR、t-PA、PAI-1、及PAI-2的mRNA基因層次表現。尤其是 u-PAR的mRNA表現，並進一步探討soluble u-PAR及membrane u-PAR的差異及比例。並 進一步以病患同意授權之資料，對骨盆腔炎症的發生原因、細菌感染與否及一些臨床診 斷如WBC數目、Neutrophils數目及CRP數值等做進一步的分析。【4】利用casein zymography及酵素呈色的分析方式，分析100位骨盆腔炎症患者治療前及治療後其血漿 中t-PA及u-PA的活性表現。並進一步以病患同意授權之資料，對骨盆腔炎症的發生原 因、細菌感染與否及一些臨床診斷如WBC數目、Neutrophils數目、及CRP數值等做進一 步的分析。本計畫擬於第二年進行【1】收集100位正常人及100位骨盆腔炎症患者的血 液，萃取其genomic DNA，利用PCR-RFLP及直接定序的方式，探討正常人及骨盆腔炎 症患者的u-PA、u-PAR、t-PA、PAI-1及PAI-2不同位點的基因多型性。且進一步探討正常 人及骨盆腔炎症患者的差異性，並以病患同意授權之資料，對骨盆腔炎症的發生原因、 細菌感染與否及一些臨床診斷如WBC數目、Neutrophils數目及CRP數值等做進一步的分 析，並探討這些酵素的表現量與其預後的相關性。【2】擬探討這100位骨盆腔炎症患者 及100位正常個案的u-PA、u-PAR、t-PA、PAI-1、及PAI-2不同位點的基因多型性的基因 型，與其血漿中u-PA、soluble u-PAR、t-PA、PAI-1、及PAI-2的蛋白表現量之相關性。【3】 擬進一步挑選有差異性的基因，構築不同的表現型載體。送入HeLa 229 epithelial cell或 ECC-1 human uterine epithelial cell內，並進一步利用RT-PCR、real time RT-PCR、western blotting 及ELISA方式，觀察不同的基因表現型是否的確會影響此基因的mRNA及protein 表現量。【4】進一步探討不同基因型對HeLa 229 epithelial cell或ECC-1 human uterine epithelial cell分泌發炎因子如IL-1、TNF-alpha、IL-6等基因的表現，並進一步探討與發 炎因子活性有關的訊息傳遞因子如MAPK family ERK 1/2 、p38 、JNK 1/2、及 phosphatidylinositol 3-kinase（PI3K）/Akt pathway的表現。本計畫擬於第三年進行【1】 以IL-1，TNF-a及細菌內毒素（lipopolysaccharide, LPS）處理不同的表皮細胞株，如HeLa 229 epithelial cell、HEC 1B cell、或ECC-1 human uterine epithelial cell等，接著以RT-PCR、 real time RT-PCR、western blotting 、及ELISA等方法測量這些經過處理後的表皮細胞株 u-PA、soluble u-PAR、t-PA、PAI-1、及PAI-2的mRNA及蛋白質表現情形。【2】進一步分 析IL-1，TNF-a及LPS改變表皮細胞株分泌u-PA、soluble u-PAR、t-PA、PAI-1、及PAI-2 的機制，如其相關的訊息傳遞因子MAPK family ERK 1/2 、p38 、JNK 1/2、及 phosphatidylinositol 3-kinase（PI3K）/Akt pathway。【3】以uPAR-neutralizing antibody處 理不同的表皮細胞株，如HeLa 229 epithelial cell、HEC 1B cell、或ECC-1 human uterine epithelial cell等，以改變細胞株uPAR的表現量，並觀察u-PA、soluble u-PAR、t-PA、PAI-1、 及PAI-2的表現情形，更進一步利用RT-PCR、real time RT-PCR、western blotting、及ELISA 等方法探討表皮細胞株在處理uPAR-neutralizing antibody之後，其分泌發炎因子如IL-1、 TNF-alpha、IL-6等能力之變化情形。【4】構築uPAR的RNAi vectors以liposome或adenovirus 將RNAi殖入表皮細胞株中以改變細胞株中uPAR活性，進一步觀察在處理uPAR的RNAi vectors或uPAR的RNAi stable colnes的情況下，其表皮細胞株分泌發炎因子如IL-1、 TNF-alpha、IL-6等變化情形。【5】進一步分析uPAR inhibitor 及RNAi系統改變表皮細胞 株分泌發炎因子如IL-1、TNF-alpha、IL-6的機制，如影響發炎因子活性有關的訊息傳遞 因子MAPK family ERK 1/2、p38、JNK 1/2、及phosphatidylinositol 3-kinase（PI3K）/Akt pathway。【6】以electrophoretic mobility shift assay（EMSA）、Western blotting 探討uPAR inhibitor 及RNAi系統是否抑制上述基因的相關轉錄因子表現，或抑制與其target binding sequence的結合能力，如：IL-1的轉錄因子CREB、AP-2、SP-1，IL-6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。骨盆腔炎症纖維蛋白溶解系統基因多型性Pelvic inflammatory diseasePlasminogen activatorplasma systemgenepolymorphismProject