取代2H-苯并吡喃-3-甲酰苯胺化合物的合成及抗辐射活性评价

目的 发现新结构的活性分子,为抗辐射药物研究提供候选化合物。方法 使用构象固定和官能团转换策略对Ex-RAD进行结构改造,设计并合成取代2H-苯并吡喃-3-甲酰苯胺化合物。通过照射细胞存活模型进行抗辐射活性筛选,并考察活性化合物的细胞毒性。优选活性化合物进行Western印迹实验,考察对照射细胞凋亡相关蛋白表达的影响,并通过照射小鼠模型评价其抗辐射活性。结果 合成目标化合物21个,通过筛选发现8个化合物可以显著提高照射细胞的存活,选择4个化合物进行复筛和细胞毒性评价。优选化合物D19进行Western印迹实验,发现D19可以影响照射AHH-1细胞凋亡相关蛋白的表达。与辐射组相比,D19可以使全身8.6 Gy照射小鼠30 d存活率提高70%。D19对非致死剂量照射小鼠的血液系统具有保护作用。结论 新结构的化合物D19具有明确的抗辐射活性,值得进一步深入研究。     

西妥昔单抗对人肝癌细胞HepG2的体外效应

目的探讨西妥昔单抗（爱必妥,cetuximab,Erbitux）对人肝癌细胞HepG2的体外效应。方法流式细胞术检测HepG2细胞表面表皮生长因子受体（EGFR）的表达。细胞划痕及Transwell实验检测西妥昔单抗对HepG2细胞迁移能力的影响。Western印迹检测西妥昔单抗对HepG2细胞EGFR、AKT及ERK表达及活化的影响。碘化丙啶（propidium lodide,PI）染色检测西妥昔单抗对细胞周期的影响。结果流式细胞分析结果显示,HepG2细胞表面存在较高水平的EGFR表达;细胞划痕及Transwell结果表明,西妥昔单抗对HepG2细胞迁移能力具有明显的抑制效应;Western印迹结果证明西妥昔单抗能显著降低HepG2细胞内EGFR、AKT及ERK的磷酸化水平;细胞周期分析显示西妥昔单抗作用24 h剂量依赖性影响HepG2细胞周期进程,并将其阻抑在G0/G1期。结论西妥昔单抗可以抑制高表达EGFR的肝癌细胞系HepG2胞内关键信号蛋白的活化,阻滞其细胞周期,抑制肝癌细胞的迁移能力。     

Ad—HGF转染对HepG2细胞的生长抑制作用

目的：探讨Ad—HGF转染对HepG2生长抑制作用的影响。方法：用Ad—HGF转染HepG2细胞，检测HGF蛋白表达及对细胞凋亡的影响；并用裸鼠致瘤试验体内观察Ad—HGF对HepG2细胞致癌的影响。结果：转染后24hHGF即有表达，48h达高峰。并观察到Ad—HGF可促进HepG2凋亡，体内Ad—HGF可抑制HepG2细胞生长成瘤。结论：Ad—HGF在体外可抑制HepG2细胞增殖、促进其凋亡，体内抑制其致癌作用。     

一种合成的纳米粒高分子材料的细胞毒性研究

目的 检测本实验室新合成的准备应用于静脉注射的系列高分子材料poly(ethylene glycol)-D,L-lactic and glycolic acid-poly(ethylene glycol)(mPEG-PLGA-mPEG,PELGE)以及由其制成的纳米粒对多种细胞的细胞毒性.方法 利用快速评定细胞增殖率和细胞毒性的噻唑蓝比色法(MTT),不仅参照美国药典用小鼠成纤维细胞L929细胞株进行评价,还根据该材料将来可能的应用领域,用Chang氏正常肝细胞株,原代人肾血管内皮细胞,原代人胚成骨细胞,原代人胚成肌细胞评价其细胞毒性.结果 结果表明该系列聚合物对L929细胞株24 h内和内皮细胞48 h内的毒性为0或I级,即无细胞毒性,而对其它不同种细胞具有不同程度的细胞毒性.结论 该材料具有应用于静脉注射的可能性.该实验为进一步的动物试验提供依据,同时为该新型材料应用于实际提供了前提检测方法.     

