氮氧自由基R-1对人肝细胞L-02的辐射防护作用

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.

Abstract：

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.     

Bcl-2基因特异性小干涉RNA增强食管癌细胞辐射敏感性的实验研究

目的 探讨Bcl-2基因特异性的小干涉RNA (siRNA)对食管癌细胞的辐射增敏作用。方法 构建表达Bcl-2基因特异性siRNA的真核表达载体,并借助脂质体将其转入食管癌细胞EC109。Western blot检测Bcl-2蛋白在食管癌细胞的表达,流式细胞仪检测食管癌细胞的凋亡,克隆形成实验和裸鼠移植瘤生长抑制实验检测Bcl-2基因特异性siRNA真核表达载体联合X射线照射对食管癌的生长抑制作用。结果 Bcl-2基因特异性siRNA真核表达载体可抑制Bcl-2蛋白在EC109细胞中的表达,诱导EC109细胞凋亡,增强X射线照射对EC109细胞克隆形成的抑制能力,增强X射线照射对EC109裸鼠移植瘤生长的抑制作用。结论 Bcl-2基因特异性siRNA真核表达载体可显著增强食管癌细胞对X射线照射的敏感性,具有良好的临床应用前景。     

参与式教学法在防原医学教学中的应用及体会

作为传统教学法的一种重要补充，参与式教学是适应当今社会形式的一种新的教育理念，它没有固定的形式，给予了教育工作者极大的发挥空间。将参与式教学法与传统教学法有机结合，取长补短，是提高防原医学教学质量的有效途径，必将成为防原医学教学改革的方向之一。     

电磁脉冲暴露对雄性BALB/c小鼠子代性别比的影响

目的 探讨雄性BALB/c小鼠电磁脉冲（electromagnetic pulse,EMP）暴露对子代性别比的影响。方法 BALB/c小鼠雄性33只,雌性66只。雄性小鼠按照体重随机分为10 000次、100 000次脉冲辐照组和假辐照组。雌性小鼠相同环境分隔饲养,用以合笼。辐照组给予场强35 kV/m,重复频率50 Hz,脉冲数分别为10 000次和100 000次的辐照,每日1次,连续辐照2周。辐照后按雌雄比2：1合笼1周,1周后处死雄鼠,对比分析3组雄鼠的体重和睾丸重量,计算体睾指数;游离附睾中精子进行计数。孕鼠于孕20 d解剖取胎鼠组织进行PCR,鉴定子代性别比。结果与假辐照组相比,10 000次辐照组和100 000次辐照组雄鼠的体重、睾丸重量和体睾指数差异不大;精子计数均有下降,但差异无统计学意义。10 000次辐照组子代性别比（雄性：雌性）为0.298明显低于假辐照组0.871（χ²=9.37,P<0.01）;100 000次辐照组为0.826,与假辐照组相比差异无统计学意义。结论 雄性小鼠在场强35 kV/m,重复频率50 Hz,脉冲次数10 000次的EMP中暴露,可能导致子代雄性与雌性性别比降低。     

紫杉醇纳米粒对乏氧MCF-7细胞的辐射增敏作用

目的 制备出具有生物活性并可控制释放的紫杉醇纳米粒,探讨其对乏氧人乳腺癌细胞(MCF-7)的辐射增敏作用。方法 采用单乳溶剂挥发法制备聚乳酸-聚羟基乙酸共聚物［Poly (D,L- lactide-co-glycolide),PLGA］包裹的紫杉醇纳米粒,高效液相色谱分析纳米粒的载药率、包封率和模拟体外释药,扫描电镜研究纳米粒的形态。经乏氧处理的MCF-7细胞与紫杉醇纳米粒共培养,通过细胞形态和细胞周期变化来分析从纳米粒中释放出来的紫杉醇的生物活性,采用平板克隆形成实验检验其辐射增敏作用。结果 成功制备出的紫杉醇纳米粒呈光滑球形,粒径分布为200～800 nm,载药率为4.5%,包封率为85.5%,模拟体外释药曲线呈双相,即在暴发释放之后为缓慢释放。同紫杉醇纳米粒共培养的乏氧MCF-7细胞形态发生改变,极性增加,呈梭形,并且G₂期细胞比例升高,克隆形成能力明显降低。结论 本研究证实具有生物活性的紫杉醇从纳米粒中以可控的方式被释放,有效地增加了乏氧MCF-7细胞的辐射敏感性,为辐射增敏剂的临床应用提供了一种新的给药方式。     

氮氧自由基R-1对人肝细胞L-02的辐射防护作用

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.     

