黄芪总黄酮对人体正常细胞和肿瘤细胞辐射防护作用的差异性研究

Different roles of total flavonoids of astragalus on human normal mesenchymal stem cells and hepatoma cells in radiation protection

目的研究黄芪总黄酮（totalflavonoidsofastragalus，TFA）对60Coγ射线辐射损伤的人体正常骨髓间充质干细胞（humanmesenchymalstemcells，hMSCs）和肝癌细胞HepG-2辐射防护作用的差异性。方法MTT法检测TFA处理组与单纯照射组hMSCs和HepG-2的细胞活性；HepG-2细胞克隆形成实验检测细胞的辐射敏感性；流式细胞技术分析细胞凋亡率；Westernblot技术分析凋亡相关蛋白Fas，Bcl-2，Bax的表达。结果MTT检测结果显示，当给予6Gyγ射线一次性照射后，TFA预处理组hMSCs细胞活性分别比单纯照射组提高了1．15～1．95倍；经相同浓度TFA预处理的肝癌细胞HepG-2的细胞活性仅为单纯照射组的53％～23％；TFA的给药浓度与细胞存活率（survivalrate）之间显现出良好的量效关系。细胞克隆形成实验结果显示：TFA＋照射组能够明显抑制HepG-2细胞增殖，作用强于单纯TFA给药组和单纯照射组。流式细胞分析表明，经6Gyγ射线-次性照射6、24和48h后，TFA预处理组hMSCs的细胞凋亡率分别为23．3％，11．2％和2．9％，单纯照射组hMSCs的细胞凋亡率相应为29．3％，24．9％和13．6％；TFA预处理组肝癌细胞HepG-2的细胞凋亡率分别为11．6％，17．3％和20．1％，单纯照射组HepG-2的细胞凋亡率分别为6．9％、9．3％和15．8％。Westernblot分析显示，在肝癌细胞HepG-2中，TFA预处理照射组促凋亡蛋白Fas和Bax的表达量显著高于单纯照射组和对照组（t=-11．17～-2．8，-12．35～-3．4，P〈0．05）；凋亡抑制蛋白Bcl-2的表达量，TFA预处理照射组明显低于单纯照射组和对照组（t〈6．36～17．61，P〈0．05）。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凋亡促进作用；TFA对肝癌细胞的促凋亡作用，主要通过上调促凋亡蛋白Fas和Bax的表达与下调凋亡抑制蛋白Bcl-2的表达，从而大大增强了60Coγ射线对肝癌细胞的凋亡诱导作用。

Objective To investigate the different radioprotective effects of total flavonoids of Astragalus （TFA） on human normal mesenchymal stem cells（hMSCs） and hepatoma cells injured by 60Co γ-ray radiation. Methods hMSCs and HepG-2 cells were cultured and randomly divided into TFA-treated and untreated groups. The cells of different groups were irradiated with 60Co γ-rays at the dose of 6 Gy. MTT method was utilized to detect the survival rates of the hMSCs and HepG-2 cells pretreated or untreated with TFA before irradiation. Cell clone formation test was used to measure the cellular radiosensitivity. The apoptosis rates of different groups were determined by flow cytometer assay. The expression rates of the apoptosis-promoting proteins Fas and Bax and the apoptosis-inhibiting protein Bcl-2 were analyzed by Western blotting. Results MTT showed that the survival rates of hMSCs pretreated by TFA were 1. 15 - 1.95 times higher than that of the pure irradiation group. On the contrary, the survival rates of the TFA pretreated HepG-2 cells were only 0. 53 -0. 23 times that of the pure irradiation group. There was a gooddose-effect relationship between the cell survival rate and the TFA concentration. Cell clone formation rate indicated that combined treatment of TFA and radiation inhibited the cell proliferation more effectively than single TFA or pure radiation. Flow cytometry showed that 6, 24 and, 48 h post-irradiation to 6 Gy, the apoptosis rates of the hMSCs were 23.3% , 11.2% , and 2. 9% , respectively in the TFA pretreated group and were 29.3% , 24.9% , and 13.6% in the pure radiation group. However, the apoptosis rates of the HepG-2 cells at 6, 24, and 48 h post-irradiation to 6 Gy were 11.6% , 17.3% , and 20.1% , respectively in the TFA pretreated group and were 6.9% , 9.3% , and 15.8% , respectively in the direct radiation group. Western blotting showed that the expression levels of Fas and Bax proteins in the HepG-2 cells were significantly higher in the TFA pretreated group than in the pure radiation group. On the contrary, the expression level of the apoptosis inhibiting protein Bcl-2 was significantly lower in the TFA pretreated group than in the pure radiation group. Conclusions TFA has obvious effects of radiological protection on human hMSCs and has no effects of radiological protection but effects of apoptosis enhancement on hepatoma cells. The promotion of apoptosis of TFA on hepatoma cells is primarily through increasing the expression of apoptotic proteins such as Fas and Bax and reducing the expression of anti-apoptotic protein Bcl-2.