葛根素联合5-FU对肝癌细胞SMMC7721作用的研究

发布时间：2018-03-12 23:05

本文选题：化疗　切入点：5-FU　出处：《武汉大学》2015年博士论文　论文类型：学位论文

【摘要】：第一部分葛根素对人肝癌细胞系SMMC7721增殖和凋亡作用的影响及机制研究背景与目的：多个试验研究结果显示葛根素具有抗肿瘤效果,但目前无研究证实其对肝癌细胞的作用,该部分实验的目的是证明葛根素具有抗肝癌细胞的活性,与5-FU联合有协同效应,并探讨其可能的机制。方法：该实验选择的是肝癌细胞系SMMC7721,首先进行细胞的复苏及培养。然后采用CCK-8活体细胞染色吸收法分别检测不同剂量葛根素和5-FU单独或联合治疗对SMMC7721细胞的生长抑制作用。并通过中效原理来评价联合效果。采用细胞凋亡-Hoechst 33258染色试剂盒和annexin V/PI细胞凋亡检测试剂盒分别检测凋亡细胞的形态学特征和凋亡百分率。采用Western blot检测Bax、Bcl-2蛋白表达量。结果：6400μM浓度的葛根素治疗SMMC7721肝癌细胞平均(标准差)生长抑制率达到95.56(0.81)%,而640 μM浓度的葛根素治疗SMMC7721肝癌细胞平均(标准差)生长抑制率也达到了75.91(3.54)%。葛根素与5-Fu联合治疗,当有效率在0.2555和0.7420之间时,联合指数值(CI)小于1。葛根素或5-FU药物治疗组中可以看到亮的染色质凝聚和核破裂；与正常对照组相比,葛根素或5-FU单药治疗组中细胞凋亡率显著增加。而与葛根素或5-Fu单独治疗相比,联合治疗组凋亡细胞的百分率增加更显著。分子水平检测结果显示,Bax蛋白表达量在联合用药组最高,而Bcl-2正好与之相反。结论：葛根素具有抗肝癌细胞的活性,联合5-FU用药能有效抑制肝癌细胞SMMC7721的生长,促进其凋亡。低剂量的葛根素可明显增强5-FU对肝癌细胞抑制作用的敏感性。其可能的机制与Bax蛋白表达量增高,Bcl-2蛋白表达量减少有关。第二部分葛根素联合5-FU对肝癌细胞作用的体内实验研究目的：第二部分实验从体内水平验证了葛根素和5-FU分别具有抗肝癌细胞系SMMC7721的作用,其联合治疗SMMC7721肝癌细胞的疗效更优于葛根素或5-FU单独治疗。方法：本部分研究通过模拟体内实验完成。建立裸鼠SMMC7721肝癌细胞肿瘤种植模型。将葛根素或5-FU分别单独和联合注射到此肝癌种植模型裸鼠,检测此裸鼠肿瘤的体积及重量,计算其肿瘤生长抑制率。HE染色观察形态学变化,通过TUNEL原理用原位细胞凋亡检测试剂盒检测细胞凋亡。同时还观察药物的毒副作用。结果：体内实验结果表明,与葛根素或5-FU单独治疗相比,联合治疗组凋亡细胞的百分率显著增加。葛根素单独治疗组肿瘤体积抑制率是70.58%,5-FU单独治疗组肿瘤体积抑制率为76.26%,而联合治疗组肿瘤体积抑制率为93.11%；葛根素单独治疗组肿瘤重量抑制率是46.20%,5-FU单独治疗组肿瘤重量抑制率为49.86%,而联合治疗组肿瘤重量抑制率为75.21%。AST、ALT等血清学指标没有统计学差异,无明显毒副作用产生。结论：与单独治疗组相比,葛根素联合5-FU对SMMC7721肝癌细胞有显著的生长抑制作用,并促进其凋亡,同时没有增加其毒副作用。这说明葛根素联合5-FU治疗SMMC7721肝癌细胞有协同作用,可增强5-FU对肝癌细胞作用的敏感性,为临床应用葛根素治疗肝癌提供了一定的理论基础。
[Abstract]:The first part of Puerarin effects on human hepatocellular carcinoma cell line SMMC7721 proliferation and apoptosis and its mechanism research background and objective: a number of test results showed that puerarin has anti-tumor effect, but no studies have confirmed on HCC cells, part of the experiment is to prove that puerarin has anti hepatoma activity. And 5-FU has a synergistic effect, and to explore its possible mechanism. Methods: the experiment is the choice of hepatocellular carcinoma cell line SMMC7721 cells were first subjected to recovery and training. Then the CCK-8 cell staining method was used to detect the in vivo absorption of different doses of puerarin and 5-FU treatment alone or in combination on the growth inhibition of SMMC7721 cells and through. The effect of principle to evaluate the combined effect of cell apoptosis by -Hoechst. Apoptotic cells were detected in 33258 staining kit and annexin V/PI apoptosis detection kit The morphological characteristics and the percentage of apoptosis by Western blot detection of Bax, the expression of Bcl-2 protein. Results: 6400 M concentration of Puerarin in the treatment of hepatocellular carcinoma cell SMMC7721 mean (standard deviation) growth inhibition rate reached 95.56 (0.81)%, and 640 M concentration of Puerarin in the treatment of SMMC7721 liver cancer cells mean (standard deviation) the growth inhibition rate reached 75.91 (3.54)% of Puerarin Combined with 5-Fu treatment, when the efficiency between 0.2555 and 0.7420, the joint index (CI) less than 1. puerarin or 5-FU drug treatment group can be seen in chromatin