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目的:探讨蛞蝓提取物(Limax Extracts,LE)联合5-氟尿嘧啶(5-fluorouracil,5-Fu)对人肝癌细胞株HCCLM3的增殖抑制及凋亡诱导作用。方法:以人肝癌细胞株HCCLM3为研究对象,分为正常对照组、LE组、5-Fu组、LE+5-Fu联合组。采用四甲基偶氮噻唑蓝(MTT)比色法检测细胞活性,倒置显微镜下观察细胞凋亡形态学变化,异硫氰酸荧光素标记的膜联蛋白V/碘化丙啶(Annexin V-FITC/PI)双染流式细胞术定量检测细胞凋亡率,Western blot法检测凋亡蛋白Caspase-3的活化表达。结果:(1)LE和5-Fu单用或联用组对正常人肝细胞L-02未见明显抑制作用,但可抑制肝癌细胞HCCLM3的增殖并明显诱导其凋亡。(2)LE和联用组作用HCCLM3细胞早期凋亡率比5-Fu组高,而中晚期凋亡及坏死率却相反。(3)联用组在低浓度时具有很好的促凋亡作用,其促凋亡活力优于LE或5-Fu单用组,而且联用时可起到显著减毒增效作用。(4)LE和5-Fu单用或联用均可促进Caspase-3蛋白的活化,具有时间正效应,且LE组和联合组cleaved Caspase-3表达量高于5-Fu组。结论:LE和5-Fu联合或单用均能有效抑制HCCLM3细胞增殖,诱导其凋亡,其机制可能与激活Caspase-3表达有关。
Objective: To investigate the effects of Limax Extracts (LE) combined with 5-fluorouracil (5-Fu) on the proliferation and apoptosis induction of human hepatoma cell line HCCLM3. Methods: Human hepatocellular carcinoma cell line HCCLM3 was divided into normal control group, LE group, 5-Fu group and LE + 5-Fu group. Cell viability was measured by MTT assay. Morphological changes of apoptosis were observed under inverted microscope. Annexin V- FITC / PI) double staining flow cytometry quantitative detection of apoptosis rate, Western blot detection of apoptotic protein Caspase-3 activation expression. Results: (1) LE and 5-Fu alone or in combination group had no obvious inhibitory effect on normal human hepatocyte L-02, but inhibited the proliferation of hepatocellular carcinoma cell HCCLM3 and induced its apoptosis obviously. (2) Effect of LE and combination group The early apoptosis rate of HCCLM3 cells was higher than that of 5-Fu group, while the rate of apoptosis and necrosis was the opposite in the late stage. (3) The combination group had a good pro-apoptotic effect at low concentration, and its pro-apoptotic activity was better than that of LE or 5-Fu alone group, and synergistic effect could be achieved when used in combination. (4) LE and 5-Fu alone or in combination could promote the activation of Caspase-3 protein with time-positive effect, and the expression of cleaved Caspase-3 in LE group and combined group was higher than that in 5-Fu group. CONCLUSION: The combination of LE and 5-Fu alone or in combination can effectively inhibit the proliferation and induce apoptosis of HCCLM3 cells, which may be related to the activation of Caspase-3 expression.