HIF依赖性表达的自杀基因抑制肾细胞癌生长的实验研究

目的探讨依赖乏氧诱导因子(hypoxiainduciblefactor.HIF)依赖性表达的自杀基因治疗系统对肾细胞癌的治疗效果。方法借助DNA重组技术构建HIF依赖性表达的重组腺病毒载体Ad-5HRE/hCMVmp-BCD。用Westernblot检测细菌胞苷脱氨酶(bacterialcytosinedeaminase,BCD)的表达,细胞生长抑制试验检测人肾细胞癌786-0细胞对5-氟胞嘧啶(5-fluorocytosine,5-FC)的敏感性,裸鼠移植瘤试验观察Ad-5HRE/hCMVmp-BCD/5-FC对786-0细胞移植瘤的抑制效应。结果786-0细胞感染Ad-5HRE/hCM-Vmp-BCD后,可诱导BCD蛋白的表达,并显著提高细胞对5-FC的敏感性。裸鼠移植瘤试验结果显示,Ad-5HRE/hCMVmp-BCD/5-FC可抑制肾细胞癌移植瘤的生长。结论HIF依赖性表达的Ad-5HRE/hCM-Vmp-BCD/5-FC自杀基因系统可显著抑制肾细胞癌生长,具有良好的临床应用前景。     

电磁脉冲对小鼠脾细胞Fas和FasL mRNA表达的影响

目的　观察电磁脉冲对小鼠脾细胞 Fas和 Fas Lm RNA表达的影响 .方法　 40只体质量 (18± 2 ) g的健康雄性 BAL B/c小鼠随机分为未照射组 (8只 )和全身照射组 (32只 ) .小鼠连续 2 d接受每日 2 5次脉冲的电磁脉冲全身照射 ,每次照射间隔约 2 min.于照射后 2 ,4,8和 12 h分别取 2只未照射小鼠和 8只照射小鼠的脾脏 .采用 RNA打点杂交法检测小鼠脾细胞 Fas和 Fas L RNA的表达水平 ,并对杂交信号进行灰度扫描后计算 RNA表达指数 .结果　受电磁脉冲照射后 2 h,小鼠脾细胞 Fas和 Fas L 的 RNA表达指数即分别从未照射的 0 .5 9± 0 .2 6和 0 .5 6± 0 .2 3升高到 0 .97± 0 .2 5和 0 .96± 0 .2 1,4h又下降至 0 .75± 0 .2 4和 0 .6 9± 0 .2 2 ,8h再度上升达最高峰 1.2 2± 0 .2 2和 1.32± 0 .5 0 (与对照组相比P<0 .0 0 1) ,于 12 h时下降至接近正常水平 .结论　电磁脉冲辐照可引起小鼠脾细胞 Fas和 Fas L RNA表达水平波动性升高 .     

乏氧靶向性自杀基因治疗系统增强胰腺癌放疗效果的实验研究

目的 探讨乏氧靶向性自杀基因治疗系统对胰腺癌放射治疗的增强效应。方法 借助DNA重组技术构建乏氧依赖性表达的重组腺病毒载体Ad-5HRE/hCMVmp-BCD。用Westernblot检测细菌胞苷脱氨酶(BCD)的表达,细胞生长抑制实验检测人胰腺癌MIA-PACA2细胞对5-氟胞嘧啶(5-FC)的敏感性,裸鼠移植瘤实验观察Ad-5HRE/hCMVmp-BCD/5-FC单独或联合放射治疗对MIA-PACA2细胞移植瘤的杀伤效应。结果 MIA-PACA2细胞感染Ad-5HRE/hCMVmp-BCD后,乏氧处理可诱导BCD蛋白的表达,并显著提高细胞对5-FC的敏感性。裸鼠移植瘤实验结果显示,Ad-5HRE/hCMVmp-BCD/5-FC与放射治疗均可抑制胰腺癌移植瘤的生长,但两者联合可显著增强对移植瘤的抑制效应。结论 乏氧靶向性的Ad-5HRE/hCMVmp-BCD/5-FC自杀基因系统可显著增强胰腺癌细胞的放疗效果,具有良好的临床应用前景。