表没食子儿茶素没食子酸酯对肝癌细胞增殖的抑制作用研究

目的 探讨表没食子儿茶素没食子酸酯(EGCG)对肝癌细胞HepG2增殖和凋亡的影响及其可能机制.方法 采用不同浓度(0、25、50、100、200、400mg/L)EGCG作用于HepG2细胞,于24、48h后采用MTT方法检测细胞增殖抑制率.以不同浓度(0、50、100、200mg/L)EGCG作用于HepG2细胞,24h后采用流式细胞术检测细胞凋亡率、细胞周期、细胞分裂周期蛋白25A(CDC25A)及Smad3蛋白的表达,RT-PCR检测CDC25A和Smad3 mRNA的表达.结果 MTT检测结果显示,不同浓度EGCG对HepG2细胞均有生长抑制作用,且具有时间和剂量依赖性(P＜0.01).随着EGCG浓度升高,细胞增殖指数(PI)明显降低(P＜0.01),细胞凋亡率明显增高(p＜0.01),CDC25A蛋白和mRNA表达水平下降(p＜0.05),Smad3蛋白和mRNA表达水平上升(p＜0.05).结论 EGCG可能通过下调CDC25A、上调Smad3的表达,抑制HepG2细胞增殖并诱导其凋亡,从而对肝癌细胞起到生长抑制作用.     

pSUPER-Racl-siRNA质粒的构建表达及对肝癌细胞HepG2体外增殖的抑制

目的 使用pSUPER作为产生siRNA的载体,构建针对Racl的siRNA表达载体,并观察其对肝癌HepG2细胞中Racl蛋白表达的抑制作用,同时研究Racl蛋白表达抑制后肝癌细胞HepG2的体外增殖情况.方法 合成用于产生针对Racl的发卡状RNA的寡核苷酸,含64个碱基,退火后插入线性化pSUPER载体的H1启动子下游.重组载体经限制性酶切和测序,构建载体pSUPER-Racl-siRNA,并将其转染肝癌细胞系HepG2.空载体pSUPER-Control-siRNA作为阴性对照.Western blotting检测转染质粒后细胞内Racl基因的变化,MTT法检测Racl蛋白表达后体外细胞增殖情况.结果 成功构建了可以表达针对Racl的siRNA表达载体pSUPER-Racl-siRNA.转染该载体能有效地下调HepG2细胞中Racl蛋白的表达,受抑制率为82%.MTT检测结果显示,抑制Racl蛋白表达后,细胞增殖速度明显减慢.结论 成功构建了针对Racl的发卡状RNA表达载体pSUPER-Racl-siRNA,其可以高效而特异地下调HepG2细胞内靶基因Racl的表达.Racl蛋白沉默对肝癌细胞增殖具有明显的抑制作用,说明其在细胞的体外生长过程中具有重要作用.     

抑制星形胶质细胞丝裂原活化蛋白激酶14减轻谷氨酸兴奋性毒性神经元损伤的机制

目的:探讨抑制星形胶质细胞丝裂原活化蛋白激酶14（MAPK14）表达对减轻谷氨酸兴奋性毒性进而发挥神经元保护的作用机制。方法:慢病毒介导MAPK14干扰载体由上海吉凯基因化学技术有限公司合成,星形胶质细胞从出生48 h的SD大鼠获取,通过慢病毒介导转染体外培养的星形胶质细胞,按照随机数字表法分为三组：（1）未转染组,为...     

