壁虎多肽混合物对肝癌SMMC7721细胞增殖及其自噬的影响

Effect of Gecko peptides mixture on proliferation and autophagy in hepatocellular carcinoma SMMC7721 cells

目的研究壁虎多肽混合物(GPM)对人肝癌SMMC7721细胞自噬的影响。方法取对数生长期细胞种板,用不同质量浓度的GPM(0,0.04,0.06,0.09,0.14,0.20,0.30,0.45 mg·m L^(-1))处理SMMC7721细胞24 h,通过噻唑蓝比色法(MTT)检测细胞增殖,根据MTT结果,分别作为正常组(0)、低中高3个质量浓度(GPM:0.1,0.15,0.22 mg·m L^(-1))实验组,对照组(西罗莫司40μg·m L^(-1))。经单丹磺酰尸胺(MDC)荧光染色观察不同质量浓度GPM对SMMC7721细胞自噬的影响;免疫组化法检测细胞内Beclin1及LC3表达的变化;免疫印迹法检测细胞自噬标志物Beclin1、LC3-Ⅱ蛋白的表达。结果 GPM可抑制SMMC7721细胞的增殖,并具有质量浓度依赖性,作用24 h后其IC50值为0.16 mg·m L^(-1)。GPM处理SMMC7721细胞6 h后,在胞质中可见大量自噬小泡的形成,在显微镜下呈亮绿色荧光。正常组、对照组、低中高3个质量浓度实验组Beclin1阳性细胞数占总细胞数的百分比分别为(15.70±0.26)%,(63.47±0.54)%,(17.09±0.37)%,(70.66±0.56)%,(78.48±0.68)%;与正常组相比,对照组与中高2个质量浓度实验组差异有统计学意义(均P<0.05)。这5组LC3-阳性细胞数占总细胞数的百分比分别为(35.84±0.36)%,(82.41±0.82)%,(60.83±0.61)%,(69.66±0.70)%,(74.61±0.75)%,与正常组相比,各组差异均具有统计学意义(均P<0.05)。这5组的Beclin1与β-actin灰度比值分别为(6.87±0.68)%,(11.26±0.87)%,(6.46±1.34)%,(11.58±0.95)%,(15.82±1.58)%;与正常组相比,对照组、中高2个质量浓度实验组差异有统计学意义(均P<0.05)。这5组LC3-Ⅱ与βactin灰度比值分别为(26.70±0.41)%,(103.17±0.88)%,(30.29±0.52)%,(36.32±0.52)%,(64.34±0.48)%;与正常组相比,各组差异均具有统计学意义(均P<0.05)。结论壁虎多肽混合物能够影响人肝癌SMMC7721细胞自噬现象的发生,可能通过诱导其自噬而使细胞死亡。

Objective To investigate the possible molecular mecha- nisms of Gecko peptides mixture （GPM） and research to human hepatocellular carcinoma SMMC7721 cells on autophagy with GPM. Methods SMMC7721 cells were put into plates in its logarithmic phase, and they were treated with different concentration of GPM （0, 0.04, 0.06, 0.09, 0.14, 0.20, 0.30, 0.45 mg · mL^-1） for 24 h, and then detected corresponding indicators with respective methods. The viability of SMMC7721 cells was detected with 3 -（4,5 -dimethyl-2 - thiazolyl） - 2,5 - diphenyl - 2 - H - tetrazolium bromide （MTT） . The concentration of normal group, GPM low-dose, middle- dose and high- dose experimental groups was separately 0, 0. 1, 0. 15, 0.22 mg · mL^-1, according to the results of MTF. Rapamycin was chosen as the positive control drug, which concentration was 40 μg · mL^-1. The autophagy of different concentration GPM on SMMC7721 cells was detected by monodansylcadaverine （MDC） staining. Expression levels of Beclinl and LC3 in SMMC7721 cells were measured by immunohistochemical method and Western blot assay. Results GPM could significantly inhibit the proliferation of SMMC7721 cells in a dose - dependent manner, and the ICs0value was 0. 16 mg · mL^-1 for 24 h. MDC staining showed that there are plenty of autophagosome in cytoplasm, emerging bright green fluorescent in the fluorescence microscope after treatment with GPM for 6 h. The percentage number of positive cells of total cells number in normal group, control group, GPM low, middle and high - dose experimental groups were （15.70 ± 0.26）%, （63.47 ± 0.54）%, （17.09 ± 0.37）%, （70. 66 ± 0. 56） %, （78.48 ± 0. 68 ） %, respectively. Compared with the normal group, control group, GPM middle and high - dose experimental groups increased, the differences were statistically significant （P 〈 0. 05 ）. The indicators of LC3 in that five groups were （ 35. 84 ± 0. 36 ） %, （ 82. 41 ± 0. 82 ） %, （ 60. 83±0. 61 ） %, （ 69. 66 ± 0. 70 ） %, （74. 61 ± 0. 75 ）%, respectively. The differences between each group and normal group were statistically significant （P 〈 0. 05 ）. The grayscale average ratio of Beclinl and 13 aetin in normal group, control group, GPM low, middle and high - dose experimental groups were （6. 87 ± 0. 68 ） %, （ 11.26 ± O. 87） %, （6. 46 ± 1.34 ） %, （ 11.58 ± 0. 95 ） %, （15.82 ± 1.58 ）%, respectively. Compared with the normal group, control group, GPM middle and high -dose experimental groups increased, the differences were statistically significant （ P 〈 0. 05 ）. The indicators of LC3 Ⅱ and β actin in that five groups were （26. 70 ± 0. 41 ） %, （ 103.17 ± 0. 88 ） %, （ 30.29 ± 0. 52 ） %, （ 36. 32 ± 0. 52 ） %, （64. 34±0. 48 ）% The differences between each group and normal group were statistically significant （P 〈 0. 05 ）. Conclusion GPM could have an effect on the autophagy in human hepatocellular carcinoma SMMC7721 ceils. The possible mechanism may be GPM induced autophagy of SMMC7721 cells, causing cell death ultimately.