- 期刊
人工合成腐植酸對人類臍帶靜脈血管內板細胞之存活率及血液凝固相關因子ET-1、PAI-1及t-PA的影響
Effects of Synthetic Humic Acid on the Viability and Coagulant Factors: ET-1, t-PA and PAI-1 of Human Umbilical Vein Endothelial Cells
摘要
背景 烏腳病是一種閉塞性血栓末梢血管病變，流行於台灣西南沿岸地區。烏腳病區井水中高含量的腐植酸（humic acid, HA）被認為是烏腳病可能致病因子；但是，烏腳病區地下井水所純化之腐植酸不可避免地皆有金屬離子或其它分子的污染。本研究目的在探討三種人工合成之純腐植酸對人類臍帶靜脈血管內皮細胞之存活率及其分泌之血液凝固相關因子的影響。方法 本研究利用三種單一酚酸（protocatechuic acid 、venillic acid、ferulic acid）聚合而成之多酚酸聚合物即人工合成腐植酸（synthetic humic acid, SHA），作用於細胞培養之人類臍帶靜脈血管內皮細胞。內皮細胞以2.3×105個細胞為起始密度，種在含20%胎牛血清之M199之6-孔盤培養皿之中培養24小時後，分別加入不同濃度之人工腐植酸(0-200 μg/mL)，作用24, 48, 72小時後計算細胞數目，測量腐植酸所造成之細胞毒性；且於作用72小時後取上清液，利用酵素免疫分析法（enzyme link immunocorbent assay, ELISA）測定內皮細胞之內皮素(entothelin-1, ET-1)、胞漿素原活化劑之抑制劑（plasminogen activator inhibitor, PAI-1）及組織型胞漿素原活化劑（tissue plasminogen activator, t-PA）的分泌量。結果 本研究發現三種人工合成腐植酸都會抑制內皮細胞生長，造成細胞毒性；且會隨著腐植酸作用時間和濃度的增加，細胞數目越減少毒性越強。本研究亦發現三種人工合成腐植酸作用內皮細胞後，可增加血管收縮素的ET-1分泌量；且ET-1含量隨腐植酸濃度的增加而增加。相同實驗條件下，亦可增加抗溶血因子PAI-1以及減少溶血因子t-PA的分泌量；且PAI-1含量隨腐植酸濃度的增加而增加，而t-PA含量隨腐植酸濃度的增加而減少。三種單一酚酸類則無以上的現象。結論 人工合成腐植酸作用於人類臍帶靜脈血管內皮細胞，可促進血液凝固的作；用故推測腐植酸作用於血管內皮細胞的各種效應可能是烏腳病造成血栓性血管疾病的致病因子久一。
並列摘要
Background. Blackfoot disease is a thrombotic peripheral vascular disease causally related to humic acid (HA) found in the drinking water of wells along the southwestern coast of Taiwan. To better define the causative chemical components of the endemic artesian well water, synthetic humic acid (SHA) was used to investigate effects on the viability and coagulant properties of human umbilical vein endothelial cells (HUVECs). Methods. Three SHAs of well defined phenolic derivatives, including monomeric protocatechuic acid, venillic acid and ferulic acid, were incubated with the HUVECs. The cells were maintained at 2.3×105 cell/well in 6-well plates and cultured in M-199 medium containing 20% fetal bovine serum for 24 hr before treatment. Cell viability of each confluent monolayer was checked before and after treatment with various concentrations of the three SHAs (25-200 μg/ mL) at 24 hr, 48 hr and 72 hr, respectively, using trypan blue and examined using phase contrast microscopy. The anount of endothelin-1 (ET-1) antigen, tissue plasminogen activator (t-PA) antigen and plasminogen activator inhidtor (PAI-1) antigen in the endothelial cells containing medium after 72 hr of treatment was determined using a double antibody sandwich enzyme-linked immunosorbent assay. Results. SHA was found to inhibit the growth and viability of subconfluent HUVECs in both dose and time dependent conditions. Furthermore, after 72 hr of treatment a dose dependent effect occurred when SHA stimulated HUVEC to produce ET-1 and PAI-1, and to reduce t-PA. Conclusions. These results suggest that the biological effects of SHA on HUVECs may lead to local procoagulant states or thrombotic disorders and that vascular endothelial cell damage may contribute partly, at least, to the thrombotic disorders of blackfoot disease.