condensation and nuclear bright burst; compared with the normal control group, the apoptosis of Puerarin cytokines or 5-FU monotherapy group was significantly increased. Compared with Puerarin or compared to 5-Fu treatment alone, the percentage of apoptotic cells in the combined treatment group increased significantly. The detection results of molecular level showed that Bax protein expression was the highest in the combination group, and Bcl -2 is the opposite. Conclusion: Puerarin has anti hepatoma activity, in combination with 5-FU can effectively inhibit the growth of SMMC7721 cells and promote its apoptosis. Low dose of Puerarin can significantly enhance the sensitivity of inhibitory effect of 5-FU on liver cancer cells. The possible mechanism and the expression of Bax protein increased and Bcl-2 protein expression the second part to reduce the amount of Puerarin Combined with 5-FU on hepatocellular carcinoma cells in vivo Objective: the second part experiment of puerarin and 5-FU respectively with SMMC7721 against hepatocellular carcinoma cell lines from the body level, the combined treatment of human hepatocellular carcinoma cell line SMMC7721 is better than that of Puerarin or 5-FU alone treatment. Methods: this part the research completed by simulation experiments in vivo. Nude mice SMMC7721 cell tumor model. The puerarin or 5-FU separately and combined injection to liver cancer planting mode Type of nude mice. The volume and weight of the detection of tumors in nude mice, the growth inhibition rate of.HE staining was used to observe the morphological changes of the tumor is calculated through the principle of TUNEL, the cell apoptosis was detected by in situ cell death detection kit. We also observed the side effects of drugs. Results: in vivo experimental results show that with Puerarin or 5-FU alone treatment compared to the percentage the combined treatment group of apoptotic cells increased significantly. Puerarin alone treatment group, the inhibition rate of tumor volume was 70.58% 5-FU, separate the tumor volume inhibition rate was 76.26%, and the combined treatment group tumor volume inhibition rate was 93.11%; puerarin alone treatment group, tumor weight inhibition rate is 46.20%, 5-FU alone treatment group, tumor weight inhibition rate 49.86%, and the combined treatment group tumor weight inhibition rate was 75.21%.AST, no statistically significant differences between the serological markers of ALT, no significant side effects. Conclusion: alone The treatment group compared with Puerarin Combined with 5-FU significantly inhibited the growth of SMMC7721 cells and promote its apoptosis, but did not increase its toxicity. This shows that Puerarin Combined with 5-FU in the treatment of hepatocellular carcinoma SMMC7721 cells have a synergistic effect, can enhance the sensitivity of 5-FU for liver cancer, provides a theoretical basis for the clinical the application of Puerarin in the treatment of liver cancer.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.7

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