适用于基因转染的细胞培养板的研制

目的:研制用于基因转染的细胞培养板.方法:用0.2%的明胶稀释LPEI, BPEI, Superfect,Lipofectamine 2000等转染试剂并将它们固定在96孔细胞培养板上.以绿色荧光蛋白(GFP)基因和荧光素酶基因(Luciferase)为报告基因检测培养板的转染效率,用MTT法检测转染24 h后的细胞毒性,将LPEI包被的培养板放置在37℃环境下进行稳定性研究.结果:所有试剂包被的培养板均有一定的基因转染效率,聚合物类试剂的效率高于脂质体类的Lipofectamine 2000.其中,LPEI包被的培养板的转染效率最高、细胞毒性最低.当LPEI的包被量为3.2 μg/孔时,293细胞在转染24 h后的荧光素酶活性达3.0×108 RLU/mg,细胞存活率为85%左右.LPEI包被的培养板在37℃保温14 d后基因转染效率无明显变化.结论:LPEI包被的转染平板具有转染效率高、细胞毒性低、稳定性好的优点.该平板具有操作简便、节省时间的优点,可用于进行大规模的基因转染研究.     

重组长效人精氨酸酶抑制肿瘤细胞生长的研究

 目的 研究重组长效人精氨酸酶对黑色素瘤、喉癌和肝癌等肿瘤细胞的抑制效果,探讨该酶作为抗癌药的应用前景.方法 以离体培养的6株肿瘤细胞(人黑色素瘤细胞MV3、 M14、A875、A375,喉癌细胞Hep2和人肝癌细胞HepG2)为实验对象,培养至90%汇片时收获细胞,转种到96孔板,加入不同浓度的重组长效人精氨酸酶共同培养72 h.采用MTT法,测定重组长效人精氨酸酶对肿瘤细胞生长的抑制情况.结果 所有被测细胞的活力随着该酶浓度的增加而降低,且该酶对两株黑色素瘤细胞的IC50小于0.1 U/ml.被测药物对人肝癌细胞HepG2的IC50是0.835 U/ml,对人喉癌细胞Hep2的IC50是2.269 U/ml.结论 重组长效人精氨酸酶对体外培养的黑色素瘤、喉癌和肝癌细胞具有细胞毒性,是有潜力的抗癌药物候选物.     

葡萄糖水平对CEES染毒16 HBE细胞能量代谢的影响及其机制

目的：比较在高糖和低糖培养条件下,经半硫芥（CEES）染毒后支气管上皮细胞（16HBE）能量代谢的变化。方法采用高糖（4．5 mg/ml）和低糖（1．1 mg/ml）两种不同培养基培养16HBE细胞。 MTS法比较细胞增殖和CEES染毒后6 h细胞活性差异;用不同剂量CEES染毒两组细胞,24 h后HPLC检测胞内ATP、ADP、AMP含量,并计算ATP/ADP比值、腺苷酸池（总腺嘌呤核苷酸, total adenine nucleotides , TAN）及能荷值（能量负荷值,energy charge, EC）,Western印迹检测线粒体能量代谢功能酶COX-10和ISCU变化;流式细胞仪检测0．5 mmol/L CEES染毒后5、8、12、24 h时相点线粒体膜电位（MMP）变化。结果低糖培养的16HBE细胞的增殖明显增加,能量代谢指标ADP、TAN、COX-10和ISCU显著高于高糖组。高于0．5 mmol/L染毒剂量的CEES能显著降低高糖、低糖培养的16HBE细胞的活性,且在1．0 mmol/L下两组间差异有显著意义。高糖组在0．5、1．0 mmol/L染毒24 h后,ATP、ADP、TAN显著增高,而ATP/ADP比值及EC显著降低。低糖组在1．0 mmol/L染毒24 h后,ADP、AMP、TAN显著低于染毒前,而ATP/ADP比值及EC显著高于染毒前。在0．5 mmol/L CEES染毒下,高糖组线粒体膜电位在8～12 h显著增加,其后至24 h时恢复至正常水平。低糖组MMP在5 h有一过性显著降低,8 h后与染毒前已无显著差异。高糖培养时,16HBE细胞的氧化磷酸化相关蛋白COX-10和ISCU蛋白水平显著低于低糖培养的细胞;0．5～1．0 mmol/L CEES染毒24 h后,两者的水平显著升高,与低糖组已无显著差异。1．0 mmol/L CEES染毒24 h后,低糖组COX-10和ISCU水平显著降低。结论糖浓度差异会影响16 HBE细胞的能量代谢、增殖和CEES损伤后能量应激反应。高糖可能通过增加细胞的应激反应能力抵抗CEES的细胞毒性作用。     

