论文部分内容阅读
目的:评估细菌脂多糖(LPS)在体外环境下对中华绒螯蟹血细胞凋亡的影响,并探讨其诱导血细胞凋亡的机制。创新点:首次在体外条件下实验证明了LPS可诱导中华绒螯蟹血细胞的凋亡,并确定了活性氧自由基在细胞凋亡过程中的变化和作用。方法:在体外26°C条件下,培养中华绒螯蟹血细胞。用浓度1μg/ml的LPS处理血细胞0、4、8和16 h后,利用4’,6-二脒基-2-苯基吲哚(DAPI)染色、荧光显微镜和透射电子显微镜观察血细胞的形态变化,用DNA Ladder试剂盒检测脱氧核糖核酸(DNA)的损伤程度。分别用浓度1和10μg/ml的LPS处理血细胞0、1和2 h,经膜联蛋白V-异硫氰酸荧光素(V-FITC)/碘化丙啶(PI)染色后,用流式细胞仪检测血细胞凋亡数量。收集血细胞制成细胞悬浮液,加入LPS至终浓度为10μg/ml,同时加入2’,7’-二氯荧光黄双乙酸盐(DCFH-DA)探针至终浓度为1μmol/L;对照组加入等量生理盐水和DCFH-DA探针。在26°C孵育不同时间后,用流式细胞仪和激光共聚焦显微镜检测血细胞活性氧的产生情况。结论:LPS可诱导中华绒螯蟹血细胞出现典型的凋亡特征,包括染色质浓缩和边聚、核膜破裂、线粒体肿胀、凋亡小体形成等(图1和2),形成明显的DNA梯形条带(图3),且细胞凋亡数量与LPS浓度呈正相关,大多数凋亡细胞处于凋亡晚期(图4)。活性氧检测结果显示:经LPS刺激后,细胞内活性氧自由基信号显著增强,随后又迅速减弱(图5和6)。综上所述,体外LPS可诱导中华绒螯蟹血细胞产生明显的凋亡反应,且凋亡过程伴随着显著的活性氧爆发。
OBJECTIVE: To evaluate the effect of bacterial lipopolysaccharide (LPS) on the apoptosis of Eriocheir sinensis haemocyte in vitro and to explore its mechanism of apoptosis induced by blood cells. Innovative point: the first in vitro experiments confirmed LPS can induce Eriocheir sinensis blood cell apoptosis, and to determine the active oxygen free radicals in the process of apoptosis and the role of change. Methods: Eriocheir sinensis blood cells were cultured in vitro at 26 ° C. Blood cells were treated with 4 ’, 6-diamidino-2-phenylindole (DAPI) staining, fluorescence microscope and transmission electron microscope at 0, 4, 8 and 16 h after treatment with LPS at a concentration of 1 μg / ml The DNA Ladder kit was used to determine the degree of deoxyribonucleic acid (DNA) damage. Blood cells were treated with LPS at concentrations of 1 and 10 μg / ml for 0, 1 and 2 h, respectively, stained with annexin V-FITC / PI, Cytometry to detect the number of apoptotic cells. The blood cells were collected and the cell suspension was prepared. LPS was added to a final concentration of 10 μg / ml, and the 2 ’, 7’-dichlorofluorescein diacetate (DCFH-DA) probe was added to a final concentration of 1 μmol / Equal amounts of saline and DCFH-DA probe. After incubating at 26 ° C for different times, the production of reactive oxygen species in blood cells was detected by flow cytometry and laser confocal microscopy. CONCLUSION: LPS can induce the typical apoptotic characteristics of hemocytes of Eriocheir sinensis, including chromatin condensation and edge aggregation, rupture of nuclear membrane, mitochondrial swelling and formation of apoptotic bodies (Figures 1 and 2), leading to significant DNA ladder (Figure 3). The number of apoptotic cells was positively correlated with LPS concentration, and most apoptotic cells were in late stage of apoptosis (Fig. 4). The result of reactive oxygen species test showed that after LPS stimulation, the signal of intracellular reactive oxygen species significantly increased and then rapidly weakened (Figure 5 and 6). In summary, in vitro LPS induced significant apoptotic response in blood cells of Eriocheir sinensis, and the apoptotic process accompanied by a significant active oxygen burst.