Porphyromonas gingivalis感染誘發人類 主動脈平滑肌細胞發生程序性壞死機制之相關研究

Porphyromonas gingivalis Infection Induces Necroptosis of Human Aortic Smooth Muscle Cells

黃文馨(Wen-Hsin Huang)

指導教授 ： 林哲堂

臺北醫學大學/口腔醫學院/牙醫學系碩博士班/碩士(2015年)

摘要

目的
人類主動脈瘤發生率高，以腹主動脈瘤尤為普遍，高齡與抽菸更是提高疾病發生
風險，而其主要特徵為主動脈膨大超過正常管徑之50%，此現象成因與主動脈管
壁之平滑肌細胞死亡喪失有關。過去動物實驗與臨床心血管疾病檢體相關研究指
出牙周病致病菌Porphyromonas gingivalis (P.g.)感染會誘發血管瘤生成或惡
化，然而確切造成疾病之途徑與機制並未釐清。本篇研究比較感染不同菌株的
P.g. 對於人類主動脈平滑肌細胞之生物現象，以及測試P.g. 感染是否透過
necroptosis 誘發平滑肌細胞死亡，以期進一步探討牙周病與心血管疾病之關
聯。
方法
以antibiotic protection assay 及傳統菌落培養比較不同P.g.菌株對於人類
主動脈侵入平滑肌細胞能力差異；另外利用即時聚合酶鏈式反應驗證P.g.能夠
貼附與入侵平滑肌細胞。平滑肌細胞感染P.g.後之型態藉由穿透式電子顯微鏡
觀察；另外利用西方墨點法偵測平滑肌細胞蛋白質表現分析細胞表型差異；細胞
活性評估則利用結晶紫染色以及Hoechst/PI 染色；而TUNEL assay 併流式細胞
儀偵測用於分析比較P.g.誘發之平滑肌細胞死亡現象。
結果
菌落培養以及即時聚合酶鏈式反應皆證實P.g.貼附與侵入人類主動脈平滑肌細
胞。兩菌株感染平滑肌細胞後發生不同的生物現象，fimA type III P.g.造成細
胞表型轉化，而fimA type I P.g.誘發細胞死亡。主動脈平滑肌細胞感染fimA
type I P.g.後，細胞膜受損，穿透式電顯下細胞膜破裂，細微胞器型態被破壞，
4
細胞核型態完整，與典型細胞凋亡型態背離；感染之後24 小時內細胞發生大量
死亡。若於感染P.g.之前，分別利用下列製劑前處理人類主動脈平滑肌細胞，
Z-DON （transglutaminase 2, TG2 之抑制劑）， necrostatin 1（receptor
interacting protein kinase 1, RIPK1 之抑制劑)，以及PJ-34 （inhibitor of
poly(ADP-ribose) polymerase 1, PARP1 之抑制劑），細胞死亡情況發生減緩；
而使用Z-VAD-FMK（pan-caspase 抑制劑）前處理對於提升細胞存活率之效果則
較不顯著，顯示P.g.感染會誘發之人類主動脈平滑肌細胞發生necroptosis。
結論
P.g.感染藉由細胞膜受體（TG2）進而活化細胞內訊號傳遞（RIPK1 與 PARP1）
導致人類主動脈平滑肌細胞發生necroptosis。此研究幫助探討牙周疾病與心血關
疾病相關機制與提供臨床治療新的思考方向。

關鍵字

牙周病 ； 平滑肌細胞 ； 心血管疾病 ； 主動脈瘤 ； 程序性壞死

並列摘要

Objectives
This study investigates whether Porphyromonas gingivalis(P.g.) infection induces loss
of vascular smooth muscle cells through necroptosis. Progression of aortic aneurysms
involves depletion of vascular smooth muscle cells. P.g., a major periodontal
pathogen, was identified in cardiovascular specimens from patients with aneurysms in
previous studies, suggesting the possible correlation between the disease and the
bacteria.
Methods
P. gingivalis (ATCC 33277；ATCC49417) and human aortic smooth muscle cells
(HAMSCs) were obtained from certified vendors. Antibiotic protection assay for
bacterial invasion was performed with colony formation and polymerase chain
reaction-based 16s rDNA quantification. HASMC morphology was documented with
light microscopy and transmission electron microscopy. Cell viability was
quantitatively assessed with crystal violet staining/methanol elution and propidium
iodide (PI) exclusion assay. Cell membrane integrity and nuclear DNA breaks were
measured with the flow cytometry.
Results
P.g. invasion into HAMSCs was confirmed by the antibiotic protection assay and
real-time polymerase chain reaction. fimA type I & III P.g. infection induce different
biological effects on HASMCs. While fimA type III P.g. caused phenotype switch of
HASMCs, cell viability assay by crystal violet staining and Hoechst/PI staining
2
revealed significant cell loss by fimA type I P.g. infection. Besides, terminal
transferase-mediated dUTP nick end-labeling (TUNEL) assay by flow cytometry
showed a dose-dependent effect with more cell death noted in undiluted P.g. infection.
Reduction of cell death caused by P.g. infection was observed in HAMSCs pretreated
with TS92, the neutralizing antibody of oxidized low-density lipoprotein receptor-1
(OLR1) or Z-DON, the transglutaminase 2 (TG2) inhibitor, necrostatin1 (inhibitor of
RIPK1), or PJ-34, (inhibitor of poly(ADP-ribose) polymerase, PARP-1). However,
pretreatment with Z-VAD-FMK (pan-caspase inhibitor ) only marginally prevented
the death of HASMCs.
Conclusions
Our results indicate that P.g. infection induces necroptosis of HASMCs through
engaging specific surface receptors (TG2) and intracellular signaling pathways
(RIPK1 and PARP1). Targeting necroptosis may be a new strategy for treating
periodontitis-related cardiovascular diseases.

並列關鍵字

Porphyromonas gingivalis ； periodontitis ； cardiovascular disease ； aortic aneurysm ； vascular smooth muscle cell ； necroptosis

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