Genitourinary toxicity after permanent iodine-125 seed implantation: The nationwide Japanese prostate cancer outcome study of permanent iodine-125 seed implantation (J-POPS)

PurposeThe purpose of this study was to evaluate acute and late genitourinary (GU) toxicity and to elucidate factors associated with GU toxicity in patients with prostate cancer treated with permanent seed implantation (PI) enrolled in a nationwide prospective cohort study in Japan.Methods and MaterialsOf 2,354 patients enrolled in this study, GU toxicity was evaluated in 2,339 patients at 3, 12, 24, and 36 months after PI. To elucidate independent factors predictive of acute and late Common Terminology Criteria for Adverse Events Grade 2 or higher (Grade ≥2) GU toxicity, multivariate logistic regression analyses were carried out. Regarding acute urinary retention (AUR), the incidence rate and the recovery rate for AUR were estimated using the Kaplan–Meier curve.ResultsApproximately 53% of the patients treated with PI alone and 42% of those treated with combination therapy with PI therapy and external beam radiation therapy showed urinary frequency/urgency at 3 months. The multivariate analysis revealed that age, prostate volume, pretreatment international prostate symptom score, drinking status, and PI were independent predictors of acute GU toxicity Grade ≥2. Of all patients, 53 (2.3%) suffered from AUR, and 49 (92.5%) recovered from AUR with a median time of 4.3 months during the followup period.ConclusionsThe results of GU toxicity in Japanese patients who underwent low-dose-rate brachytherapy were acceptable and comparable to those previously reported in U.S. patients. The patients treated with PI alone showed a significantly higher incidence rate of GU toxicity than did those undergoing combination therapy with PI and external beam radiation therapy in the acute phase.     

Low-dose hyper-radiosensitivity in human hepatocellular HepG2 cells is associated with Cdc25C-mediated G2/M cell cycle checkpoint control

Purpose: Although the significance of cell cycle checkpoints in overcoming low-dose hyper-radiosensitivity (HRS) has been proposed, the underlying mechanism of HRS in human hepatocellular cells remains unclear. Therefore, the aim of this study was to characterize HRS inhuman hepatocellular HepG2 cells and to explore the molecular mechanism(s) mediating this response.

Materials and methods: HepG2 cells were exposed to various single doses of γ radiation (from 0?Gy to 4?Gy), and then were assayed at subsequent time-points. Survival curves were then generated using a linear-quadratic (LQ) equation and a modified induced repair model (MIRM). The percentage of cells in the G1, G2/M, and S phases of the cell cycle were also examined using propidium iodide (PI) staining and flow cytometry. Levels of total cell division cyclin 25C (Cdc25C) and phosphorylated Cdc25C were examined by Western blotting.

Results: Low-dose γ radiation (<0.3?Gy) induced HRS in HepG2 cells, while doses of 0.3, 0.5, and 2.0?Gy γ radiation significantly arrested HepG2 cells in the G2/M phase. While total Cdc25C levels remained unchanged after irradiation, levels of phosphorylated Cdc25C markedly increased 6, 16, and 24?h after treatment with 0.5 or 2.0?Gy radiation, and they peaked after 16?h. The latter observation is consistent with the G2/M arrest that was detected following irradiation.

Conclusions: These findings indicate that low-dose HRS in HepG2 cells may be associated with Cdc25C-mediated G2/M cell cycle checkpoint control.     

Regional Hyperthermia in Conjunction with Definitive Radiotherapy against Recurrent or Locally Advanced Prostate Cancer T3 pN0 M0

