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目的:探讨大蒜素对急性单核细胞白血病细胞株THP-1凋亡及凋亡基因Fas/FasL表达的影响。方法:采用四甲基偶氮唑蓝(MTT)法测定大蒜素对THP-1细胞增殖抑制率;AO/EB染色观察细胞凋亡形态;Annexin V-FITC/PI双染法流式细胞术(FCM)检测凋亡率的变化;FCM检测细胞凋亡率和细胞周期分布;免疫细胞化学法和Western blot观察细胞Fas、FasL蛋白表达。结果:MTT显示大蒜素能抑制THP-1细胞增殖,呈时间-剂量依赖性,72h中效浓度(IC50)为12.8mg/L。Annexin V-FITC/PI检测凋亡率随大蒜素浓度增加而增加。6.4、12.8mg/L大蒜素作用THP-1细胞72h后,FCM检测G0/G1期细胞比率明显上升,而S期比率明显下降,均出现凋亡峰,凋亡率分别为(22.93±2.93)%和(36.73±2.88)%,呈剂量依赖性,与对照组比较有显著差异(P<0.01)。在荧光显微镜下可区分早、晚期凋亡细胞、活细胞和坏死细胞。免疫组化显示大蒜素作用后Fas/FasL蛋白上调,主要分布在细胞浆和细胞膜。Western blot结果显示Fas/FasL蛋白表达量随大蒜素浓度的升高而增加,呈剂量依赖性。0、6.4、12.8mg/L大蒜素分别作用后Fas蛋白表达水平(相对灰度值)分别为0.2874±0.009,0.427±0.008和0.597±0.011;FasL蛋白表达水平分别为0.212±0.014,0.299±0.028和0.409±0.027;差别均有显著性意义(P<0.01)。结论:大蒜素可诱导THP-1细胞发生凋亡,诱导凋亡的机制可能部分与其上调Fas及FasL表达有关。
Objective: To investigate the effect of allicin on the apoptosis of acute monocytic leukemia cell line THP-1 and the expression of Fas / FasL gene. Methods: MTT assay was used to determine the inhibitory effect of allicin on the proliferation of THP-1 cells. AO / EB staining was used to observe the apoptotic morphology. Flow cytometry was performed with Annexin V-FITC / PI double staining FCM) were used to detect the changes of apoptosis rate. FCM was used to detect the apoptosis rate and cell cycle distribution. The expressions of Fas and FasL protein were detected by immunocytochemistry and Western blot. Results: MTT showed allicin could inhibit the proliferation of THP-1 cells in a time-and dose-dependent manner. The IC50 of 72h was 12.8 mg / L. The apoptosis rate of Annexin V-FITC / PI increased with the increase of allicin concentration. The apoptosis rate of THP-1 cells induced by allicin at 6.4 and 12.8 mg / L for 72 h was significantly higher than that of control group (22.93 ± 2.93) % And (36.73 ± 2.88)%, respectively, in a dose-dependent manner compared with the control group (P <0.01). Under the fluorescence microscope can distinguish early and late apoptotic cells, living cells and necrotic cells. Immunohistochemistry showed that Fas / FasL protein was up-regulated after allicin treatment, mainly distributed in cytoplasm and cell membrane. Western blot results showed that the expression of Fas / FasL protein increased with the increase of allicin concentration in a dose-dependent manner. The expression levels of Fas protein (relative gray value) after treated with 0, 6.4, 12.8 mg / L allicin were 0.2874 ± 0.009, 0.427 ± 0.008 and 0.597 ± 0.011, respectively; the expressions of FasL protein were 0.212 ± 0.014 and 0.299 ± 0.028 And 0.409 ± 0.027 respectively, with significant difference (P <0.01). CONCLUSION: Allicin can induce THP-1 cell apoptosis and the mechanism of apoptosis induction may be related to its up-regulation of Fas and FasL expression.