硒酸酯多糖联合羟基喜树碱对HepG-2细胞生长及细胞周期的影响

发布时间：2018-06-18 02:16

本文选题：硒酸酯多糖(KSC) + 羟基喜树碱(10-HCPT)　；参考：《吉林大学》2017年硕士论文

【摘要】：在肿瘤化疗过程中,化疗药物的单独应用往往不能有效地杀死肿瘤,为了提高治疗效果,人们在临床上多使用联合用药方案,主要是通过选择两种或多种针对不同目标靶点的药物联合来遏制肿瘤。硒是一种非金属,它是机体每日所摄入的必要微量元素之一,它可以抗氧化,还会对恶性肿瘤细胞死亡和代谢有影响,从而发挥防癌、抗癌的重要作用。硒酸酯多糖(Kappa-selenocarrageenan,KSC))是一种人工制备的卡拉胶的硒化产物,具有非常显著的抗肿瘤活性。羟基喜树碱(10-hy droxycamptothecin,10-HCPT)具有较强的抗癌作用。10-HCPT的作用机制通常是通过影响恶性肿瘤细胞的拓扑异构酶I(Topo-Ⅰ),所以也被称为Topo-Ⅰ特异抑制剂。拓扑异构酶I能使DNA的一条链发生断裂和再连接。10-HCPT抑制拓扑异构酶I在DNA合成中的作用,阻碍DNA的生成,最后使双链DNA发生断裂等情况。从而抑制肿瘤细胞的生存。本实验以人类肝癌Hep G-2细胞为研究对象,检测KSC与羟基喜树碱联合对Hep G-2细胞的效应,为探讨肝癌综合治疗方案提供新的理论依据。本研究采用MTT法考察硒酸酯多糖与羟基喜树碱单独使用及联合使用对Hep G-2细胞的生长抑制作用,建立回归方程并求中效浓度IC50,同时通过计算q值判断联合用药效果;使用倒置显微镜及荧光显微镜来观察两种药物单独使用及联合使用对细胞形态的影响;使用流式细胞仪分析细胞周期及凋亡率;使用流式细胞仪及Western Blot法检测细胞S期周期蛋白Chk2、Cyclin A、Cdc25A和Cdk2含量的变化。MTT结果显示硒酸酯多糖与羟基喜树碱均具有明显的抑制Hep G-2细胞生长作用,且具有时间—剂量的依赖性,联合用药组较2种药物单独组作用更加显著。硒酸酯多糖和羟基喜树碱单独作用72小时时IC50分别为46.79mg/L和1.70nmol/L,1nmol/L 10-HCPT和30mg/L KSC联合作用72小时后IC50降至27.89mg/L和0.15nmol/L。硒酸酯多糖和10-HCPT联合使用后得出的q值大于0.85小于1.15,这说明联合用药具有协同效应。倒置显微镜观察结果显示对照组细胞基本都贴壁生长,胞集落生长特性明显。给药组使用药物48h之后,恶性肿瘤细胞体积变小、形状边缘、细胞间隙变大、甚至出现细胞脱落等情况。还有一部分的细胞体积骤然增大,细胞膜破裂呈现坏死状态。其中联合给药组活细胞数少于单独给药组。荧光显微镜观察细胞结果显示,药物作用于细胞48小时后,可以发现一些细胞出现的典型的形态学改变:细胞核凝固缩小,染色质出现凝集甚至片段化,致密强荧光及凋亡小体形成等;但联合给药组与单独给药组凋亡细胞数并无明显差异。流式细胞仪得出的结论是,硒酸酯多糖和10-HCPT主要影响Hep G-2细胞细胞周期的G1期下降、S期上升,而G2/M期无明显的变化。联合用药之后,G1期会下降,而S期明显上升,这不难看出硒酸酯多糖及10-HCPT均能影响细胞S期。硒酸酯多糖组24小时时凋亡率为零,但48小时时出现凋亡峰。羟基喜树碱组细胞死亡率会受药物浓度和作用时间的影响,即时间—剂量依赖性。联合用药组的细胞死亡个数要低于单独给药组,但是联合用药组S期细胞比例数值比单独给药组要高,说明硒酸酯多糖与羟基喜树碱联合用药主要通过诱导细胞周期阻滞从而抑制肿瘤细胞生长。在硒酸酯多糖与羟基喜树碱联合诱导Hep G-2细胞S期阻滞机制研究中,流式细胞仪检测结果显示用药48小时后,空白组Cyclin A蛋白表达量为52.2%,1nmol/L10-HCPT、120mg/L KSC和1nmol/L 10-HCPT+120mg/L KSC各组Cyclin A蛋白表达分别为61.5%、89.5%和91.1%;空白组Chk2蛋白表达为49.6%,其余各组Chk2蛋白表达分别为58.1%、91.3%和92.2%,和对照组进行比较,可以看出存在一定的差异,具有统计学意义(P0.01),联合用药组和羟基喜树碱单独用药组具有明显差异,和硒酸酯多糖组没有显著改变。Western Blot结果显示给药后细胞内Cdc25A和Cdk2蛋白表达量均下降,联合给药组表达量最低。综上所述:硒酸酯多糖与羟基喜树碱联合对Hep G-2细胞具有相加的生长抑制作用,并且可诱导Hep G-2细胞S期阻滞。硒酸酯多糖与羟基喜树碱诱导Hep G-2细胞S期阻滞的原因为联合用药激活了细胞周期S期检控点,Chk2被激活后催化Cdc25A,并使其磷酸化,甚至出现降解。这样Cdc25A就无法去掉Cdk2上的位点,Cdk2激活受到阻碍,不能很好的结合Cyclin A,由此引发S期被抑制的情况,这就影响了整个细胞周期。
[Abstract]:In the course of tumor chemotherapy, the individual use of chemotherapeutic drugs can not effectively kill the tumor. In order to improve the therapeutic effect, people use a combination of drugs in clinical practice, mainly by choosing two or more drugs combined with different target targets to prevent cancer. Selenium is a non metal, it is the daily intake of the body. One of the essential trace elements, it can be antioxidant, and also affects the death and metabolism of malignant tumor cells, which plays an important role in cancer prevention and cancer. Selenate polysaccharide (Kappa-selenocarrageenan, KSC)) is a prepared carrageenan selenide product with very significant antitumor activity. Hydroxycamptothecin (10-hy droxyc). Amptothecin, 10-HCPT), which has a strong anti-cancer effect of.10-HCPT, is usually known as a specific inhibitor of I (Topo- I) affecting the malignant tumor cells, so it is also known as a specific inhibitor of Topo- I. Topoisomerase I can break a chain of DNA and re connect the.10-HCPT inhibition of topoisomerase I in DNA synthesis. Hinder the formation of DNA and finally make the double strand DNA break and so on. Thus inhibiting the survival of tumor cells. In this experiment, the effects of KSC and hydroxycamptothecin on Hep G-2 cells were detected by the combination of KSC and Hydroxycamptothecin in human hepatoma Hep G-2 cells, and a new theoretical basis was provided to explore the comprehensive treatment scheme for liver cancer. This study was used to investigate selenate ester by MTT method. The inhibitory effect of polysaccharide and hydroxycamptothecin on the growth of Hep G-2 cells, the regression equation was established and the medium effect concentration IC50 was established, and the effect of combined use of Q was calculated by calculating the value of Q; the effects of the two drugs alone and combined use on the cell morphology were observed by the inverted microscope and the fluorescence microscope. Cell cycle and apoptosis rate were analyzed by cytometer. The changes of S periodic protein Chk2, Cyclin A, Cdc25A and Cdk2 content