【作者】 赵文静；

【导师】 牛凤兰；

【作者基本信息】 吉林大学 ， 卫生毒理学， 2009， 博士

【摘要】 肝癌是发生于肝细胞或胆管细胞的恶性肿瘤,为全世界范围内常见、高发恶性肿瘤之一,具有难发现、难诊断、难治疗、发展快和预后差等特点,近年的研究发现肝癌发病率和死亡率有向小年龄组推移的趋势,而且肝癌和其他恶性肿瘤相比,具有更强的耐药性,因此寻找靶点明确、低毒、高效的抗肝癌药物具有重要意义。3,4,5-三羟基苯甲酸(Trihydroxybenzoic acid, TBA)是广泛存在于植物中的多酚类化合物,具有抗炎、抗氧化、抗过敏、抗诱变及抗肿瘤等多种生物学活性,但其体内、体外抗肝癌作用及作用机制尚未见报道。本研究采用药理学、细胞学和分子生物学技术,体内和体外实验相结合,从整体、细胞与分子水平三个层次,证实TBA的抗肝癌作用并探讨其作用机制。体内实验以H22肝癌细胞荷瘤小鼠为研究对象,TBA不同剂量灌胃给药,观察其对肿瘤生长的抑制作用及对荷瘤小鼠生存时间的影响。体外实验以3种人肝癌细胞株SMMC-7721细胞、HepG2细胞和Bel-7402细胞为研究对象,观察TBA对肝癌细胞生长的抑制作用和诱导凋亡作用并探讨其作用机制。研究结果显示TBA 0.50 g/kg、0.25 g/kg和0.13 g/kg灌胃给药可以不同程度抑制H22肿瘤的生长及延长荷瘤小鼠生存时间。体外实验结果显示TBA对实验中3种肝癌细胞增殖具有明显的抑制作用,并呈剂量依赖性,其中SMMC-7721细胞对TBA的作用最敏感。TBA可以通过增加细胞ROS水平,上调Bax mRNA表达,下调Bcl-2 mRNA表达,降低细胞线粒体膜电位,促进线粒体中细胞色素c释放入胞浆,激活caspase-9、caspase-3蛋白诱导SMMC-7721细胞凋亡实现其抑制肝癌细胞增殖作用。本研究首次证实了TBA的体内和体外抗肝癌作用,为寻找高效低毒的抗肝癌药物的先导化合物和开发新药提供了实验基础和理论依据。更多还原

