【作者】 胥正敏；

【导师】 鄢佳程；

【作者基本信息】 川北医学院 ， 药理学， 2010， 硕士

【摘要】 目的：本实验以川产膜夹黄芪(Astragalus)为原料,以乙酸乙酯改良萃取技术分离提取黄芪总黄酮(Total Flavonoids of Astragalus TFA),主要研究TFA对放射损伤人正常骨髓间充质细胞(human mesenchymal stem cells hMSCs)和人肝癌细胞HepG-2的不同放射防护效应；从DNA和蛋白水平上深入探讨TFA对正常细胞和肿瘤细胞放射防护作用差异性的分子机理。方法：以肝癌细胞HepG-2和人体正常细胞hMSCs为实验对象,设置空白对照组(不照射,不给药),单纯照射组(只照射,不给药)TFA用药照射组(TFA处理+照射)三个实验组；当细胞生长至对数生长期时,给予照射剂量为6Gy的60Coγ射线一次性照射。MTT法检测不同浓度TFA处理组与未处理组hMSCs和HepG-2经60Coγ射线照射后细胞的存活率；流式细胞技术分析照射后6h、24h、48h各实验组细胞的凋亡率；琼脂糖电泳技术分析照射后24h和72h各实验组细胞DNALadder的形成；Western blot方法分析照射后24h和72h各实验组HepG-2细胞凋亡相关蛋白Fas, Bcl-2, Bax的表达。结果：MTT检测结果显示在照射后24h,经0.05mg/ml、0.10mg/ml、0.15mg/ml、0.20mg/ml不同浓度TFA预处理的hMSCs,其细胞存活率分别是单纯照射组的1.15、1.38、1.80、1.95倍；与此相反,经相同浓度TFA预处理的肝癌细胞HepG-2,其细胞存活率分别只有单纯照射组的0.53、0.51、0.41、0.23倍；说明TFA对人体正常细胞具有良好的放射防护作用,对肿瘤细胞具有显著的放射增敏效应,并呈现出良好的剂量依赖性。琼脂糖电泳结果显示,TFA对正常细胞和肿瘤细胞核DNALadder形成具有不同的影响。TFA用药照射组hMSCs核DNA形成的DNA Ladder明显低于单纯照射组；而TFA用药照射组HepG-2核DNA形成的DNA Ladder明显高于单纯照射组。流式细胞分析显示,经60Coγ射线照射6h、24h、48h后,单纯照射组hMSCs的凋亡率分别为29.3%、24.9%、13.6%,TFA用药照射组hMSCs的凋亡率分别为23.3%、11.2%、2.9%；单纯照射组HepG-2的凋亡率分别为6.9%、9.3%、15.8%；TFA用药照射组HepG-2的凋亡率分别为11.6%、17.3%、20.1%。结果提示,TFA预处理能够降低60Coγ射线对正常细胞的凋亡诱导,促进60Coγ射线对肿瘤细胞的凋亡诱导,说明TFA对细胞凋亡具有双重调节作用。Western blot结果显示,在肝癌细胞HepG-2中,单纯照射组Fas蛋白表达较弱与空白对照组Fas表达量相似；TFA用药照射组Fas表达量明显高于单纯照射组和空白对照组。各组细胞Bax的表达与Fas表达基本一致,Bcl-2的表达刚好与Bax和Fas的表达相反。结论：TFA对人正常骨髓间充质细胞具有明显的放射防护作用,对肝癌细胞不仅没有放射防护作用反而具有促凋亡作用,TFA通过调控凋亡相关蛋白表达从而增强γ射线对肝癌细胞的杀伤作用。更多还原

【Abstract】 Objective:In this study, we use Astragalus Membranaceus from Sichuan as raw material and make use of the improved ethyl acetate extraction technology to separate the total Flavonoids of Astragalus(TFA) from Astragalus. Then, we focus our research priorities on the different radioprotective effects of TFA on normal human mesenchymal stem cells (hMSCs) and hepatoma cells injured by irradiation and explore the molecule mechanism of the different radioprotective effects of TFA on normal cells and tumor cells in DNA and protein level.Methods:The HepG-2 hepatocellular carcinoma cells and hMSCs were used as experimental subject. The experiment was set up three experimental groups:blank control group (no irradiation, no TFA), direct irradiation group (radiation only, no TFA), TFA treated irradiation group (TFA treatment+radiation). The experiment cells were irradiated by 60Coγ-ray with a irradiation dose of 6Gy one time when it reached logarithmic growth phase. MTT assay was used to detect the survival rates of hMSCs and HepG-2 cells which were pretreated with different concentrations of TFA before irradiation, and flow cytometer was used to detect the cell apoptosis rates of all the groups in 6h、24h、48h time points. Agarose gel electrophoresis was utilized to examine the DNA ladders of HepG-2 and hMSCs cells which were irradiated by 60Co y-ray before 24 hour or 72 hour. Western blot technique was applied to detect the expression of apoptotic protein on HepG-2 cells such as Fas, Bcl-2 and Bax in all the experiment groups.Results:The MTT results showed that the hMSCs survival rates of TFA treated irradiation group were respectively as 1.15,1.38,1.80 and 1.95 times as the direct irradiation group when the treated concentration of TFA were 0.05mg/ml, 0.10mg/ml,0.15mg/ml and 0.20mg/ml. Inversely, under the same pretreatment, the HepG-2 survival rates of TFA treated irradiation group were respectively as 0.53,0.51,0.41 and 0.23 folds as the direct irradiation group. It indicated that TFA had a strong radioprotection on human normal cells and a significant radiosensitization on tumor cells as well as an evident dose dependent. Agarose gel electrophoresis showed that TFA had different effects on the DNA Ladder of normal cells and tumor cells. Compared with the direct radiation group, the apoptotic DNA ladders of hepatoma cells in TFA treated irradiation group were apparently higher, on the other hand the apoptotic DNA ladders of hMSCs in TFA treated irradiation group were obviously lower. Flow cytometry showed that after being irradiated 6h、24h、48h by 60Coγray the apoptosis rates of hMSCs in the direct radiation group were 29.3%、24.9%、13.6% and the apoptosis rates of hMSCs in the TFA treated irradiation group were 23.3%、11.2%、2.9%; however, the apoptosis rates of HepG-2 in the direct radiation group were 6.9%、9.3%、15.8% and the apoptosis rates of HepG-2 in TFA treated irradiation group were 11.6%、17.3%、20.1%. These results indicate that TFA can decrease the apoptosis in normal cells and promote the apoptosis in tumor cells induced by 60Coγray. It indicated that TFA had a dual role on the regulation of the apoptosis of the cells. The results of Western blot demonstrated that the expression of apoptotic protein Fas on hepatoma cells both in blank control group and in direct irradiation group were poor and similar. However, the expression of Fas in TFA treated irradiation group was higher than that of the direct radiation group and the blank control group significantly. The expressions of Bax in all the groups were the same as Fas. As for the expression of Bcl-2, it was just the opposite expression of Bax and Fas.Conclusion:TFA has obvious effects of radiological protection on human hMSCs, and has not effects of radiological protection and also has effects of apoptosis enhancement on hepatoma cells. TFA can strengthen the lethal effect of 60Coγray on hepatoma carcinoma cell through the expression regulation of apoptosis associated protein.更多还原