两种藤梨根制剂促凋亡作用的实验研究

Experimental research on apoptosis-promoting effect of two kinds Actinidiae preparations

目的研究两种藤梨根制剂促进人食管癌Eca-109细胞凋亡的机制及差异。方法采用MTT比色法检测藤梨根正丁醇药液及乙酸乙酯药液在不同浓度(1μg/mL、10μg/mL、100μg/mL等)及不同时间(24h、48h、72h等)对Eca-109细胞生长的抑制作用;采用TUNEL法检测其对癌细胞生长的诱导凋亡效应;采用免疫组化SP法检测凋亡相关蛋白Bax、Bcl-2及caspase的表达。结果藤梨根正丁醇药液对人食管癌Eca-109细胞的生长抑制作用,随着药物浓度的升高和作用时间的延长而增强,其生长抑制率可达87.2%。两种藤梨根制剂对瘤细胞有明显的凋亡效应,而在对照组未见有明显凋亡现象。瘤细胞作用24h、48h、72h后分别与对照组比较,用药组Bax蛋白表达明显增强(P<0.01);同时伴有Bcl-2表达减弱,72h后最明显,差异有显著意义(P<0.01)。藤梨根正丁醇药液促进caspase-3、caspase-9表达明显增强,而对caspase-8表达影响较小。藤梨根乙酸乙酯药液作用于肿瘤细胞时也可观察到类似的效应,且正丁醇药液对肿瘤细胞生长的抑制率高于乙酸乙酯药液。结论藤梨根可通过下调Bcl-2表达,激活caspase-9、caspase-3诱导Eca-109细胞凋亡。

Objective To study the mechanism and difference of two kinds Actinidia preparations on the apoptosis induction in human carcinoma of esophagus Eca-109 cells. Methods MTT colonmetric assay was used to examine the growth inhibition of concentration-effect （1 μg/mL, 10 /μg/mL, 100 μg/mL ere） and time-effect （24 h,48 h, 72 h etc） of extracts from Actinidia arguta by n-butyl alcohol and ethyl acetate on Eca-109 cells. TUNEL test were performed to observe the apoptosis induced by the extracts and the level of expression of Pax, BcP2 and caspase in tumor cells were measured by immunohistochemical method （SP technique）. Results The inhibitory effect of the extracts from Actinidia arguta by n-butyl alcohol on Eca-109 cells increased step by step to concentration and time, the highest rate of inhibition was 87.2%. The extract can significantly induced apoptosis of Eca-109 cells, but in control group, was not observed apoptosis. Compared with control group, the level of expression Bax in tumor cells increased（P〈0.01）, but the level of ex- pression of Bcl-2 decreased after 24 h, 48 h and 72 h（P〈0.01）, particularly after 72 h. The expressions of caspase-3 and easpase-9 were significantly enhanced by Actinidia arguta, but Little effect on caspase-8. The similar effect was be observed while Actinidia arguta by ethyl acetate on Eca-109 cells, and the tumor cell growth inhibition rate of Aetinidia arguta by n-butyl alcohol was higher than Actinidia arguta by ethyl acetate. Conclusion Actinidia arguta may induces apoptotic Eca-109 ceils death through suppressing bcl-2 and then activating caspase-9 and caspase-3.