本文關鍵詞： 細胞凋亡 毛細管電泳 谷胱甘肽 木糖醇-亞硒酸酯　出處：《華中師范大學》2016年碩士論文　論文類型：學位論文

【摘要】：實驗目的：研究木糖醇-亞硒酸酯誘導人肝癌細胞SMMC-7221的凋亡過程中還原型谷胱甘肽(GSH)水平的變化,并探究GSH含量變化與藥物濃度及作用時間之間的關系,以此推測該藥物可能的抗癌機理。實驗方法：首先用木糖醇-亞硒酸酯作用體外培養(yǎng)的SMMC-7221細胞,通過倒置熒光顯微鏡的形態(tài)學和Hoechst 33258的染色觀察,尋找到細胞和細胞核形態(tài)的變化,進而驗證木糖醇-亞硒酸酯具有誘導癌細胞凋亡的作用。再用毛細管電泳-激光誘導的熒光檢測方法(CE-LIF)測定不同濃度的藥物(0.0.5 mg/L、1mg/L)作用的各組細胞中GSH的含量,比較其含量的差異,找出細胞凋亡與GSH含量之間的關系,以此推測該藥物的可能的抗癌機理。實驗結果：0.5mg/L木糖醇-亞硒酸酯作用于細胞凋亡的檢測實驗中,對細胞和細胞核的形態(tài)學觀察,發(fā)現0.5 mg/L藥物能使SMMC-7221細胞及其細胞核發(fā)生形態(tài)學變化,且觀察到正在斷裂的細胞核等。單因素實驗和正交試驗應用到CE-LIF實驗條件的優(yōu)化中,選定的最終的實驗條件如下：內徑大小75μm,有效的長度43 cm,非涂層的毛細管；衍生時間為2 h；緩沖液為10 mM pH 1 1.4 Na2HP04；5-IAF濃度為0.4 mM;分離電壓為18 kV；卡盒溫度為25℃；進樣時間為5s。在該實驗條件下,GSH在0-70μM濃度范圍內與GSH/NAC面積之比顯示出很好的線性關系,確定了GSH/NAC面積之比與GSH濃度的標準曲線。曲線方程為：y =0.06++0.02(R2=0.99).最后,采用非涂層毛細管電泳法,對未加藥的對照組,分別加入0.5 mg/L和1mg/L木糖醇-亞硒酸酯的實驗組的SMMC-7221細胞進行GSH含量的測定實驗。結果發(fā)現,在12 h、36h、60 h三個時間段,兩實驗組中GSH的含量都明顯少于對照組,且呈現極顯著性差異(**p0.01)。0,0.5 mg/L,1 mg/L三組細胞內GSH的含量分別為: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(單位為nmol/mgprotein)。兩實驗組對比發(fā)現,1 mg/L組在每一個時間段的GSH含量都少于0.5 mg/L組,但差異并不明顯(p0.05)。細胞內GSH含量的降低率受藥物濃度的影響(GSH減少率在69.95±1.87%-100%范圍內)大于藥物作用時間(GSH減少率在0.22±0.20%-100%范圍內)的影響。實驗結論：1.木糖醇-亞硒酸酯具有誘導SMMC-7221細胞凋亡的作用,是一種很有潛力的抗癌藥物。2.木糖醇-亞硒酸酯誘導SMMC-7221細胞凋亡的過程中伴隨著GSH的耗竭,其受藥物濃度的影響大于其作用的時間。3.木糖醇-亞硒酸酯可能通過耗竭細胞內GSH而啟動相關凋亡信號,誘導癌細胞凋亡。
[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.

【學位授予單位】：華中師范大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：O658.9;R96

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