不同来源Lfcin诱导Jurkat细胞凋亡的对比研究

Apoptosis in Jurkat Cells Induced by Lfcin from Different Sources

摘要:

本文以牛乳铁蛋白素LfcinB和人乳铁蛋白素LfcinH为研究对象，研究其通过降低线粒体膜电位抑制Jurkat细胞增殖效果及两者的差异性。采用MTT法检测LfcinB和LfcinH对Jurkat细胞增殖影响；Hoechst33258染色法荧光显微镜观察Jurkat细胞核的变化；运用Annexin V-FITC∕PI双标记流式细胞术将LfcinB和LfcinH促进Jurkat 细胞凋亡的阶段进行区分；JC-1染色激光共聚焦显微镜观察细胞线粒体膜电位的改变。结果显示：LfcinH和LfcinB对Jurkat细胞有显著的抑制作用（P<0.05），呈剂量依赖性;经LfcinB和LfcinH处理后的Jurkat细胞在荧光显微镜下均可见细胞核皱缩、碎裂呈凋亡特征；激光共聚焦显微镜观察到Jurkat细胞线粒体膜电位下降；流式细胞术检测发现Jurkat细胞经LfcinB和LfcinH处理48 h时主要发生早期凋亡。研究结果表明：Jurkat细胞凋亡的机制可能是通过影响线粒体膜电位导致Jurkat细胞凋亡并抑制细胞增殖;当两者浓度小于250 μg/mL时LfcinH对Jurkat细胞抑制效果较强,浓度大于250 μg/mL时两者对Jurkat细胞抑制效果相近。

Abstract:

In this study, the inhibitory effects of bovine lactoferricin (LfcinB) and human lactoferricin (LfcinH) on the proliferation of Jurkat cells were explored and compared, by reducing the mitochondrial membrane potential in the cells. The effect on Jurkat cell proliferation was measured by MTT assay, and nuclear changes were observed by fluorescence microscopy with Hoechst 33258 staining. A flow cytometry of double-labeling by Annexin V-FITC/PI was used to distinguish the apoptosis stages in the Jurkat cells that were induced by LfcinH and LfcinB. The results showed that LfcinB and LfcinH significantly inhibited the proliferation of Jurkat cells (P < 0.05) in a dose-dependent manner. A decreased mitochondrial membrane potential in Jurkat cells was observed by JC-1 staining using laser scanning confocal microscopy. The early phase of apoptosis occurred after Jurkat cells were treated by LfcinH and LfcinB for 48 h; characteristics of apoptosis such as nuclear shrinkage and debris were observed using fluorescence microscopy. The results suggest that the change in mitochondrial membrane potential may have led to apoptosis in Jurkat cells and, eventually, the inhibition of proliferation. When the concentration of both, LfcinH and LfcinB was less than 250 μg/mL, the inhibitory effect of LfcinH on Jurkat cells was noted to be stronger; whereas at concentrations higher than 250 μg/ mL, LfcinH and LfcinB showed similar inhibitory effects.