were detected by flow cytometer and Western Blot method. The results showed that both selenate polysaccharide and hydroxycamptothecin had obvious inhibition of Hep G-2 cell growth and had time and dose dependence and combined use. The effect of the drug group was more significant than that of the 2 drugs alone. IC50 was 46.79mg/L and 1.70nmol/L respectively when selenate polysaccharide and hydroxycamptothecin acted alone for 72 hours. After the combination of 1nmol/L 10-HCPT and 30mg/L KSC for 72 hours, IC50 was reduced to 27.89mg/L and 0.15nmol/L. selenate polysaccharide was combined with 10-HCPT, and the Q value was less than 0.85 less than 1.15, This shows that the combined use of drugs has synergistic effects. The results of the inverted microscope show that the cells in the control group are basically adhered to wall growth and the colony growth characteristics are obvious. After the use of drug 48h in the drug group, the volume of the malignant tumor cells becomes smaller, the shape edge, the cell gap becomes larger, even the cell fall off. However, the number of living cells in the combined drug group was less than that of the single drug group. The results of the fluorescence microscope observation showed that after 48 hours the drug acted on the cell, the typical morphological changes of some cells could be found: the nuclear coagulation was reduced, the chromatin was agglutinated and even fragmented, and the density was strong. Fluorescent and apoptotic bodies were formed, but there was no significant difference in the number of apoptotic cells in the combined administration group and the individual group. The flow cytometer concluded that selenate polysaccharide and 10-HCPT mainly affected the G1 phase of the cell cycle of Hep G-2 cells, the S phase increased and the G2/M phase had no obvious changes. After the combination, the G1 phase decreased and the S phase was obvious. Up, it is not difficult to see that both selenate polysaccharide and 10-HCPT can affect the cell S phase. The apoptosis rate of the selenate polysaccharide group is zero at 24 hours, but the apoptotic peak is found at 48 hours. The cell mortality of HCPT group is affected by the drug concentration and time, that is, time dose dependence. Only drug group, but the proportion of S phase cells in the combination group is higher than that in the single drug group. It shows that the combination of selenate polysaccharide and hydroxycamptothecin can inhibit the growth of tumor cells mainly by inducing cell cycle arrest. In the study of the S phase blocking mechanism of Hep G-2 cells induced by selenate polysaccharide and hydroxycamptothecin, flow cytometry After 48 hours, the expression of Cyclin A protein in blank group was 52.2%, 1nmol/L10-HCPT, 120mg/L KSC and 1nmol/L 10-HCPT+120mg/L KSC were expressed as 61.5%, 89.5% and 91.1%, and Chk2 protein in the blank group was 49.6%. The expression of Chk2 protein in the other groups was 58.1%, 91.3% and 92.2%, respectively, and the control group entered the control group. Comparison, we can see that there is a certain difference, with statistical significance (P0.01), the combination group and hydroxycamptothecin alone group has obvious differences, and selenate polysaccharide group has no significant changes in.Western Blot results showed that the expression of Cdc25A and Cdk2 protein in the cells decreased, the combination of drug group expression was the lowest. The combination of selenate polysaccharide and hydroxycamptothecin can inhibit the growth of Hep G-2 cells, and induce the S phase block of Hep G-2 cells. The reason for the S phase arrest of Hep G-2 cells induced by selenate polysaccharide and hydroxycamptothecin is to activate the S phase control point of the cell cycle by combined use of drugs, and Chk2 is activated and catalyzes Cdc25A, and its phosphorus is activated. Acidification, even degradation. So Cdc25A can not remove the site on Cdk2, Cdk2 activation is hindered, not a good combination of Cyclin A, resulting in the inhibition of the S phase, which affects the whole cell cycle.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

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