【作者】 刘洪瑞；

【导师】 李智；

【作者基本信息】 中国医科大学 ， 药理学， 2005， 博士

【摘要】 目的 滨蒿内酯(scoparone,Sco)是传统中药茵陈中的主要有效成分,近年来的研究表明,Sco具有抗突变和抑制肺癌细胞增殖的作用,但有关Sco在体抗肿瘤的作用及其机制的研究尚未见报道。随着对细胞凋亡现象的进一步认识及其发生机制的研究,人们逐渐认识到大多数抗肿瘤药物具有诱导肿瘤细胞凋亡的作用,而在凋亡的发生与调控机制中,Caspase是关键性的蛋白,也是有关凋亡研究中的主要热点问题之一。本研究一方面采用动物模型和体外细胞实验观察Sco在体内外抗肿瘤的作用,另一方面,采用通过分子生物学手段探讨Sco抗肿瘤作用的机制。 实验方法 1.小鼠肿瘤模型的建立 将0.05ml浓度为1×10~8个/ml的sacoma 180细胞悬液接种于BALB/c小鼠左侧腹背部皮内,制备实体瘤动物模型。将0.2ml浓度为1×10~7个/ml的EAC细胞悬液接种于BALB/c小鼠腹腔中,制备腹水瘤动物模型。 2.动物分组及给药方法 Sacoma 180细胞悬液接种一周后,根据肿瘤大小将BALB/C小鼠随机分为5组,对照组、阳性药物对照组(CTX 10.0mg/kg,ip)和Sco给药组(2.5,5.0和10.0mg/kg,ip),每组15只,连续给药10天,每2～3d测量一次肿瘤大小,治疗10日后,将小鼠称重,在乙醚麻醉下,剥离皮内的移植肿瘤组织并称重,计算抑瘤率(%)=(给药组平均瘤重/空白对照组平均瘤重)×100。接种EAC肿瘤细胞后的BALB/C小鼠进行同样的分组,连续给药14天后停药,记录小鼠生存天数,到第30天仍存活者按30天记。计算小鼠生命延长率(%)=(处理组小鼠平均存活天数/空白对照组小鼠平均存活天更多还原

