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目的:观察γ-分泌酶抑制剂DAPT阻断Notch信号通路对食管鳞状细胞癌细胞株增殖和凋亡的影响,并初步探讨其作用机制。方法:采用γ-分泌酶抑制剂DAPT处理培养的食管鳞癌Eca109和TE-1细胞株以阻断Notch信号,分别应用CCK-8试剂和流式细胞术检测两种食管鳞癌细胞株的增殖与凋亡情况;同时采用定量PCR检测细胞Notch受体及靶基因Hes-1mRNA的表达,蛋白质印迹法检测与细胞增殖、凋亡相关蛋白Cyclin D1和Bcl-2的表达。结果:DAPT处理明显抑制了食管鳞癌Eca109和TE-1细胞株增殖,5μmol/L DAPT处理Eca109细胞株72h、TE-1细胞株96h后增殖抑制率分别为(61.8±5.3)%和(59.8±2.9)%,与DMSO对照组(17.2±2.6)%和(7.0±2.1)%相比差异有统计学意义,P=0.000 2和P<0.000 1,且抑制作用呈时间和浓度依赖关系。DAPT可诱导Eca09和TE-1细胞株凋亡,5μmol/L DAPT处理组Eca109细胞凋亡率在24和48h分别为(18.24±2.60)%和(21.77±5.82)%,与相应对照组(10.49±2.27)%和(9.74±3.38)%相比凋亡率增加,差异有统计学意义,P值分别为0.017 8和0.036 4;处理组TE-1细胞凋亡率在24和48h分别为(20.21±5.90)%和(32.14±5.92)%,与相应对照组(8.00±2.84)%和(12.59±3.72)%相比凋亡率增加,差异有统计学意义,P值分别为0.032 1和0.008 4。DAPT处理下调两种细胞株Notch2受体、Notch信号靶基因Hes-1及Cyclin D1的表达水平。DAPT处理可上调Eca109细胞Bcl-2水平,但下调了TE-1细胞中Bcl-2水平。结论:γ-分泌酶抑制剂DAPT可抑制食管鳞癌细胞株增殖并促进其凋亡,其机制可能与DAPT阻断Notch信号活化、调控Cyclin D1和Bcl-2的表达有关,阻断Notch信号通路有望成为食管鳞状细胞癌治疗的新靶点。
OBJECTIVE: To observe the effect of DAPT, a γ-secretase inhibitor, on the proliferation and apoptosis of esophageal squamous cell carcinoma cell line, and to explore its possible mechanism. Methods: The esophageal squamous cell carcinoma Eca109 and TE-1 cell lines were treated with γ-secretase inhibitor DAPT to block Notch signaling. The proliferation of two esophageal squamous cell carcinoma cell lines were detected by CCK-8 reagent and flow cytometry respectively And apoptosis. Meanwhile, the expression of Notch receptor and target gene Hes-1 mRNA was detected by quantitative PCR. The expressions of Cyclin D1 and Bcl-2 were detected by Western blotting. RESULTS: DAPT treatment significantly inhibited the proliferation of Eca109 and TE-1 cell lines. The proliferation inhibition rate of Eca109 cells treated with 5μmol / L DAPT for 72h and (61.8 ± 5.3)% and (59.8 ± 2.9%, respectively, which was significantly different from that of DMSO control group (17.2 ± 2.6)% and (7.0 ± 2.1)%, P = 0.000 2 and P <0.0001 respectively. The inhibition was time- and dose-dependent. DAPT induced the apoptosis of Eca09 and TE-1 cell lines. The apoptotic rates of Eca109 cells treated with 5μmol / L DAPT for 24 and 48h were (18.24 ± 2.60)% and (21.77 ± 5.82)%, respectively. Compared with the corresponding control group ± 2.27)% and (9.74 ± 3.38)%, the difference was statistically significant (P = 0.017 8 and 0.036 4 respectively). The apoptotic rates of TE-1 cells in treated group were ( 20.21 ± 5.90)% and (32.14 ± 5.92)%, respectively. Compared with the corresponding control group (8.00 ± 2.84)% and (12.59 ± 3.72)%, the apoptotic rate increased significantly with P values of 0.032 1 and 0.008 4. DAPT treatment down-regulated the expression of Notch2 receptor, Notch signaling target genes Hes-1 and Cyclin D1. DAPT treatment up-regulated the Bcl-2 level in Eca109 cells but down-regulated the Bcl-2 level in TE-1 cells. CONCLUSION: DAPT, a γ-secretase inhibitor, can inhibit the proliferation and promote the apoptosis of esophageal squamous cell carcinoma. The mechanism may be related to DAPT blocking the activation of Notch signal, regulating the expression of Cyclin D1 and Bcl-2, blocking the Notch signaling pathway Is expected to become a new target for the treatment of esophageal squamous cell carcinoma.