CAJ | 학술논문

目的探讨氮氧自由基R-1(简称R-1)对人肝细胞L-02的辐射防护作用.方法在L-02细胞培养液中,加入终浓度为0、0.125、0.25、0.5、1、2、4、8、16、32μmol/L的R-1,作用24、48和72 h.用MTT法测定R-1的毒性作用,以筛选合适的R-1浓度.后续实验选择0.125、0.25、0.5、1 μmol/L 4个浓度组检测其防护作用.设终浓度4 mmol/L的细胞保护剂WR2721为阳性对照组.采用60Coγ射线照射,吸收剂量为0、1、2、4、8 Gy,照射72 h后,进行MTT比色法.L-02细胞分为2组:照射前30 min加药组和照射后立即加药组.终浓度均为0.25 μmol/L,吸收剂量为4 Gy,照射后72 h进行MTT比色实验.照射后10 d,用克隆形成实验检测不同浓度的R-1对L-02细胞活力的影响.选用防护效果最佳的0.25μmol/L浓度对细胞进行预处理,分别在4 Gy照射后的24、48和72 h,用倒置显微镜观察细胞形态的变化,Hoechst 33258荧光染色法和流式细胞仪检测细胞凋亡.结果当R-1浓度低于1 μmoL/L时,与0 μmol/L组相比,L-02细胞各时间点的吸光度(A)值无明显变化;而浓度高于2 μmol/L时,与0 μmol/L组相比,其A值随浓度的增高而下降.选用0、0.125、0.25、0.5、1μmol/L的浓度组.与照射组相比,R-1各浓度组的A值和克隆形成率明显提高,其中0.25μmol/L组的作用最明显.与照射组相比,0.25 μmol/L预处理组的L-02细胞贴壁好,折光性强,轮廓清晰,凋亡细胞和死亡细胞明显较少.结论 R-1能有效地防护60Coγ射线对L-02细胞的辐射损伤,其防护作用可能与减少细胞凋亡有关.
목적 탐토담양자유기R-1(간칭R-1)대인간세포L-02적복사방호작용.방법 재L-02세포배양액중,가입종농도위0、0.125、0.25、0.5、1、2、4、8、16、32μmol/L적R-1,작용24、48화72 h.용MTT법측정R-1적독성작용,이사선합괄적R-1농도.후속실험선택0.125、0.25、0.5、1 μmol/L 4개농도조검측기방호작용.설종농도4 mmol/L적세포보호제WR2721위양성대조조.채용60Coγ사선조사,흡수제량위0、1、2、4、8 Gy,조사72 h후,진행MTT비색법.L-02세포분위2조:조사전30 min가약조화조사후립즉가약조.종농도균위0.25 μmol/L,흡수제량위4 Gy,조사후72 h진행MTT비색실험.조사후10 d,용극륭형성실험검측불동농도적R-1대L-02세포활력적영향.선용방호효과최가적0.25μmol/L농도대세포진행예처리,분별재4 Gy조사후적24、48화72 h,용도치현미경관찰세포형태적변화,Hoechst 33258형광염색법화류식세포의검측세포조망.결과 당R-1농도저우1 μmoL/L시,여0 μmol/L조상비,L-02세포각시간점적흡광도(A)치무명현변화;이농도고우2 μmol/L시,여0 μmol/L조상비,기A치수농도적증고이하강.선용0、0.125、0.25、0.5、1μmol/L적농도조.여조사조상비,R-1각농도조적A치화극륭형성솔명현제고,기중0.25μmol/L조적작용최명현.여조사조상비,0.25 μmol/L예처리조적L-02세포첩벽호,절광성강,륜곽청석,조망세포화사망세포명현교소.결론 R-1능유효지방호60Coγ사선대L-02세포적복사손상,기방호작용가능여감소세포조망유관.
Objcetive To investigate the protective effects of the nitroxides R-1 on human liver cells exposed to ionizing radiation.Methods Human liver cells L-02 were cultured and irradiated with 60Co γ-rays at the doses of 0,1,2,4,and 8 Gy,in order to screen the proper irradiation dose.WR2721 at the terminal concentration of 4 mmol/L was used as positive control.L-02 cells irradiated with 4 Gy were added with R-1 at the terminal concentration of 0.25 μmol/L at 30 min before irradiation or immediately after irradiation.MIT method was used to screen the proper conditions for follow-up experiment 72 h later.L-02 cell culture fluid was added with R-1 at the concentrations of 0,0.125,0.25,0.5,and 1 μmol/L,respectively for 30 min before irradiation at the doses of 0,1,2,4,and 8 Gy to ealculate clone formation rate at 10 d post-irradiation.L-02 cells were cultured and divided into 4 groups:control group without any treatment.drug group pretreated by 0.25 μmol/L R-1 only,irradiation group,irradiated at 4 Gy only,and drug + irradiation group with combination of 0.25 μmol/L R-01 and 4 Gy irradiation.The inverted microscopy and Hoechst 33258 staining and flow eytometry were used to observe the apoptosis of the cells at 24,48,and 72 h later.Results Nitroxides R-1 did not inhibit the viability of L-02 cell when its concentration was less than 1 μmol/L and it inhibited the L-02 cell growth when the concentration wu higher than 2 μmoL/L.The A value and colony formation rate of different concentration of R-1 groups were all higher than those of the irradiation group,and the effect of the 0.25 μmol/L drug concentration group was the most significant.Consequently,the concentration 0.25 μmoL/L was selected for follow-up experiment.Compared with the irradiation group,the L-02 cells of the pretreatment group showed solid adherence, increased refraction,clear outline,less apoptotic and dead cells at 4 Gy post-irradiation.Conclusions Nitroxides R-1 can protect the human liver cells from 60Coγ-ray induced injury effectively.The mechanism of its protective effect may be the reduction of apoptosis.