【Abstract】 Scoparone （Sco） is a extract from Chinese traditional herd which is called Artemisia scoparia Waldst. Et Kit. The mechanisms of Sco serving to exert its antitumor action have not been elucidated, although recent studies suggest that it has antimutation activity and the proliferation inhibition of lung cancer cells. Otherwise, depending on the further exploring on the mechanisms of apoptosis, we have confirmed that apoptosis plays an important role in antitumor action of most antitumor drugs. Moreover, Caspase is a key protein during apoptosis genesis and regulation, which has also being the hot topic in apoptosis research. This study investigated the antitumor action in vivo and in vitro. Also, the mechanisms of antitumor action were explored by molecular biology methods.METHODS1. Preparation of tumor model miceBALB/c mice were inoculated with 1 10⁸ cells/0. 05ml sarcoma 180 cells into the left flank. Then, solid tumor model mice were prepared. BALB/c mice were inoculated with 1 10⁷ cells/0. 2ml EAC cells into abdominal cavity. Then, ascites tumor model mice were prepared.After seven days, we divided the solid tumor model mice into five groups according to the sizes of tumor; control group, positive control group （ CTX 10. 0mg/kg, ip） and Sco groups （2.5, 5.0 and 10.0mg/kg, ip） , each group has 15 mice. Drugs were administered for ten days. Then, the weights of mice weredetected. The tumor were removed out with its weights detected, then the inhibition rate of tumor were calculated as following: Inhibition ratio （% ） = [1the mean weights （ drug ） / the mean weights （ control ） ] 100%. The ascites tumor model mice were the same separation into five groups. After drugs were administered for forteen days, survival days were recorded. Survival rate of mice were calculated as following: Survival ratio （% ） = [ the mean survival days （drug ） / the mean survival days （control ） ] 100%.2. HT - 29 cells growth inhibition testHT - 29 cells were seeded into 96 - well plate in triplicate at a density of 1 10³cells/ml and various concentrations of Sco （10^-7 10^-3 mol/L） were added, then cultured for 24, 48 and 72h at 37℃. MTT 10μl/well were added and cultured for further 4h, and detected by Bio - Red. The percentage of cell growth was calculated as following: Growth ratio （ % ） = [ OD₅70 （ drug） / OD₅70 （ control） ] ×l00%.3. Nuclear damage observed by Hoechst 33258 stainingHT - 29 cells were seeded into 96 - well plate in triplicate at a density of 1 x 10 cells/ml and various concentrations of Sco （ 10" mol/L） were added, then cultured for 48 h at 37℃. Then the cultured cells were fixed with Glutaral-dehyde 1% , washed twice with PBS, and stained with Hoechst 33258 lmmol/L for 12h at 4℃. At the end of incubation, nuclear morphology was observed u-sing fluorescence microscope.4. Apotosis and cell cycle assayHeLa cells were seeded into 6 wells plates in triplicate at a density of 2 x 10⁶ cells/Land Sco 10^-3 mol/L were added, then cultured for 2, 4, 6, 8, 10, 12, 18, 24 and 48h at 37℃ Sco - treated and control cells were collected after the indicated time and fixed with 75% ethanol for 24h at 4℃. After diluting the cell concentration into 1 × 10⁶cells/L, 200(xl RNase （10μg/ml） was added in 200μl sample, and kept at 4℃ for 30 min. The suspension was filtered, then PI 250μg/ml was added. The cells were kept at 4℃ for 30 min, then analyzed by flow cytometry.5. Caspase-3 activity assayHT - 29 cells were seeded into 24 - well plate in biplicate at a density of 5xlO5 and various concentrations of Sco （10⁷, 10 "5, 10"3mol/L） were added , then cultured for 24, 48 and 72h. The cells were collected by centrifugation 400 x g for 5min and regulated the concentration into 2 x 106 cells/ml. Then cell lysate 50ui was added and ice - bath for lOmin. The cells were collected centrifugation 10,000 x g for lOmin again. DTT and lmM DEVD - pNA 50ui were added into the above supernate fluid and water - bath at 37Tl for lh. Optical density was detected at 405 nM. The activity of caspase -3 was calculated as following: activity （% ） = [ OD^ （ drug） / OD^ （ control） ] x 100%.6. RT - PCR assay of bax geneHeLa cells 5 x 106 cells/ml were collected by centrifugation 1,000 x g for 5min. lml Trizol was added for lysate cells and supernate fluid was collected by centrifugation 12,000rpm for 5min. Then chloroform 200(xl was added and kept, then upper fluid was collected by centrifugation 12,000rpm for 15min. Isopropanol 0. 5ml was added into the upper fluid and kept at room temperature for lOmin. After centrifugation, RNA was precipitated in the Eppendof tube. Finally RNA was washed by 75% ethanol and diluted by water. AccessQuick? RT - PCR System was used for RT - PCR. Reaction system was prepared as direction of PCR - kit, incubated at 48°C for 45 min, then amplificate followed by 30 cycles. Agarosagarose electrophoresis was used in observing the result of amplified DNA fragments.7. Western blotting analysis of bax geneHeLa cells 5 x 106 cells/ml were collected by centrifugation 1,000 g for 5min. After being washed with ice - cold PBS twice, cells were lysed for 30min on ice in appropriate volume of ice - cold lysis buffer containing 50mmol/L HEPES, 1% TritonX -100, 2mmol/L sodium vanadate, 100 mmol/L NaF, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L PMSF, 100 mg/L aprotinin, lOmg/L leupeptin fP lOOmg/L pepstatin. Debris was removed by centrifugation for 15 min at 12,000rpm at 4X,. Soluble ptotein was separated by electrophoresis on 12% SDS polyacrylamide gels and blotted onto PVDF filter. After being blocked, the filters were reacted with monoclonal antibody anti — bax for 2h at room temperature and incubated with secondary antibody for 1 h, at last colorat-ed.8. Statistical analysisAll results were showed by mean SD. All of data were statistic by SSPS and excel 2000 software. The significant differences were evaluated according to Students t - test and one - way ANOVA.RESULT1. Inhibition of Sco on tumor model animalsThe result showed that Sco （5. 0 and 10. Omg/kg） significantly inhibited the growth of the tumor induced by inoculated with sacoma 180 cells. This result began from 3 days after drugs administered （P<0.05,n = 15） and further from 10 days （ P < 0. 01, n = 15 ）. Compared with control group, Sco groups （2. 5 mg/kg, ip. ） didn’t show remarkable difference. However, 3 doses of Sco and CTX had no difference of survival rate of ascites tumor model mice.2. Inhibition of Sco on HT - 29 cellsThe result showed that Sco （10 "5 - 10³mol/L） significantly inhibited the proliferation of HT - 29 cells from 24h and was showed at 72h. The inhibition effect was showed in a dose - and time - dependent manner.3. Inducing apoptosis activity of Sco on HT -29 cellsThe nuclear morphological changes were observed by Hoechst 33258 staining. After 48h treatment with Sco 10"Jmol/L, apoptotic bodies appeared.4. Inducing apoptosis activity and cell cycle of Sco on HaLa cellsThe result from flow cytometry showed, treated with Sco 10³mol/L, Sco induced HeLa cells apoptosis in a time - dependent manner. After the treatment of Sco for 4h, 12h and 48h, the rate of apoptosis was 1. 56% , 11.4% and 65. 3% respectively. The maximum effect showed at 48h. The result of cell cycle DNA assay showed that treatment of Sco 10"3mol/L for lOh, 24h and 48h increased the Gl fraction of HeLa cells with 62.7% , 68.5% and 85. 6% repec-tively. The maximum effect showed at 48h. However, without of Sco treatment, the proliferation of HeLa cells was normal. Our results indicated that Sco induced Gl arrest and inhibited DNA synthesis, then decreased the cell viability on HeLa cells.更多还原