膠酶在弓形蟲感染星狀膠細胞之致病機轉

Pathogenesis of gelatinase in astroglia infected with Toxoplasma gondii

盧正祐(Cheng-You Lu)

指導教授 ： 賴世展

中山醫學大學/醫學院/醫學研究所/博士(2013年)

https://doi.org/10.6834/CSMU.2013.00015

摘要

弓形蟲（Toxoplasma gondii）能感染絕大部分的哺乳類動物，包括人。當弓形蟲感染免疫不全的宿主常會誘發弓形蟲性腦炎（toxoplasmic encephalitis）。本研究利用弓形蟲速殖子（TS-4 strain）感染星狀膠細胞（astroglia）的細胞模式進行研究，初步探討基質金屬蛋白酶（matrix metalloproteinase, MMP）-2、MMP-9和其受質纖維連結蛋白（fibronectin）的變化，接著再探討細胞外信號調節激酶1/2（extracellular signal-regulated kinases 1/2, Erk1/2）-核轉錄因子-kappa B（nuclear factor-kappa B, NF-κB）訊息傳遞作用和MMP-2/ MMP-9之間的相關性。在MMP-2、MMP-9和纖維連結蛋白的實驗中，MMP -2和MMP-9蛋白產量在感染後第1小時、第6小時和第12小時的細胞均質液，與感染後第6小時、第12小時、第24小時和第48小時細胞培養的上清液中有明顯的增加。同樣的，纖維連結蛋白降解現象（degradation）也發生在相同時間點。另外，在細胞免疫化學染色的結果顯示MMP-2和MMP-9表現在細胞質，且藉由共軛焦顯微鏡也得到MMP-2和MMP-9與纖維連結蛋白重疊在相同位置。除此之外，由免疫共同沉澱法結果得知MMP-2和MMP-9也各別會與纖維連結蛋白互相作用。綜合上述結果，我們推論在弓形蟲性腦炎的過程，MMP-2和MMP-9會降解纖維連結蛋白並讓星狀膠細胞活化，且促使白血球轉移到弓形蟲複製的位置。接著我們更進一步去探討Erk1/2-NF-κB訊息傳遞作用與MMP-2和MMP-9蛋白酶之間的相關性，實驗結果顯示在感染後的細胞均質液中，磷酸化的（phosphorylated, p-）Erk1/2會在第1小時快速的產生而p-NF-κB則是表現在第1小時到第12小時。另外，由電泳移動分析法（electrophoretic motility shift assay, EMSA）結果得知NF-κB會直接與MMP-9啟動子（promoter）上的NF-κB寡核苷酸（oligonucleotides）交互作用。其次，p-Erk1/2、p-NF-κB、MMP-2和MMP-9的表現會受到PD98059（Erk1/2抑制劑）的處理而有效的降低。在給予MG132（NF-κB間接抑制劑）的試驗中，p-NF-κB、MMP-2和MMP-9的表現也會隨著給予MG132而有效的減少。綜合上述實驗結果，我們推測在弓形蟲感染星狀膠細胞的過程中，MMP-2和MMP-9的表現會受到Erk1/2-NF-κB訊息傳遞作用所調節，且若能藉由阻斷MMP-2和MMP-9訊息傳遞中繼點（signaling intermediate），或許可以作為弓形蟲感染星狀膠細胞誘發腦炎的治療方法之一。

關鍵字

基質金屬蛋白酶-2 ； 基質金屬蛋白酶-9 ； 纖維連結蛋白 ； 弓形蟲 ； 星狀膠細胞 ； 核轉錄因子-kappa B ； 細胞外信號調節激酶1/2

並列摘要

Toxoplasma gondii infection in human can induce toxoplasmic encephalitis in immune disorders. In this study, astroglia were infected with the TS-4 strain of T. gondii tachyzoite in vitro to investigate the changes of matrix metalloproteinase (MMP)-2, MMP-9 and their substrate fibronectin. Further, investigating the association between extracellular signal-regulated kinase (Erk)1/2 -nuclear factor (NF)-κB pathway and MMP-2/MMP-9 expression. In first research, we found that
MMP-2 and MMP-9 were significantly increased at 1 h, 6 h and 12 h post-infection (PI) in the cell homogenates, and increased at 6 h, 12 h, 24 h and 48 h PI in the cell-cultured supernatants. Fibronectin degradation also occurred at the same time points. In addition, immunocytochemistry showed that MMP-2 and MMP-9 localized in the cytoplasm, and confocal scanning laser microscopy revealed co-labeled patterns of MMP-2 and MMP-9 with fibronectin. MMP-2 and MMP-9 interacted with fibronectin, respectively. These results suggest that MMP-2 and MMP-9 induction from astroglia may contribute to extracellular matrix (ECM) degradation occurring in toxoplasmosis. Thus, we hypothesize that MMP-2 and MMP-9 cleave fibronectin and may contribute to the astroglia reaction and leukocyte migration to the sites of T. gondii replication during toxoplasmic encephalitis. In our next research showed that phosphorylated (p)-Erk1/2 were transiently increased on 1 h post-infection and and p-NF-κB were significantly increased from 1 h PI to 12 h PI in the cell homogenates. NF-κB was bound directly to the oligonucleotides which contained putative NF-κB binding sites of MMP-9 promoter. In addition, the expression of p-Erk1/2, p-NF-κB, MMP-2 and MMP -9 were significantly decreased by PD98059, Erk kinase inhibitor. Also, treatment with MG132, NF-κB indirectly inhibitor, efficiently reduced p-NF-κB, MMP-2 and MMP -9 expression. These results suggested that suppression of Erk1/2-NF-κB signaling pathway cause a reduction of MMP-2 and -9 activities in astroglia response to T. gondii infection. Thus, inhibiting this signaling intermediate for MMP-2 and -9 expression might offer a potential way for control inflammatory development of T. gondii-induced encephalitis.

並列關鍵字

astroglia ； Erk1/2 ； fibronectin ； MMP-2 ； MMP-9 ； NF-κB ； T. gondii

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