【作者】 严晓莺；

【导师】 王明艳；

【作者基本信息】 南京中医药大学 ， 中西医结合基础， 2012， 硕士

【摘要】 近年来,中药及其制剂的毒性报道不断增多。中药引起的肝损伤为药物性肝损伤的四大常见原因之一。实验目的：分别观察中药活性抗癌成分斑蝥酸钠、没食子酸、熊果酸对人正常肝细胞LO2的影响,为寻找有效的减毒增效拮抗剂提供线索。实验方法：采用MTT法检测这三种抗癌活性成分对细胞的增殖抑制,倒置显微镜、荧光染色、电镜检测细胞形态学改变,吉姆萨染色检测细胞微核率,流式细胞术检测细胞凋亡率,western blot检测各用药组细胞内蛋白表达的变化。实验结果：发现0.625μg/ml组、1.25μg/ml组、2.5μg/ml组斑蝥酸钠作用24h,对LO2细胞的抑制率分别为为9.1%、17.1%、23.9%；对SMMC-7721的抑制率分别为11.2%、23.5%、30.7%。荧光显微镜下0.625μg/ml组无明显改变,1.25、2.5μg/ml组LO2出现细胞染色体的断裂；吉姆萨染色后在光镜下计数0.625μg/ml组无明显改变,1.25μg/ml组微核率为46.0‰、2.5μg/ml组为52.2‰；0.625μg/ml组凋亡率为5.08%,1.25μg/ml为16.31%,2.5μg/ml组为21.86%,呈剂量依赖关系；电镜证实2.5μg/ml组出现细胞皱缩、表面微绒毛减少,线粒体肿胀、脊断裂,细胞内空泡增多,细胞核固缩碎裂,呈凋亡特征性改变。1.25ug/ml和2.5ug/ml组Caspase-3、Bcl-2含量下降,Bax升高,与阴性对照组相比有统计学意义(P<0.05),NFkb也有所升高,与阴性对照组相比无差异。熊果酸作用24h,12.5μmol/L组、25μmol/L、50μmol/L组对LO2细胞的抑制率分别为为5.4%,21.4%,49.8%,对SMMC-7721的抑制率分别为17.6%、36.1%、60.9%。荧光显微镜下50μmol/L组出现细胞核浓缩,亮度增高,形成弯月状,偏向细胞一侧,甚至出现细胞碎片及凋亡小体。流式结果显示细胞凋亡率为48.76%。没食子酸作用24h,各剂量组均对LO2和SMMC-7721细胞显示出增殖抑制作用,但是到72h后,对SMMC-7721细胞的增殖抑制作用超过了对LO2细胞的增殖抑制。没食子酸作用24h后100μmol/L组细胞出现明显的形态学改变,细胞凋亡,凋亡率为12.53%,Caspase-3和P53的表达升高。实验结论：斑蝥酸钠、熊果酸和没食子酸对肝癌细胞和正常肝细胞均有抑制作用。斑蝥酸钠对正常肝细胞的抑制低于对肝癌细胞的抑制；在中剂量时可引起正常肝细胞增殖抑制、细胞染色体的损伤和细胞的凋亡；斑蝥酸钠引起的细胞凋亡,可能与Caspase-3、Bcl-2、Bax蛋白表达的改变有关。熊果酸对正常肝细胞的抑制低于对肝癌细胞的抑制；熊果酸在25μmol/L时即可引起正常肝细胞增殖抑制,50μmol/L时不仅抑制肝细胞的增殖,还可以引细胞的凋亡；熊果酸引起的细胞凋亡,与Caspase-3蛋白的表达的改变有关。没食子酸给药给药72h后对对肝癌细胞的抑制才超过正常肝细胞的抑制；没食子酸100μmol/L时引起人正常肝细胞凋亡,考虑和P53及Caspase-3蛋白的改变有关。更多还原

【Abstract】 In recent years,toxicity reported for traditional Chinese medicine and its preparation is increasing,traditional Chinese medjcine-induced liver injury,is one of the four common causes of drug-induced liver injury.objective:To observe the traditional Chinese medicine actiVe anticancer ingredient djsodium cantharidinate,gallic acid,ursolic acid on normal human hepatocytes LO2.provide clues to find efffectiVe attenuated efficiency antagonist..Methods:MTT assay of these three anticancer constltuents of cell proliferation inhibition. inverted microscope, fluorescent staining and electron microscopy to determine the morphological changes of cells,Giemsa staining to detect micro-nuclear rate,flow cytometry to measure the apoptosis rate,Western blot to determine the expression changes of proteins within the cells of each administration group.Results:Found0.625μg/ml group1.25μg/ml group,the the2.5μg/ml grouUp cantharides of sodium for24hours,inhibition of LO2cells were9.1%,17.1%,23.9%；SMMC-7721,The inhibition rates were11.2%,23.5%,30.7%,respectiVely.No significant change in the nuorescence microscope0.625μg/ml group of1.25,2.5μg/ml group LO2cells chromosome breakage；Giemsa staining under light microscope counting0.625μg/ml group no significant changes in1.25μg/ml group of micro-nuclear rate of46.0‰,52.2‰2.5μg/ml group；0.625μg/ml apoptotic rate of5.08%and16-31%in1.25μg/ml the2.5μg/ml group21.86%,a dose dependent manner；electron microscopy confirmed that the2.5μg/ml group,cell shrinkage, surface microvilli,mitochondrial swelling,spinal fracture,intracellular vacuoles increased nuclei condensation and fragmentation,characterislic change was apoptosis.1.25ug/ml and2.5ug/ml groups of Caspase-3,Bcl-2content decreased,Bax increased with the negatiVe contro1group was statistically significant(P<0.05),NFkb increase slightly,with the negative contro1group Compared to no difference.Ursolic acid for24h,the inhibitory rate of12.5μmol/L group,25μmol/L,50μmol/L group LO2cells were5.4%,21.4%,49.8%inhibition of SMMC-7721rate of17.6%,respectively,36.1%,60.9%.50μmol/L group,nuclear condensation under a fluorescence microscope,the brightness increased,the formation of crescent-shaped,bias cell side,and even cell debris and apoptotic bodies.Streaming results showed that the apoptosis rate of48.76%.Gallic acid for24h,each dose group showed LO2and SMMC-7721cell proliferation inhibition,but affer72h,the inhibition of proliferation of SMMC-7721cells than LO2celJ proliferation inhibition.Gallic acid for24h,100μmol/L,group of cells appears obviOUS morphological changes of cell apoptosis rate was12.53%,elevated Caspase-3and P53 expression.Conclusions:The cantharides sodium, ursolic acid and gallic acid on hepatoma cells and normal liver cells was inhibited. The cantharides sodium inhibition of normal liver cells is lower than the inhibition of hepatoma cells; in dose can cause inhibition of proliferation of normal liver cells, chromosome damage and cell apoptosis; the cantharides sodium caused cell apoptosis, which may and Caspase-3, Bcl-2and Bax protein expression related to the change. Ursolic acid inhibition of the normal liver cells is lower than the inhibition of hepatoma cells; ursolic acid can be caused by the normal liver cell proliferation inhibition at25μmol/L,50μmol/L not only inhibit the proliferation of liver cells, can also lead cells apoptosis; ursolic acid-induced apoptosis, Caspase-3protein expression changes related. Gallic acid administered administered72h after the inhibition of liver cancer cells more than the suppression of the normal liver cells; gallic acid100μmol/L, caused by human normal liver cell apoptosis, consider changes in P53and Caspase-3protein.更多还原