本文選題：雙氫楊梅樹皮素　切入點：人視網(wǎng)膜母細胞瘤細胞　出處：《廣西醫(yī)科大學》2011年碩士論文　論文類型：學位論文

【摘要】：目的:通過檢測雙氫楊梅樹皮素(APS)對體外培養(yǎng)的人視網(wǎng)膜母細胞瘤細胞株(HXO-RB44)增殖的影響,觀察細胞凋亡、線粒體跨膜電位、細胞內(nèi)活性氧(ROS)的變化,探討APS誘導人視網(wǎng)膜母細胞瘤(RB)細胞凋亡的氧化作用機制。 方法:取對數(shù)生長期的人HXO-RB44進行實驗。1.觀察APS干預下細胞的生長曲線:設7個不同藥物濃度(50.00、33.33、22.22、14.84、9.90、6.60、4.40)μg/ml和空白對照組,分別作用0、24、48、72h后,繪制生長曲線圖。2.觀察APS對細胞增殖的抑制作用:7個濃度的APS干預RB細胞24h后,觀察其形態(tài)學變化, MTT法、軟瓊脂克隆形成法觀察RB細胞的體外抑制作用。3.AO/EB染色觀察細胞凋亡形態(tài)學變化:選取3個不同濃度(50.00、14.84、4.40)μg/ml的APS干預RB細胞24h后,染色,熒光顯微鏡觀察,并拍照。4.流式細胞儀檢測細胞凋亡:設3個藥物濃度組(50.00、14.84、4.40)μg/ml和空白對照組,作用24h后,用流式細胞儀檢測細胞凋亡情況。5.流式細胞儀檢測線粒體跨膜電位及ROS的變化:選取對數(shù)生長期細胞培養(yǎng),設3個藥物濃度組和空白對照組培養(yǎng)24h后,染色,流式細胞儀檢測線粒體跨膜電位及ROS的變化。 結果:1.APS干預后,RB細胞的生長受到明顯抑制,隨著藥物濃度的增加呈劑量依賴性;作用24h時抑制作用最明顯。2.通過倒置顯微鏡可觀察到RB細胞出現(xiàn)形態(tài)學的變化。細胞的IC50為14.71μg/ml;藥物濃度為9.9μg/ml以上時單細胞克隆形成抑制率為100㳠,藥物濃度為4.4μg/ml,抑制率為52.2㳠。3.AO/EB熒光染色,藥物作用后出現(xiàn)了明顯的凋亡細胞,表現(xiàn)為細胞皺縮、核染色質濃縮、核碎裂等凋亡特征性的形態(tài)學改變。4.APS對細胞凋亡有明顯的誘導作用。藥物作用24h后的凋亡率分別為88.13%、58.46%、14.83%,各濃度組凋亡率與空白對照組(4.82%)比較,差異均有顯著性(P0.05),各濃度組細胞凋亡率之間差異也均有顯著性(P0.05)。5.APS能降低RB細胞線粒體跨膜電位。APS處理24h后不同濃度組(50.00、14.84、4.40、0)μg/ml的平均熒光強度分別為20.70、55.63、88.57和146.53,藥物組與空白對照組比較差別有統(tǒng)計學意義( P均 0. 01),三個濃度之間差異有統(tǒng)計學意義( P 0. 01)。6.APS能促進RB細胞內(nèi)ROS的產(chǎn)生。APS處理24h后不同濃度組(50.00、14.84、4.40、0)μg/ml的平均熒光強度分別為54.87、43.03、36.80和20.93,藥物組與空白對照組比較差別有統(tǒng)計學意義( P均 0. 01),三個濃度之間差異有統(tǒng)計學意義( P 0. 01)。 結論:1.APS對RB細胞的體外增殖有明顯的抑制作用。2.APS可誘導體外培養(yǎng)的RB細胞凋亡。3.APS誘導RB細胞凋亡與線粒體內(nèi)跨膜電位的下降、細胞內(nèi)ROS產(chǎn)生增加有關。
[Abstract]:Aim: to investigate the effects of dihydromyricetin (APS) on the proliferation of human retinoblastoma cell line HXO-RB44 in vitro, and to observe the changes of apoptosis, mitochondrial transmembrane potential and intracellular reactive oxygen species (Ros). To investigate the oxidative mechanism of APS induced apoptosis of human retinoblastoma (RBB) cells. Methods: human HXO-RB44 in logarithmic growth period was taken for experiment. 1. Observe the growth curve of human HXO-RB44 in logarithmic growth phase. Observe the growth curve of cells treated with APS: set up 7 different concentrations of 5 0.0033X 33.33C 22.2222 14.84 渭 g / ml and control group 6.604.40) 渭 g / ml and control group, respectively, after 72 hours of treatment, the cells were exposed to 0.24 渭 g / ml and 0.24 渭 g / ml, respectively. To observe the inhibitory effect of APS on the proliferation of RB cells: 7 concentrations of APS were used to observe the morphological changes of RB cells after 24 hours of intervention, and MTT method was used to observe the effects of APS on the proliferation of RB cells. The inhibitory effect of RB cells in vitro was observed by soft Agar Clone formation method. 