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目的:研究重组荞麦胰蛋白酶抑制剂(rBTI)对HepG2细胞增殖、凋亡、迁移及胞内活性氧(ROS)的影响。方法:以不同浓度的rBTI蛋白(1.25 10μmol/L)作用肝癌HepG2细胞,通过MTT比色法检测rBTI蛋白对HepG2细胞增殖抑制作用;DAPI荧光染色法观察凋亡细胞的发生及形态学变化,流式细胞术(FCM)检测细胞内活性氧变化、划痕擦伤实验检测rBTI蛋白对HepG2细胞迁移的影响。结果:rBTI蛋白对HepG2细胞的生长具有明显的抑制作用,并呈剂量依赖效应,但对正常细胞HL-7702影响很小;DAPI染色发现细胞产生凋亡小体;细胞内活性氧水平检测结果表明rBTI可以诱导HepG2细胞内ROS水平显著升高;划痕擦伤实验显示rBTI在一定浓度范围内可以抑制HepG2细胞迁移。结论:rBTI蛋白能够诱导HepG2细胞发生凋亡并抑制细胞增殖和迁移。
Objective: To investigate the effect of recombinant rat buckwheat trypsin inhibitor (rBTI) on proliferation, apoptosis, migration and intracellular reactive oxygen species (ROS) in HepG2 cells. Methods: HepG2 cells were treated with different concentrations of rBTI protein (1.25 10μmol / L), and the inhibitory effect of rBTI protein on the proliferation of HepG2 cells was detected by MTT colorimetric assay. The occurrence and morphological changes of apoptotic cells were observed by DAPI staining. The changes of reactive oxygen species (ROS) in cells were detected by flow cytometry (FCM). Scratch-scratching test was used to detect the effect of rBTI protein on the migration of HepG2 cells. Results: The rBTI protein had a significant inhibitory effect on the growth of HepG2 cells in a dose-dependent manner, but had little effect on the HL-7702 cells. The apoptotic bodies were found by DAPI staining. The results of intracellular reactive oxygen species rBTI could induce a significant increase of ROS in HepG2 cells. Scratch-scratching experiments showed that rBTI could inhibit the migration of HepG2 cells in a certain concentration range. Conclusion: rBTI can induce HepG2 cell apoptosis and inhibit cell proliferation and migration.