熊果酸诱导结肠癌HT-29细胞凋亡的实验研究

目的探讨熊果酸（UA）诱导结肠癌HT-29细胞凋亡的作用及机制。方法用不同浓度的UA处理HT-29细胞,采用四甲基偶氮唑蓝（MTT）比色法,检测UA对HT-29细胞的增殖抑制效应;采用形态学、TUNEL法和流式细胞术,检测细胞凋亡的发生;应用免疫组织化学S-P法,检测凋亡相关基因caspase-9和bcl-2的表达,并用病理图像分析软件进行半定量分析。结果UA在体外对HT-29细胞有中度增殖抑制效应,在UA作用下,HT-29细胞出现显著的细胞凋亡征象。TUNEL法显示细胞固缩,核染色质聚集或断裂,形成凋亡小体。流式细胞术检测结果显示,在G1期之前出现Sub—G1峰,凋亡率最高为11．63％,UA的作用具有浓度和时间依赖性。在HT-29细胞凋亡过程中,凋亡相关基因caspase-9的表达增强,bcl-2的表达减弱。结论凋亡是UA杀伤肿瘤细胞的机制之一;UA诱导结肠癌HT-29细胞凋亡主要与促进caspase-9的活化、下调bcl-2的表达有关。

Ursolic acid induces apoptosis in colon cancer HT-29 cells

Objective To study the antitumor effects of ursolic acid and mechanisms of its action. Methods The ceils of human colorectal carcinoma cell line HT-29 were treated with ursolic acid at different concentration. The proliferation inhibition was examined by MTF assay. Morphological examination, TUNEL method and flow cytometry were used to detect apoptosis. Immunohistochemical method was used to detect the expression of apoptosis related genes caspase-9 and bcl-2. The semi-quantification of protein expression was analyzed by pathological image-analysis. Results UA inhibited the proliferation of HT-29 cells moderately. Apoptosis of HT-29 cells was induced by ursolic acid treatment. The morphology of HT-29 showed changes such as nuclear chromatin condensation and fragmentation. Sub-G1 peak was found by flow cytometry. The maximal apoptosis rate was 11.63%. The expression of caspase-9 gene was enhanced. The expression of bcl-2 gene was decreased. All these effects were in a dose-dependent and time-depend manner. Conclusion Apoptosis in colon cancer HT-29 cells is one of the key mechanisms of ursolic acid action and its antitumor activiy may be applicable for the treatment of cancers.