BACKGROUND AND PURPOSE: Since long-term results of the standard treatment of locally advanced or recurrent prostatic carcinoma are unsatisfactory, the role for additional regional hyperthermia was evaluated in a phase I/II study. PATIENTS AND METHODS: From 08/1996 to 03/2000, 22 patients were treated by a standard irradiation regimen (68.4 Gy) in combination with regional hyperthermia (weekly, five to six times), and five of 22 patients received short-term (neoadjuvant) hormonal treatment. Of these, 15 patients had primary prostatic carcinoma T3 pN0 M0 and seven a histologically confirmed local recurrence after radical prostatectomy. Feasibility of hyperthermia, and acute/late toxicity as well as long-term follow-up (prostate- specific antigen [PSA] control, overall survival) were analyzed. Clinical endpoints were correlated with thermal parameters. RESULTS: Mean maximum temperatures along the urethra of 41.4 degrees C (41.0 degrees C for the recurrences), and mean T(90) values of 40.7 degrees C could be achieved. Severe acute toxicity of grade 3 occurred at the rectum in three, at the urethra in four, at the intestine in one, and a burn induced by hyperthermia in one of 22 patients. Late toxicity was only observed rectally in one patient (grade 3) and at the urethra in two patients (grade 2). There was no correlation between thermal parameters and any toxicity. The survival curves showed a PSA control for primary prostatic carcinoma > 50% after 6 years, but no long-term PSA control for the recurrences. Overall survival after 6 years was 95% for primary carcinoma, and 60% for the recurrences. There was a clear correlation between higher temperatures or thermal doses with long-term PSA control. CONCLUSION: Regional hyperthermia might be a low-toxicity approach to increase PSA control of common treatment schedules. Further evaluation, in particular employing improved hyperthermia technology, is worthwhile.     

大鼠头部暴露全氟异丁烯中毒致肺损伤实验模型的建立

目的：建立大鼠头部暴露吸入染毒致急性肺损伤实验模型以便进行全氟异丁烯(pefluoroisobutylene，PFIB)毒理及防治药物研究。方法：用设计的大鼠头部暴露吸入PFIB染毒装置致动物中毒，测定动物中毒后肺灌流液(BALF)中总蛋白含量和肺湿干比变化，在染毒过程中同时用气相色谱连续监测DFIB浓度。结果：在大鼠头部暴露吸入染毒中，PFIB染毒浓度比较稳定。大鼠吸入PFIB0．18mge／L(8—10min)致肺损伤实验模型比较明显，各项指标变化显著，中毒性肺水肿程度与HFIB浓时积(染毒浓度乘以时间，即CT值)呈正相关，动物中毒后12～24h肺灌流液中总蛋白含量和肺湿干比显著升高。结论：建立了大鼠头部暴露动态吸入PFIB中毒致急性肺损伤实验模型，该模型比较稳定，可用于观察动物PFIB中毒性肺水肿程度，也可作为：PFIB毒理及防治药物研究实验方法。大鼠PFIB中毒途径主要通过呼吸道吸入中毒，其吸入毒性至少比腹腔毒性大10倍。     

Relationships between acute reactions to radiotherapy in head and neck cancer patients and parameters of radiation-induced DNA damage and repair in their lymphocytes

PURPOSE: To study the relationship between lymphocyte radiosensitivity measured in vitro and acute reactions to radiotherapy in patients with head and neck cancer. MATERIALS AND METHODS: Acute reactions were measured in 34 patients using the Dische scale. Lymphocyte radiosensitivity was measured using the alkaline comet assay, the micronucleus assay, the nuclear division index and morphological assessment of apoptosis. RESULTS: There was a weak, statistically significant correlation between in vitro radiosensitivity measured as the rate of DNA damage repair and the cumulative radiation dose exerting the maximum acute reaction scored (r = -0.366, p = 0.039, n = 34). Subgroup analyses showed that for patients with a low level of radiation-induced DNA damage there was a statistically significant relationship between lymphocyte radiosensitivity measured as inhibition of proliferation and acute toxicity (r = -0.621, p = 0.007, n = 18). For patients with a high level of residual DNA damage, there was a relationship between lymphocyte radiosensitivity measured using the micronucleus assay and acute toxicity (r = -0.597, p = 0.023, n = 14). CONCLUSIONS: Combining two measures of radiosensitivity improves the ability to correlate in vitro lymphocyte radiosensitivity and acute radiotherapy toxicity data.     

