脱氧雪腐镰刀菌烯醇诱导血管内皮细胞凋亡及其凋亡相关因子的初步研究

Apoptosis and Relative Factors Expression of Endothelial Cells Induced by Deoxynivalenol

脱氧雪腐镰刀菌烯醇（DON）是由镰刀菌属病原真菌产生的真菌毒素之一,在自然界广泛存在,可引起人畜中毒。为了解DON对血管内皮细胞的危害及其可能机制,本试验采用不同浓度的DON（0.25-10μg/mL）作用于猪髋动脉内皮细胞（PIEC）和人脐静脉内皮细胞（HUVEC）2种细胞24 h,倒置显微镜下观察其形态变化,MTT法测定细胞增殖的变化,TUNEL法检测细胞凋亡率,免疫组织化学染色方法测定细胞凋亡过程中Caspase-9和Bax表达的变化。结果显示,高浓度DON可明显改变细胞的形态;不同浓度的DON均可抑制细胞的增殖;TUNEL凋亡检测结果显示,不同浓度的DON对这2种细胞均有诱导凋亡的作用;对于PIEC细胞,在DON浓度为0.5μg/mL时,凋亡率达到最高（71.74%）;HUVEC细胞则在DON浓度为5μg/mL时,凋亡率达到最高（26.61%）,此后随浓度的升高,凋亡率下降,可能与凋亡细胞崩解脱离细胞片有关。根据试验结果初步推断,DON诱导PIEC凋亡可能与Caspases途径有关,Bax在其中起到了一定的凋亡促进作用;而在诱导HUVEC细胞凋亡的过程中未检测到Caspase-9和Bax的变化,故认为DON诱导HUVEC细胞凋亡与Caspases途径和促凋亡因子Bax均无关。

Deoxynivalenol （DON）, a. k. a. vomitoxin, is a kind of mycotoxin mainly produced by the fungi Fusariu culrnorurn and F. graminearum while growing on crops. It is a trichothecene with relatively low toxicity. In the present experiment, five different concentrations of deoxynivalenol （DON） were added to culture PIEC and HUVEC cell lines for 24 h. Then the morphological changes of the cell lines were observed under microscope. The MTT method was performed to determine the inhibition ratio on proliferation of PIEC and HUVEC cell lines. And TUNEL method was used to assay the apoptotic rate of these cell lines. Immunohistochemical method was employed to measure the expression of Caspase-9 and Bax in apoptototic PIEC and HUVEC cell lines. The results showed that different concentration of DON can inhibit the proliferation of these cell lines and higher concentration can alter the morphology of PIEC and HUVEC cell lines.Different concentration of DON induced apoptosis of the two cell lines, for PIEC cell line, the apoptotic ratio reached the highest level at 0.5 μg/mL （71. 74%）, and HUVEC at 5 μg/mL （26.61%）. Then the apoptotic ratio decreased with increase of DON concentration, which may result from the collapse of apoptotic cells and their falling from the cell slice. The results implied that the Caspases pathway may contribute to the apoptosis of PIEC induced by DON, and the Bax may promote the process of the apoptosis. The changes of Caspase-9 and Bax were undetectable in the process of apoptosis of HUVEC cell line, which implied that the process did not relate to the Caspases pathway and Bax