【作者】 王志强；

【导师】 杨兰；

【作者基本信息】 兰州大学 ， 口腔临床医学， 2018， 硕士

【摘要】 目的:1.初步探讨红景天苷(Salidroside)对人舌鳞癌CAL27细胞增殖抑制率的影响;2.红景天苷对CAL27细胞凋亡及形态的影响;3.红景天苷对CAL27细胞周期的影响;4.红景天苷对CAL27细胞迁移的影响;5.红景天苷对CAL细胞Bcl-2、Bax、VEGF、P65蛋白表达的影响及相关机制。方法:1.利用MTT比色法观察Salidroside对CAL27细胞增殖的抑制作用并筛选药物作用浓度。2.通过Annexin V/PI双染、流式细胞术检侧红景天苷处理后的CAL27细胞早期凋亡率、晚期凋亡率及坏死率。3.通过Hoechst33258荧光染色法观察红景天苷诱导凋亡时CAL27细胞形态变化。4.利用流式细胞术检测经处理后CAL27细胞的周期分布情况。5.利用Transwell小室实验观察红景天苷对CAL27细胞迁移的作用及影响。6.通过Western blot实验检测处理后CAL27细胞中Bcl-2、Bax、VEGF、P65蛋白的表达情况。结果:1.MTT检测不同浓度组红景天苷(0、20、40、60、80、100μg/mL)对CAL27细胞增殖的影响;作用24h和48h的IC50分别为133μg/mL/70.1μg/mL;同浓度红景天苷作用48h的抑制率高于24h;在时间一定的条件下,伴随药物浓度的升高,对CAL27细胞的抑制率显著增加;其结果具有显著性统计学差异(p<0.05);结果表明红景天苷对CAL27细胞增殖抑制作用呈时间和浓度依赖性。2.通过Ho-echst33258荧光染色细胞形态观察发现随着红景天苷药物浓度的增加,凋亡细胞比例增加,细胞总数呈负相关。红景天苷干预48h后,流式细胞术检测结果表明随着药物浓度的增加,诱导CAL27细胞发生早期凋亡、晚期凋亡及总凋亡所占比例显著增加,且各药物浓度组间凋亡结果存在显著性统计学差异(p<0.05)。3.不同浓度红景天苷作用于CAL27细胞48h后,流式细胞仪检测周期变化结果表明:各药物浓度均能诱导CAL27细胞周期发生变化,且随着药物浓度增加,G0/G1期比例增加,S期及G2/M期比例下降,从而表明红景天苷可使CAL27细胞发生G0/G1期阻滞。4.采用Transwell小室实验结果发现随红景天苷药物浓度的增加,CAL27细胞穿过小室基底膜的数量明显减少,迁移能力亦受到较显著影响(p<0.05)。5.红景天苷对CAL27细胞Bcl-2、Bax、VEGF、P65蛋白表达的结果显示:不同浓度红景天苷作用CAL27细胞后,以上蛋白表达水平发生改变,随着药物浓度的增加,Bax表达水平均升高,Bcl-2、VEGF及P65表达水平均降低(p<0.05)。结论:红景天苷对人舌鳞癌CAL27细胞的增殖、凋亡、周期及迁移均产生影响,可诱导CAL27细胞显著凋亡,发生G0/G1期周期阻滞,抑制迁移能力,具有显著的体外抑制作用。更多还原

【Abstract】 Objective: 1.To explore the effect of Salidroside on proliferation of CAL27 cells.2.To explore the inducing apoptosis effect of Salidroside on CAL27 cells.3.To explore the effect of Salidroside on cell cycle distribution of CAL27 cells.4.Effect of salidroside on the migration of CAL27 cells.5.Effect of salidroside on the expression of Bcl-2,Bax,VEGF and P56 in CAL27 cells and related mechanisms.Methods: 1.MTT assay was used to evaluate the effect of Salidroside on prolif-eration of CAL27 cells and selected the effect of drug concentration.2.The early apoptosis rate,late apoptosis rate,and necrotic rate of CAL27 cells treated with salidroside were detected by Annexin V/PI double staining and flow cytometry.3.Hoechst33258 fluorescence staining observed CAL27 morphological changes of apoptosis.4.The distribution of CAL27 cells in the treated cells was detected by flow cytometry.5.Transwell chamber experiment was used to observe the effect of salidroside on migration of CAL27 cells.6.Western blot was used to detect the expression of Bcl-2,Bax,VEGF and P56 in CAL27.Results:1.The effect of different concentrations of salidroside(0,20,40,60,80,100μg/mL)on the proliferation of CAL27 cells was detected by MTT.The IC50 at 24 h and 48 h respectively were 133μg/m L/70.1μg/m L,.The inhibition rate of salidroside at the same concentration for 48 h was higher than 24 h;at the same time,the inhibitory rate of the cells was significantly increased with the increase of drug concentration;the above results were statistically significant(p<0.05);The effect of salidroside on proliferation of CAL27 cells was time and concentration dependent.2.Observed by Ho-echst33258 fluorescent staining of cell morphology observed with the salidroside drug concentration increased,the proportion of apoptotic cells also increased,the total number of cells was negatively correlated.After 48 h of salidroside treatment,the results of flow cytometry showed that the proportion of early apoptosis,late apoptosis and total apoptosis of CAL27 cells all increased significantly with the increase of drug concentration,and there was a significant difference in the apoptosis rate of each concentration group.(p<0.05).3.After 48 h of treatment with different concentrations of salidroside on CAL27 cells,the results of flow cytometry showed that the concentration of CAL27 could be changed by each drug concentration,and the proportion of G0/G1 phase increased with increasing drug concentration.Phase and G2 / M phase ratio decreased,indicating that salidroside can make CAL27 cells G0 / G1 phase arrest.4.Transwell chamber experiments showed that with the increase of salidroside drug concentration,the number of cells passing through the basement membrane significantly reduced,and the migration ability of CAL27 cells was significantly affected(p <0.05).5.The results of salidroside on the expression of Bcl-2,Bax,VEGF and P65 protein in CAL27 cells showed that the expression of above proteins was changed after Salidroside of different concentrations were treated with CAL27 cells.With the increase of drug concentration,the expression levels of Bax The expression of Bcl-2,VEGF and P65 decreased(p <0.05).Conclision: Salidroside has effects on the proliferation,apoptosis,cycle and migration of human tongue squamous cell carcinoma CAL27 cells,induces apoptosis of CAL27 cells,induces G0/G1 phase arrest,inhibits migration,and has significant inhibition effects in vitro..更多还原