【作者】 伍石华；

【导师】 罗招阳； 曹建国；

【作者基本信息】 南华大学 ， 病理学与病理生理学， 2007， 硕士

【摘要】 目的:研究8-硝基白杨素体外诱导人结肠癌细胞凋亡作用;探讨PPARγ活化介导8-硝基白杨素诱导人结肠癌细胞凋亡作用的分子机理。方法: MTT比色法和细胞计数法测定细胞增殖活性和存活率; PI染色FCM检测细胞凋亡率; AO/EB染色荧光显微镜观察凋亡细胞形态; DNA琼脂糖凝胶电泳观察细胞的基因DNA梯形条带图谱; Western Blot分析细胞PPARγ,NF-κB,Bcl-2和Bax的表达和活性。结果:MTT比色法测定显示:8-硝基白杨素抑制人结肠癌(HT-29)细胞增殖,呈浓度依赖性;其IC50值为1.78μM。细胞计数结果表明,8-硝基白杨素能显著抑制HT-29细胞生长。PI染色FCM分析、AO/EB染色荧光显微镜观察以及DNA琼脂糖凝胶电泳的结果证实:8-硝基白杨素具有诱导HT-29细胞凋亡作用。Western Blot分析发现8-硝基白杨素能显著上调HT-29细胞PPARγ和Bax蛋白表达,下调NF-κB及Bcl-2蛋白表达。PPARγ特异性阻断剂(GW9662)能部分拮抗8-硝基白杨素下调HT-29细胞Bcl-2蛋白表达作用。结论:1) 8-硝基白杨素具有抑制人结肠癌(HT-29)细胞增殖和生长作用。2) 8-硝基白杨素可诱导HT-29细胞凋亡作用。3) 8-硝基白杨素的体外抗人结肠癌作用与其活化PPARγ,抑制NF-κB活性,上调Bax蛋白表达和下调Bcl-2蛋白表达相关。更多还原

【Abstract】 Objective To investigate induction of apoptosis of human colon cancer HT-29 cells by 5,7-dihydrox-8-nitrochrysin in vitro,and its molecular mechanism.Methods The proliferative activity and survival rate of HT-29 cells treated with 5,7-dihydrox-8-nitrochrysin was measured using 3-(4,5-dimethylthiazol–2-yl)-2,5-dipheny tertrazolium blue (MTT) colorimetric assay and Cytometry; Cell apoptotic percentage were detected by PI staining flow cytometry; The fluorescence microscope using AO/EB fluorescence staining was used to observe the morphologic changes of apoptosis induced by 5,7-dihydrox-8-nitrochrysin in HT-29 cell line; DNA ladder bands were observed by DNA agarose gel electrophoresis. Effect of 5,7-dihydrox-8-nitrochrysin on PPARγ,NF-κB,Bcl-2,Bax protein expression level of HT-29 cells was detected by western blotting.Results MTT assay suggested that 5,7-dihydrox-8-nitrochrysin inhibited proliferation of HT-29 cells in a dose-dependent manner, and IC50 was 1.78μM. The cell counting data had shown that 5,7-dihydrox-8-nitrochrysin significantly inhibited the growth of HT-29 cells. FCM with PI staining, AO/EB fluorescence staining and DNA agarose gel electrophoresis indicated the induction of apoptosis on HT-29 cell lines treated with 5,7-dihydrox-8-nitrochrysin. Western blotting indicated that after 24 hours of action of 5,7-dihydrox-8-nitrochrysin, PPARγand Bax protein expression of HT-29 cells was increased and NF-κB and Bcl-2 was decreased, and the pre-incubation of GW9662, blocker of PPARγ, can partly antagonize the down-regulating effect of 5,7-dihydrox-8-nitrochrysin on Bcl-2 protein expression of HT-29 cells.Conclusion(1) 5,7-dihydrox-8-nitrochrysin can inhibit the proliferation and the growth of human colon cancer HT-29 cells(2) 5,7-dihydrox-8-nitrochrysin possess induction of apoptosis of HT-29 cells.(3) 5,7-dihydrox-8-nitrochrysin induces apoptosis of HT-29 cell line by activating PPARγ, inhibiting NF-κB ,upregulating expression of Bax protein and downregulating expression of Bcl-2 protein.更多还原