CAJ | 학술논문

目的研究黄芪总黄酮(total flayonoids of astragalus,TFA)对60°Co γ射线辐射损伤的人体正常骨髓间充质干细胞(human mesenchymal stem cells,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;Western blot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6 Gy γ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(survival rate)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6 Gy γ射线一次性照射6、24和48 h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%.Western blot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Bel-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(f<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60 Co γ射线对肝癌细胞的凋亡诱导作用.
목적 연구황기총황동(total flayonoids of astragalus,TFA)대60°Co γ사선복사손상적인체정상골수간충질간세포(human mesenchymal stem cells,hMSCs)화간암세포HepG-2복사방호작용적차이성.방법 MTT법검측TFA처리조여단순조사조hMSCs화HepG-2적세포활성;HepG-2세포극륭형성실험검측세포적복사민감성;류식세포기술분석세포조망솔;Western blot기술분석조망상관단백Fas,Bcl-2,Bax적표체.결과 MTT검측결과현시,당급여6 Gy γ사선일차성조사후,TFA예처리조hMSCs세포활성분별비단순조사조제고료1.15～1.95배;경상동농도TFA예처리적간암세포HepG-2적세포활성부위단순조사조적53%～23%;TFA적급약농도여세포존활솔(survival rate)지간현현출량호적량효관계.세포극륭형성실험결과현시:TFA+조사조능구명현억제HepG-2세포증식,작용강우단순TFA급약조화단순조사조.류식세포분석표명,경6 Gy γ사선일차성조사6、24화48 h후,TFA예처리조hMSCs적세포조망솔분별위23.3%,11.2%화2.9%.단순조사조hMSCs적세포조망솔상응위29.3%,24.9%화13.6%;TFA예처리조간암세포HepG-2적세포조망솔분별위11.6%,17.3%화20.1%,단순조사조HepG-2적세포조망솔분별위6.9%、9.3%화15.8%.Western blot분석현시,재간암세포HepG-2중,TFA예처리조사조촉조망단백Fas화Bax 적표체량현저고우단순조사조화대조조(t=11.17～-2.8,-12.35～3.4,P＜0.05);조망억제단백Bel-2적표체량,TFA예처리조사조명현저우단순조사조화대조조(f<6.36～17.61.P<0.05).결론 TFA대인정상골수간충질세포구유명현적방사방호작용,대간암세포불부몰유방사방호작용반이구유조망촉진작용;TFA대간암세포적촉조망작용,주요통과상조촉조망단백Fas화Bax적표체여하조조망억제단백Bcl-2적표체,종이대대증강료60 Co γ사선대간암세포적조망유도작용.
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 60 Coγ-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 60 Co 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 good dose-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.