毛细管电泳检测木糖醇—亚硒酸酯诱导癌细胞凋亡过程中GSH水平的变化

发布时间：2018-02-04 00:06

本文关键词： 细胞凋亡毛细管电泳谷胱甘肽木糖醇-亚硒酸酯　出处：《华中师范大学》2016年硕士论文　论文类型：学位论文

【摘要】：实验目的：研究木糖醇-亚硒酸酯诱导人肝癌细胞SMMC-7221的凋亡过程中还原型谷胱甘肽(GSH)水平的变化,并探究GSH含量变化与药物浓度及作用时间之间的关系,以此推测该药物可能的抗癌机理。实验方法：首先用木糖醇-亚硒酸酯作用体外培养的SMMC-7221细胞,通过倒置荧光显微镜的形态学和Hoechst 33258的染色观察,寻找到细胞和细胞核形态的变化,进而验证木糖醇-亚硒酸酯具有诱导癌细胞凋亡的作用。再用毛细管电泳-激光诱导的荧光检测方法(CE-LIF)测定不同浓度的药物(0.0.5 mg/L、1mg/L)作用的各组细胞中GSH的含量,比较其含量的差异,找出细胞凋亡与GSH含量之间的关系,以此推测该药物的可能的抗癌机理。实验结果：0.5mg/L木糖醇-亚硒酸酯作用于细胞凋亡的检测实验中,对细胞和细胞核的形态学观察,发现0.5 mg/L药物能使SMMC-7221细胞及其细胞核发生形态学变化,且观察到正在断裂的细胞核等。单因素实验和正交试验应用到CE-LIF实验条件的优化中,选定的最终的实验条件如下：内径大小75μm,有效的长度43 cm,非涂层的毛细管；衍生时间为2 h；缓冲液为10 mM pH 1 1.4 Na2HP04；5-IAF浓度为0.4 mM;分离电压为18 kV；卡盒温度为25℃；进样时间为5s。在该实验条件下,GSH在0-70μM浓度范围内与GSH/NAC面积之比显示出很好的线性关系,确定了GSH/NAC面积之比与GSH浓度的标准曲线。曲线方程为：y =0.06++0.02(R2=0.99).最后,采用非涂层毛细管电泳法,对未加药的对照组,分别加入0.5 mg/L和1mg/L木糖醇-亚硒酸酯的实验组的SMMC-7221细胞进行GSH含量的测定实验。结果发现,在12 h、36h、60 h三个时间段,两实验组中GSH的含量都明显少于对照组,且呈现极显著性差异(**p0.01)。0,0.5 mg/L,1 mg/L三组细胞内GSH的含量分别为:12 h:95.57±19.57.29.09±7.74.24.27±11.15,36 h:70.73±11.35. 19.54±6.39.9.35±6.69,60 h:72.63±16.94.7.43±3.84.0(单位为nmol/mgprotein)。两实验组对比发现,1 mg/L组在每一个时间段的GSH含量都少于0.5 mg/L组,但差异并不明显(p0.05)。细胞内GSH含量的降低率受药物浓度的影响(GSH减少率在69.95±1.87%-100%范围内)大于药物作用时间(GSH减少率在0.22±0.20%-100%范围内)的影响。实验结论：1.木糖醇-亚硒酸酯具有诱导SMMC-7221细胞凋亡的作用,是一种很有潜力的抗癌药物。2.木糖醇-亚硒酸酯诱导SMMC-7221细胞凋亡的过程中伴随着GSH的耗竭,其受药物浓度的影响大于其作用的时间。3.木糖醇-亚硒酸酯可能通过耗竭细胞内GSH而启动相关凋亡信号,诱导癌细胞凋亡。
[Abstract]:Objective: glutathione during apoptosis of human hepatoma SMMC-7221 cells induced by selenite of xylitol in (GSH) levels, and to explore the relationship between GSH content and the change of drug concentration and action time, and speculate the possible anticancer mechanism of drugs. Methods: first of all, using xylitol - selenite in vitro cultured SMMC-7221 cells by morphological and Hoechst inverted fluorescence microscope 33258 staining, to find the changes of cells and cell nucleus, and then verify that xylitol selenite can induce cancer cell apoptosis. The fluorescence detection method by capillary electrophoresis with laser induced (CE-LIF) determination of drugs of different concentrations (0.0.5 mg/L 1mg/L, the content of each cell) in GSH, the difference of its content, to find out the relationship between apoptosis and GSH content, in order to speculate the drug The possible anticancer mechanism. Results: 0.5mg/L xylitol - selenite on cell apoptosis detection experiment, to observe the morphology of cells and nuclei, found that 0.5 mg/L drugs can make the SMMC-7221 cell and nucleus morphological changes occurred, and observed that the nucleus is fracture. The optimization of single factor experiment and orthogonal test application the experimental conditions of CE-LIF, the final selected experimental conditions are as follows: the inner diameter of 75 m, the effective length of 43 cm, the non capillary coating; the reaction time was 2 h; buffer of 10 mM pH 11.4 Na2HP04; 5-IAF concentration was 0.4 mM; the separation voltage of 18 kV; the card box temperature is 25 C; sampling time is 5S. under the experimental conditions, GSH at 0-70 M concentration range and GSH/NAC area ratio shows a good linear relationship, the standard curve of GSH/NAC ratio and GSH concentration area is determined. Song Line equation: y =0.06++0.02 (R2=0.99). Finally, the non capillary electrophoresis method, the control group no drug, the experimental determination of SMMC-7221 cells in the experimental group were added 0.5 mg/L and 1mg/L of xylitol selenite GSH content. The results showed that in 12 h, 60 36h, three h time section two in the experimental group, the content of GSH is significantly less than control group, and showed significant difference (**p0.01).0,0.5 mg/L, 1 mg/L content in three groups of cells were GSH: 12 h:95.57 + 19.57.29.09 + 7.74.24.27 + 11.15,36 h:70.73 + 11.35. 19.54 + 6.39.9.35 + 6.69,60 h:72.63 + 16.94.7.43 + 3.84.0 (nmol/mgprotein). The experimental group found two compared to 1 mg/L group are less than 0.5 in the mg/L group, GSH content in each time period, but the difference was not significant (P0.05). To reduce the content of GSH in the cell rate is affected by drug concentration (GSH reduction rate in 69.95 + 1.87%-100% Within the scope of the drug action time (GSH) is greater than the decrease in the rate of 0.22 within 0.20%-100%). The effects of the experimental results: 1. xylitol - selenite induced SMMC-7221 cell apoptosis, the process is a potential anticancer drug.2. xylitol - selenite induced SMMC-7221 cell apoptosis accompanied by GSH the exhaustion, the influence of the drug concentration is greater than the effect of time.3. xylitol ester selenite may initiate apoptosis related signal by depletion of intracellular GSH, induced apoptosis of cancer cells.

【学位授予单位】：华中师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：O658.9;R96

【相似文献】