【作者】 钟秀宏；

【导师】 孙东植；

【作者基本信息】 延边大学 ， 病理与病理生理学， 2006， 硕士

【摘要】 目的：在体外实验中，观察桦褐孔菌提取物对人胃癌BGC-823细胞株的抗增殖和诱导凋亡作用及凋亡相关基因表达的影响，为其成为防治肿瘤的菌类药物提供实验依据。 方法：选用人胃癌BGC-823细胞株进行体外培养，用MTT比色法测定不同方法提取的桦褐孔菌提取物对胃癌BGC-823细胞株的抗增殖作用。应用倒置显微镜、HE染色光学显微镜、吖啶橙-溴化乙锭(AO／EB)双染色荧光显微镜及透射电镜技术观察细胞凋亡形态学变化。通过脱氧核甘酸末端转移酶介导的缺口末端标记法(TUNEL)和AO／EB双染色法检测细胞凋亡率。应用流式细胞术进行定量并作细胞周期分析。采用免疫细胞化学染色法检测药物处理前后Ki-67抗原及凋亡相关基因产物bax，bcl-2，caspase-3，caspase-9蛋白的表达。 结果：1)MTT实验结果表明：桦褐孔菌的水提取物、乙酸乙酯提取物、石油醚提取物在10μg／ml～80μg／ml范围内，于12h、24h、48h、72h对胃癌BGC-823细胞株均有抑制生长作用，并表现出浓度、时间依赖性关系，上述提取物在终浓度80μg／ml、于48h时，体外最大抑制率分别达到51％、62％、60％，桦褐孔菌的石油醚提取物、乙酸乙酯提取物对人胃癌BGC-823细胞株生长抑制作用高于水提取物。2)HE染色光镜下，实验组BGC-823肿瘤细胞体积变小、变圆，核染色质浓缩或染色质块形成，有的细胞膜起泡形成凋亡小体；倒置显微镜下，实验组BGC-823肿瘤细胞体积变小、变圆，核染色质凝集，细胞间连接疏松，贴壁能力减弱；AO／EB双染色荧光显微镜下，实验组BGC-823肿瘤细胞胞核染色质呈橘红色，胞质内的橘红色或橘黄色荧光消失，细胞周围可见亮绿色荧光的凋亡小体；透射电镜观察，80μg／ml桦褐孔菌水提取物处理后的凋亡肿瘤细胞核染色质固缩、边集，胞质内有空泡出现，还可见凋亡小体凸出细胞表面。3)AO／EB双染色细胞凋亡半定量方法结果，80μg／ml桦褐孔菌水提取物作用BGC-823胃癌细胞12h、24h、48h、72h，凋亡率分别为3.5％、10％、25％、15％，不同时间段相比有统计学差异(p＜0.01或更多还原

【Abstract】 OBJECTIVE To observe the effects of anti-proliferation and inducing apoptosis on BGC-823 human gastric cancer cell line induced by extracts from Inonotus obliquus and the protein expressions of apoptosis-related gene in vitro, and provide the experimental evidence for Inonotus obliquus extracts as a new anti-tumor fungus drug.METHODS BGC-823 human gastric cancer cell line was cultured in vitro. MTT assay was used to determine the effect of extracts from Inonotus obliquus extracted by different methods on BGC-823 human gastric cancer cells growth inhibitory. Morphological changes of the apoptosis of BGC-823 gastric cells were observed by using inverted, light and fluorescent microscope, and the ultrastructure of BGC-823 cells was observed by using transmission electronic microscope. Apoptotic rate was calculated with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and apoptotic half-quantitative method through AO/EB double fluorescent staining. Flow cytometry was used to detect cell cycle distribution of BGC-823 cells and apoptotic rate. Furthermore, the protein expressions of Ki-67 and the apoptosis-related gene such as bax, bcl-2, caspase-3 and caspase-9 were detected by using immunocytochemistry before and after exposure to aqueous extract from Inonotus obliquus.RESULTS 1) The MTT test showed that BGC-823 gastric cells exposure to aqueous extract from Inonotus obliquus, petroleum ether extract from Inonotus obliquus and acetic ether extract from Inonotus obliquus at final concentrations of 10~80μg/ml were inhibited for 12h, 24h, 48h and 72h in dose and time dependent manner. The MTT test showed the inhibitory rate was 51%, 60% and 62%, respectively at 80μg/ml final concentration for 48h. The effect of petroleum ether and acetic ether extracts from Inonotus obliquus inhibiting growth of human BGC-823 gastric carcinoma cell line in vitro was more marked than that of aqueous extract from Inonotus obliquus. 2) The BGC-823 human gastric cancer cells in experimental group by HE staining更多还原