2015-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/659142摘要：Pregnane X receptor (PXR)是一種核受體(nuclear receptor)。其最為人所知的功能，就是作為異生物質(xenobiotics)的sensor，以及藥物代謝酶基因的促進劑。尤其對於肝臟與小腸內的藥物代謝酶基因，都有極關鍵之調控作用。PXR之配合體(ligand)非常多樣，包括抗生素(如rifampicin)、steroids、bile acid、藥物(如RU486)等。近年來，科學家發現，PXR並非僅表現於肝臟與小腸，它也表現於T 淋巴球，且PXR一旦被其activating ligand活化後，就會抑制T lymphocyte之增殖、活化及分泌interferon-γ之功能，也會減少T lymphocyte內之NF-kB。同時，PXR與NF-kB有互相抑制之作用。而且亦發現HIV患者T lymphocyte之PXR及其他的核受體(如FXR、RXR)之表現，有大幅減少。主持人2013年執行有關PXR基因多型性與抗結核藥物性肝炎的研究時發現，PXR基因多型性不但與抗結核藥物性肝炎有關，也與結核病之易感性(susceptibility)有關。再加上上述的文獻報告，使主持人決定研究PXR受體與肺結核患者細胞免疫機轉之間的關係。計畫內容為肺結核預後與 PXR、其他核受體及NF-kB 轉錄體表現之關係，以及PXR基因多型性與結核病之易感性之間的相關性。本計畫之假設(hypothesis)為:1) 肺結核病人淋巴球之核受體PXR、retinoic X receptor (RXR)、farnesoid X receptor (FXR)以及NF-kB之mRNA表現會受到結核病之影響而與正常人不同；血漿中與這些核受體有關之趨化因子及細胞因子濃度(包括Rantes、MIG、IL-18、MMP-8及IL-10)亦會不同，且預後(治療兩個月時痰培養陰轉率)與上述parameters有關；2) PXR與NF-kB之間有互相抑制之現象; 3)若用生物體外(ex-vivo)方法，即作病人與正常人之全血細胞培養(whole blood culture assay)，在培養基中以結核菌抗原刺激血球，亦可觀察到類似現象，即淋巴球之PXR、RXR、FXR、NF-kB之表現在肺結核病人與正常人不同，且培養基上清液中，Rantes、MIG、IL-18、MMP-8及IL-10之濃度在肺結核病人與正常人亦會不同。4) 在上述之ex-vivo系統中加入PXR之活化配合體(activating ligand) rifampicin時，因PXR被活化，其對RXR、FXR、NF-kB、T 淋巴球 activation marker CD25之表現，以及上清液中Rantes、MIG、IL-18、MMP-8及IL-10之濃度有更大的影響。5) PXR基因多型性與結核病之易感性有關。本計畫為期三年，PXR、NF-kB、RXR、FXR之mRNA表現以real time qPCR來分析；病人血漿及細胞培養上清液中Rantes、MIG、IL-18、MMP-8及IL-10濃度由螢光磁珠免疫分析法(bead-based immunoassay)測定；T淋巴球CD25表現由流式細胞儀測定；PXR基因多型性由DNA定序法分析。預定收案150位肺結核病人，150位健康對照組。<br> Abstract: The pregnane X receptor (PXR) is also known as the steroid and xenobiotic sensing nuclear receptor (SXR). It is a member of the nuclear receptor superfamily. PXR is activated by a broad range of ligands, including endogenous and exogenous chemicals such as steroids, antibiotics (e.g. rifampicin), bile acids and drugs (e.g. RU 486). PXR is a transcrition factor. It is well known for its key role in regulating the metabolism of prescription drugs and environmental xenobiotics, and is primarily expressed in the liver and gastrointestinal tract.However, recent studies showed that PXR is also expressed in immune cells such as T-lymphocytes. Mouse PXR activation by PXR ligand dramatically decreased the ability of mouse T lymphocytes to proliferate, the expression of interferon-γ (IFN-γ), and phosphorylated NF-kB. In human T cells, activation of PXR by rifampicin reduced T-cell activation marker CD25 expression. These results showed that PXR had an immune-inhibition role in T lymphocytes. In HIV-(+) patients, the mRNA expression of nuclear receptors including PXR in PBMC was markedly reduced.Thus we decided to investigate the role of PXR in the immune profile of patients with pulmonary TB. The aims of our project are the association between TB prognosis (2-month sputum culture conversion rate) and transcriptome of PXR, retinoic X receptor (RXR), farnesoid X receptor (FXR), NF-kB, related chemokines & cytokines, and the association between PXR gene SNPs and TB susceptibility.Our hypotheses are: 1) In TB patients, MTB infection alters the expression of PXR, RXR, FXR, NF-kB in lymphocytes, chemokines & cytokines levels. The expression of PXR, RXR, FXR, NF-kB, and chemokines & cytokines levels are associated with 2-month sputum culture conversion rate. 2) There is reciprocal repression between PXR and NF-kB. 3) In ex-vivo cell culture system, M. tuberculosis antigen stimulation on whole blood culture obtained from TB patients alters expression of PXR, RXR, FXR, NF-kB, and the production of chemokines and cytokines in cell culture supernatant. 4) In ex-vivo system PXR activation by rifampicin suppresses CD25 (T lymphocyte activation marker) expression on T lymphocytes, and further influences the expression of PXR, RXR, FXR, NF-kB, chemokines & cytokines. 5) PXR gene single nucleotide polymorphisms (SNPs) are associated with susceptibility to TB.It is a 3-year proposal. The expression of nuclear receptors PXR, RXR, FXR and NF-kB is analyzed by real-time qPCR. Concentrations of chemokines and cytokines in the plasma and cell culture supernatant are determined by bead-based immunoassay. T-lymphocyte CD-25 expression is analyzed by flow cytometry. PXR SNPs are analyzed by sequencing. We plan to enroll 150 TB patients and 150 healthy controls.PXRRXRFXRNF-kB轉錄體T淋巴球RantesMIGIL-18MMP-8IL-10全血細胞培養活化配合體rifampicinCD25螢光磁珠免疫分析法流式細胞儀基因多型性結核病之易感性TBPXRNF-kBnuclear receptorsT-lymphocytetranscriptomecytokinechemokinewhole blood cell cultureCD25flow cytometrysusceptibility