藤梨根提取物对宫颈癌的抑制作用

Inhibition of Actinidia Arguta Extract on Cervical Cancer

目的:通过一系列分子生物学方法研究藤梨根提取物对人宫颈癌Hela细胞的抑制,探索其发生抗肿瘤细胞作用可能的原理。方法:采用体外细胞培养方法,以不同浓度(1、10、100μg/mL)的藤梨提取物作用于HeLa细胞12、24 h,荧光显微镜观察细胞凋亡;荧光染色观察细胞核内变化;应用MTT法检测其对宫颈癌Hela细胞的抑制效果;流式术检测宫颈癌HeLa细胞的周期分布和凋亡率情况;琼脂糖凝胶电泳法观察HeLa细胞的DNA Aladder形成情况;免疫组化SP法观察Bcl-2、Bax、Caspase-3的表达情况;Real time-PCR法观察宫颈癌HeLa细胞内的COX-2和PCNA mRNA含量变化。结果:低、中、高剂量组作用于HeLa细胞12、24 h后,可见明显的细胞凋亡现象,且凋亡现象明显高于对照组,其中24 h藤梨根(100μg/mL)组凋亡率最高,达38.8%;细胞的增殖逐渐减少,细胞的生长周期主要集中在G0/G1期;DNA Aladder的合成受阻,高剂量组凋亡条带明显可见;较对照组相比,Bcl-2、Cox-2、Pcna基因表达明显减少,Bax及Caspase-3 mRNA表达显著增加,高剂量组(100μg/mL)尤为明显(均P<0.05)。结论:藤梨根提取物抑制宫颈癌HeLa细胞的生长效果良好,与HeLa细胞增殖减少和细胞凋亡的促进有关,而降低Bcl-2、Cox-2、Pcna基因表达、增强Bax基因表达,活化Caspase-3 mRNA也可能是其促进细胞凋亡机制之一。

Objective: In this experiment, a series of molecular biological methods were used to study the inhibition of Actinidia argutaextract on HeLa cellsand explore the possible principle of anti-tumor cell action. Methods: In vitro cell culture method was used to treat HeLa cells in different concentrations(1, 10, 100 μg/mL) for 12 h and 24 h. The apoptosis was watched by fluorescence microscope. The intracellular changes were observed by fluorescence staining. The inhibitory effect of HeLa cells on cervical cancer was detected by MTT assay. The cycle distribution and apoptosis rate of HeLa cells were detected by flow cytometry. The contents of DNA Aladder in HeLa cells were watched by agarose gel electrophoresis. The immunohistochemical SP method was used. The expressions of Bcl-2, Bax and Caspase-3 were observed. The changes of COX-2 and PCNA mRNA in HeLa cells of cervical cancer were observed by Real time-PCR. Results: The low, medium and high dose groups of HeLa cells showed obvious apoptosis after 12 h and 24 h, and the apoptosis phenomenon was significantly higher than that of the control group. The 24 h group(100 μg/mL) had the highest apoptosis rate(38.8%). The cell proliferation gradually decreased and the cell growth cycle was mainly concentrated in G0/G1 phase. DNA Aladder synthesis was blocked. The high-dose group’s was clearly visible. Compared with the control group, the expressions of Bcl-2, Cox-2 and Pcna were significantly decreased, the expressions of Bax and Caspase-3 mRNA were significantly increasedand those of the high dose group(100 μg/mL) was particularly obvious(P<0.05).Conclusion: Actinidia arguta extract inhibited the growth of cervical cancer HeLa cells, which was associated with decreasing proliferation and apoptosis of HeLa cells. Reducing expressions ofBcl-2, Cox-2 and Pcna, enhancing Bax gene expression, and activating Caspase-3 mRNA may also be one of its mechanisms to promote apoptosis.