肝細胞生長因子在由超細微碳黑所誘發的急性和亞急性肺部傷害所扮演的角色

中文摘要可吸入性環境空氣污染物的暴露與罹患慢性肺部疾病的危險性增加有關，包括氣喘和慢性阻塞性肺病。我們先前的研究也證明了超微細碳黑的急性暴露會造成急性的肺部傷害。新近研究顯示，肝細胞生長因子可能對由博萊酶素所誘發的肺部傷害具有的保護功能。本研究的主要目的是探討肝細胞生長因子在由超細微碳黑所誘發的急性和亞急性肺部傷害中所扮演的角色。本研究使用5週大、體重25-28公克的老鼠，單次或兩天一次以 100μg之超細微碳黑(14nm)進行肺支氣管內灌流。測量支氣管沖洗液中的總蛋白質和肝細胞生長因子及白血球的分類計數。反應性有氧物種以2'7'-二氯熒光乙酰乙酸鹽測量。c-Met的表現量以西方點墨法分析。以BrdUrd一級抗體去偵測DNA的合成量和細胞的增生。單次暴露超細微碳黑能顯著增加支氣管沖洗液的總蛋白量和發炎細胞數，肝細胞生長因子的增加能持續至暴露後96小時。單核球及上皮細胞細胞株實驗確認超細微碳黑能增加肝細胞生長因子的生成，而抗氧化劑能降低其生成量。然而，纖維母細胞只有腫瘤壞死因子- α(TNF-α)可增加其肝細胞生長因子的產生。在兩天一次暴露超細微碳黑的亞急性模式下，嗜中性球和淋巴球的滲透及總蛋白質和肝細胞生長因子也會顯著的增加。和對照組相較下，重複4次暴露超微細碳黑會增加細支氣管上皮細胞的增生，在細支氣管上皮層和第二型的肺泡細胞中有更多BrdUrd-陽性的染色結果被發現。至於肝細胞生長因子接受器(c-Met)只在第一次暴露後有顯著增加。超細微碳黑暴露後，肝細胞生長因子的生成有其細胞特異性。重要的是肝細胞生長因子能減少由於暴露超細微碳黑所產生的氧化壓力，因此在急性暴露傷害，肝細胞生長因子能減少反應性氧化物種的產生，增加細胞的存活及增生，而促進組織的修復。亞急性的超細微碳黑暴露也能夠增加肝細胞生長因子的產生和肺部上皮細胞的增，這可能與肺部組織重塑有關。關鍵字:超細微碳黑，肝細胞生長因子，BrdUrd，細胞增生，組織重塑

關鍵字

超細微碳黑；肝細胞生長因子； BrdUrd ；細胞增生；組織重塑

並列摘要

英文摘要 High levels of ambient respirable pollutants have been linked to the increased risk of chronic lung diseases, including asthma and chronic obstructive pulmonary disease. Our previous study demonstrated that acute exposure to ufCB cause acute lung injury. It has recently been shown that HGF might exhibit a potent function for resolution of bleomycin induced lung injuries. The aim of this study was to explore the role of HGF in ufCB induced acute and subacute lung injury. Animal ICR mice, aged 5 wks and weighted 25-28 gm. Particle ufCB, 14 nm in diameter. Animal were intratracheal instilled with 100 μg of ufCB very other day. Quantitations of total proteins and HGF and differential analysis of leukocytes were performed. ROS production was determined using 2’, 7’-dichlorofluorescein diacetate (DCFH-DA). The level of c-Met expression was determined using Western blot analysis. Primary antibody against bromodeoxyuridine (BrdUrd) was used to detect the levels of DNA synthesis and cell proliferation. A single exposure to ufCB significantly increased total proteins and inflammatory cells in BAL fluid. In conjunction, HGF in BAL was increased and maintained at significantly high level even 96 hr after exposure, compared to PBS control. The level of HGF in THP-1 (cultured macrophages) reached the maximal level at 72 hr after exposure to 100 μg ufCB. The increase of HGF in THP-1 after 6-hr exposure to ufCB could be reversed by antioxidant (SOD) treatment. Likewise, the increase of HGF in A549 by a 6-hr exposure to ufCB was reversed by SOD. However, a 6-hr or longer exposure to ufCB did not induce HGF production in cultured fibroblasts. Cultured fibroblasts increased HGF production in response to TNF-α challenge. In subacute model with ufCB exposure every other day, neutrophil and lymphocyte infiltrations, the production of total proteins and HGF were significantly increased in BAL fluid after each exposure. Repeated exposure four times to ufCB increased the epithelial cell proliferations in bronchioles, compared to the control group. More BrdUrd-positive stainings were found in the epithelial layers of bronchioles and type II cells of alveoli. The expression of HGF receptor (c-Met) was highly elevated in lung after first exposure. Then, the levels of c-Met were not significantly altered after repeated exposure, when compared with PBS control. Our data suggest that the induction of HGF after exposure to ufCB is cell-specific. Most intriguing, HGF could reduce the oxidative stress level incurred by ufCB exposure. Therefore, in acute exposure to ufCB, HGF could increase cell survival by reducing ROS production and increase cell proliferation to facilitate tissue repair. Subacute exposures to ufCB could also increase the production of HGF and the proliferation of lung epithelial cells. This may cause lung tissue remodeling. Key Words: ultrafine carbon black, Heptocyte growth factor, BrdUrd, cell proliferation, tissue remodeling.