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目的:探讨中药半枝莲提取物(Scutellaria Barbata extract,ESB)含药血清对肝癌H22细胞凋亡率及线粒体膜电位的影响。方法:体外培养小鼠H22肝癌细胞,以ESB高、中、低剂量含药血清培养液作用于H22细胞,并设立空白血清及5-氟尿嘧啶(fluorouracil,5-Fu)组(阳性对照组)。应用四甲基偶氮唑盐法检测药物对细胞增殖的抑制作用;透射电镜下观察肿瘤细胞的超微结构变化;碘化丙啶标记,流式细胞术检测肿瘤细胞周期及细胞凋亡情况;采用荧光探针罗丹明123负载,激光扫描共聚焦显微镜下观察H22细胞的荧光强度,分析H22细胞线粒体膜电位的大小。结果:ESB含药血清有一定的抑制H22细胞增殖的作用,高、中剂量组96 h抑制率分别为(47.5±6.4)%和(36.3±4.5)%。透射电镜下可见部分细胞呈典型凋亡形态学改变。流式细胞仪检测结果显示,ESB中、高剂量组有明显的诱导凋亡作用,其凋亡率分别为(3.15±1.12)%和(7.83±2.25)%,与空白组相比差异有统计学意义(P<0.01)。空白对照组、5-Fu组和ESB低、中、高剂量,H22细胞线粒体膜电位分别为(245.45±67.37)、(127.42±41.35)和(213.68±65.52)、(186.34±56.37)、(142.65±39.44),各组线粒体膜电位与细胞凋亡率呈负相关(r=-0.826,P<0.01)。结论:ESB含药血清可有效诱导肿瘤细胞凋亡,其机制可能与降低肿瘤细胞线粒体膜电位有关。
Objective: To investigate the effect of serum containing Scutellaria Barbata extract (ESB) on apoptosis and mitochondrial membrane potential of hepatocellular carcinoma H22 cells. Methods: Mouse H22 hepatocarcinoma cells were cultured in vitro. H22 cells were treated with high, medium and low doses of serum containing ESB, and blank serum and fluorouracil (5-Fu) group (positive control group) were established. The inhibitory effect of drugs on cell proliferation was detected by MTT method. The ultrastructural changes of tumor cells were observed under transmission electron microscope. The cell cycle and apoptosis of tumor cells were detected by propidium iodide labeling and flow cytometry. The fluorescence intensity of H22 cells was observed under laser scanning confocal microscopy with the fluorescent probe rhodamine 123, and the mitochondrial membrane potential of H22 cells was analyzed. Results: ESB serum had a certain inhibitory effect on the proliferation of H22 cells. The 96 h inhibition rates of high and medium dose groups were (47.5 ± 6.4)% and (36.3 ± 4.5)%, respectively. Some cells showed typical apoptotic morphological changes under transmission electron microscope. The results of flow cytometry showed that the apoptosis rate of medium and high dose ESB groups was significantly (3.15 ± 1.12)% and (7.83 ± 2.25)%, respectively, which was statistically different from that of the blank group Significance (P <0.01). The mitochondrial membrane potential of H22 cells were (245.45 ± 67.37), (127.42 ± 41.35) and (213.68 ± 65.52), (186.34 ± 56.37) and (142.65) in blank control group, 5-Fu group and ESB low, ± 39.44). The mitochondrial membrane potential and apoptosis rate were negatively correlated (r = -0.826, P <0.01). Conclusion: ESB-containing serum can effectively induce tumor cell apoptosis, and its mechanism may be related to reducing the mitochondrial membrane potential of tumor cells.