【Abstract】 Liver cancer is the world’s sixth most common cancer, it has the character of difficult to find, diagnose and treat, it has the features of poor prognosis. In recent years, the study has found that morbidity and mortality of liver cancer has the trend to the small age group. Compared with other malignant tumors it has more resistant to anti-cancer drug. Drug therapy in liver cancer treatment plays an important role, so it is very important to find clear target, low toxicity and highly effective drugs. TBA is polyphenols which widely present in plants, study shows that it has anti-inflammatry, anti-oxidation, anti-allergic, anti-mutagenic and anti-tumor effect. But its anti-hepatoma effect and mechanism in vivo and in vitro has not been reported.The study was designed to study the anti-hepatoma role of TBA and its mechanism, provided theoretical basis and experiment evidence for researching and developing effective drug for the treatment of liver cancer.In vivo experimemt, H₂₂ tumor bearing mice were used as model to observe the inhibition effect of TBA on tumor and the extension effect of TBA on survival time of tumor bearing mice. In vitro experiment, SMMC-7721 cells, HepG2 cells and Bel -7402 cell lines were used as a model, the anti-proliferation effect of TBA were detected by MTT assay ; Switzerland - Giemsa and AO staining were observed the apoptosis of SMMC-7721 cells; DNA agarose electrophoresis was used to detected SMMC-7721 cell apoptosis; Apoptosis rate were detected by flow cytometry with AnnexinV-FITC/PI staining; Activity of caspase-3,caspase-9 and caspase-8 were detected by spectrophotometric ; Mitochondria membrane potential were detected by flow cytometry with Rh123 probe staining; The expression of Cyt-c in cytosol and mitochondrial were detected by Western blot; The expression of Bcl-2 and Bax mRNA were detected by RT-PCR; The level of ROS were detected by flow cytometry with DCFH-DA probe staining.1. Tumor inhibition and survival time extension effect of TBA on H₂₂ tumor bearing miceThe average tumor weight reduced in TBA group, in which TBA middle dose group compared with the control group , the differences were significant （P <0.05）, the inhibitory rate of TBA group were 24.83%, 45.52% and 15.86% respectively; TBA group mean survival time were longer than the control group ,in which TBA middle dose group compared with the control, the differences were significant （P <0.05）.The life extension of TBA group were 9.65%, 25.48% and 5.79% respectively. The results showed that TBA could inhibit H₂₂ tumor growth and prolong survival time of tumor bearing mice by oral administration with 0.50 g/kg, 0.25 g/kg and 0.13 g/kg dose.2. The anti-proliferation effect of TBA on SMMC-7721 cells、HepG2 cells、Bel-7402cells and PBMCSMMC-7721、HepG2 and Bel-7402 cells treated with TBA were inhibited significantly in a dose-dependent manner . In contrast, TBA had little effect on human PBMC at the same concentration. At the same concentration, the inhibitory effect of TBA on SMMC-7721 cells were considerably stronger compared to that of the TBA on HepG2 and Bel-7402 cells.In control group, SMMC-7721 cells were in a good condition: grew to polygon with excellent refraction ability; while cells in TBA group showed shrink, bad refraction, and more dead cells in culture medium.3. The apoptosis-inducing effect of TBA on SMMC-7721 cells SMMC-7721 cells were treated with 12.5、25.0、50.0 and 100.0μg/mL TBA for 48 h, morphological features of apoptosis were stained by Switzerland-Giemsa, and apoptotic changes were observed by light microscopy, cell shrinkage, bubbling with membrane , chromatin condensation and nuclear pieces can be seen.AO fluorescent staining results showed that control cells had a regular nuclear with green fluorimetric stain, while 12.5μg/mL TBA group and control group no significant differences for the green fluorescent nuclei; 25.0μg/mL TBA group, part of cell cytoplasm condensed, the volume of narrow , nuclear shrinkage, chromatin increased, more brightly fluorescent, condensed to form dense green fluorescent apoptotic features such as; 50.0 and 100.0μg/mL TBA group, some cells in the nucleus can be seen as crescent-shaped shrinkage, rosary-like performance for late apoptosis and characteristics of the shape of the nucleus.DNA agarose gel electrophoresis results found that DNA of 50.0 and 100.0μg/mL TBA cells showed typical DNA Ladder.The early apoptosis rate was measured by flow cytometry with AnnexinⅤ-FITC/PI double staining. The results showed that the percentage of early apoptotic cells was increased significantly with increasing dose （P<0.05）, and there were significantly diffrences among the exposure groups （P<0.05）. The terminal apoptosis showed the same results .The results showed that TBA can induce apoptosis in SMMC-7721 cells.4. The activity changes of caspase-3, caspase-9 and caspase-8 in SMMC-7721 cellsThere were significantly differences between TBA group and negative control in caspase-3 A405 （P<0.05）. The caspase-9 A405 showed the same results .While there was no significant difference between TBA group and control group in caspase-8 A405 （P>0.05）.The result showed that caspase-3 and caspase-9 in SMMC-7721 cells was activated by TBA, while TBA had no effect on caspase-8.5. The effect of TBA on the mitochondrial membrane potential of SMMC-7721 cellsThe SMMC-7721 cells treated with TBA for 24h, the results showed that the mitochondrial membrane potential was decreased with the increasing dose .There were significantly difference between TBA group and control group （P<0.05） ,and there were significantly diffrences among the TBA groups （P<0.05）.6. The effects of TBA on the expression of cytochrome c in cytosol and mitochondrialThe SMMC-7721 cells treated with TBA for 48 h, Western blot results showed that the cytochrome c in cytosol was increased and the cytochrome c in mitochondrial was decreased. The mitochondrial membrane potential of SMMC-7721 treated with TBA decreased significantly and cytochrome c in mitochondrial released to cytosol.7. The effect of TBA on the expression of Bcl-2 and Bax mRNAThe SMMC-7721 cells treated with TBA for 48 h, RT-PCR results showed that Bax mRNA expression was increased while Bcl-2 expression was decreased with the increasing dose.The results showed that TBA could induce Bcl-2 mRNA down-regulated and Bax mRNA up-regulated in SMMC-7721 cells.8. The effect of TBA on the level of ROS in SMMC-7721 cellsThe SMMC-7721 cells treated with TBA for 6 h, the ROS level was increased with increasing dose. There were significantly difference between TBA group and control group （P<0.05）. The SMMC-7721 cells treated with TBA for 12 h,the same results was showed.The results showed that TBA induced the production of ROS in SMMC-7721 cells in dose-dependent manner.In a word, TBA was observed the inhibitory effect on hepatoma both in vivo and vitro experimemts, and TBA inhibited the proliferation of SMMC-7721 cells by induction of SMMC-7721 cells apoptosis. Caspase-3, caspase-9, cytochrome c, ROS, Bcl-2 and Bax are involved in the inhibition effect of TBA on SMMC - 7721 cells proliferation. TBA may be increase ROS level of SMMC-7721 cells, down-regulate Bcl-2 mRNA expression and up-regulate Bax mRNA expression , decrease mitochondrial membrane potential and induced cytochrome c in mitochondrial of SMMC-7721 cells release to cytosol , activate caspase reaction and then induce SMMC-7721 cell apoptosis, and ultimately inhibited SMMC-7721 cell proliferation.更多还原