3. AO- / EB staining was used to observe the morphological changes of apoptosis. Three different concentrations of APS (50.004 ~ 14.84 渭 g / ml) were selected to interfere with RB cells for 24 h, then stained and observed by fluorescence microscope. Cell apoptosis was detected by flow cytometry: three drug concentration groups were divided into three groups (50.0044.40) 渭 g / ml and blank control group (control group). After 24 hours of treatment, the cell apoptosis was detected by flow cytometry. Cell apoptosis was detected by flow cytometry .5.The changes of mitochondrial transmembrane potential and ROS were detected by flow cytometry. The cells were cultured in logarithmic growth phase and cultured for 24 hours in three drug concentration groups and blank control group. The changes of mitochondrial transmembrane potential and ROS were detected by flow cytometry. Results 1. The growth of RB cells was significantly inhibited after intervention with APS in a dose-dependent manner with the increase of drug concentration. Morphological changes of RB cells were observed by inverted microscope. The IC50 of RB cells was 14.71 渭 g / ml, and the inhibition rate of single cell clone formation was 100 渭 g / ml when drug concentration was more than 9.9 渭 g / ml. The drug concentration was 4.4 渭 g / ml and the inhibition rate was 52.2? 3. AO- / EB fluorescence staining showed that apoptotic cells appeared after drug treatment, which showed cell shrinkage and nuclear chromatin concentration. The apoptotic characteristic morphologic changes such as nuclear fragmentation. 4. APS could induce apoptosis obviously. The apoptotic rate of APS was 88.13 ~ 58.46% and 14.83% respectively after 24 hours of treatment. The apoptotic rate of each concentration group was compared with that of the blank control group (4.82%). There were significant differences in apoptosis rate between different concentration groups. P0.05N. 5. APS could decrease mitochondrial transmembrane potential of RB cells. APS treatment for 24 hours, the average fluorescence intensity of different concentration groups was 20.7055.63 88.57 渭 g / ml and 146.53 渭 g / ml, respectively. There was significant difference between the white control group and the control group (P = 0.01, P = 0.01, P < 0.01). 6. APS could promote the production of ROS in RB cells. After 24 hours of treatment with APS, the average fluorescence intensity of the three concentration groups was 50.0014. 844.40%) 渭 g / ml, respectively. The difference between the drug group and the blank control group was statistically significant (P all 0.01), and the difference between the three concentrations was statistically significant (P 0.01). Conclusion: 1. APS can inhibit the proliferation of RB cells in vitro. 2. APS can induce RB cell apoptosis in vitro. 3. APS induced RB cell apoptosis is related to the decrease of mitochondrial transmembrane potential and the increase of intracellular ROS production.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R739.7
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本文編號：1602743