莪术油配伍椒目仁油对小鼠移植性肿瘤的抑制作用

目的观察不同剂量莪术油配伍椒目仁油对小鼠移植性肿瘤S180的抑瘤作用,以及对人源性肝癌细胞株HepG2和大肠癌细胞株SW480的体外抑瘤作用。方法 72只二级昆明小鼠采用肿瘤移植法建立小鼠荷瘤动物模型;椒莪软胶囊灌胃给药,剂量分别为0.3、0.6、1.2 ml/kg;氟尿嘧啶22.5 mg/kg作为阳性对照药腹腔注射给药;连续给药15 d,观察药物对肿瘤生长的抑制作用。对人源性肝癌细胞株HepG2和大肠癌细胞株SW480进行传代培养后,制备不同剂量(椒莪软胶囊0.015 625、0.031 25、0.062 5、0.125、0.25、0.5、1、2、4 mg/ml)含药血清、氟尿嘧啶阳性药血清和肿瘤移植模型不用药对照血清,作用于人源性肝癌细胞株HepG2和大肠癌细胞株SW480,四甲基偶氮唑蓝比色法观察血清对两种细胞的抑制作用。椒莪软胶囊0.0、6.25、12.5、25.0μg/ml给药15 h后,经流式细胞仪测定对人源性肝癌细胞株HepG2细胞凋亡的影响。结果①椒莪软胶囊0.3、0.6、1.2 ml/kg肿瘤抑制率分别为19.73%、32.22%和46.68%。随着给药剂量的增加,坏死的肿瘤细胞所占比例也相应增加,生长活跃的肿瘤细胞逐渐减少。②抑制作用随剂量升高而作用增强,对人源性肝癌细胞株HepG2体外最低药物物浓度为0.062 5 mg/ml,对大肠癌细胞株SW480体外最低药物浓度为0.125 mg/ml。③椒莪软胶囊可明显促进人源性肝癌细胞株HepG2的细胞凋亡。结论椒莪软胶囊对小鼠移植性肿瘤有明显的抑制作用,对人源性肝癌细胞株HepG2和大肠癌细胞株SW480有明显的抑制作用。椒莪软胶囊在临床具有广阔的应用前景。     

IL-8通过整合素调控肝癌HepG2细胞迁移

目的：研究IL-8对肝癌细胞HepG2增殖、迁移的影响,探讨整合素α亚基在调控HepG2细胞迁移中的作用。方法以不同浓度（0～125 ng／ml）的IL-8刺激HepG2细胞,分别采用MTT法检测IL-8对肝癌细胞的增殖作用;细胞划痕损伤模型测定不同处理组在0、4、8、12和24 h各时间点对HepG2细胞水平迁移的影响;Transwell法检测刺激24 h后HepG2细胞纵向迁移能力;免疫荧光检测0、125 ng／ml IL-8刺激HepG2细胞8 h后细胞骨架的变化;Western印迹测定整合素α亚基的表达变化规律。结果与对照组相比,IL-8促进HepG2细胞增殖,但各浓度间无明显差异;细胞划痕损伤实验和Transwell实验均表明IL-8可促进HepG2细胞迁移,且具有浓度依赖性; IL-8诱导HepG2细胞骨架重排,使丝状伪足样凸起数目增多;Western印迹结果显示,IL-8上调整合素α亚基的表达,但各亚基具有浓度性差异。结论 IL-8可通过上调整合素α亚基的表达促进HepG2细胞迁移,各α亚基调控作用可能具有差异性。     

Spred2基因对人肝癌细胞凋亡韵影响

目的观察Spred2对人肝癌细胞（HepG2）凋亡的影响。方法用阳离子脂质体瞬时转染pcDNA3．0,pcDNA3．0-Spred2到HepG2细胞,建立过表达Spred2的细胞模型。膜联蛋白（annexlFl）V．FITC／PI（7．AAD）双标通过流式细胞仪检测细胞凋亡;蛋白质印迹检测SprcdZ的表达水平。结果特柒Spred2基因能明显诱导HepG2细胞凋亡。另外,转染Spred2基因能显著地增强5一FU诱导HepG2细胞凋亡。结论Spred2对人肝癌细胞凋亡有调控作用。