【作者】 冯阿磊；

【导师】 刘基巍；

【作者基本信息】 大连医科大学 ， 肿瘤学， 2008， 硕士

【摘要】 目的:探讨人参皂甙Rg3对人乳腺癌细胞株MCF-7表面LeY寡糖抗原及其合成关键酶(FUT1/4)的作用,探讨人参皂甙Rg3抑制肿瘤细胞生长、转移潜能的机制,为临床肿瘤辅助治疗中应用人参皂甙Rg3提供理论依据。方法:体外培养人乳腺癌细胞株MCF-7,采用四甲基偶氮唑蓝比色(MTT)法检测人参皂甙Rg3对人乳腺癌细胞株MCF-7增殖活力的影响;反转录聚合酶链反应(RT-PCR)方法检测人参皂甙Rg3作用后FUT1、FUT4表达情况;细胞免疫荧光检测人参皂甙Rg3作用后LeY寡糖抗原变化情况。结果:1.不同浓度的人参皂甙Rg3作用24h、48h后,对MCF-7细胞增殖的抑制作用:人参皂甙Rg3在浓度为12.5μg/ml、25μg/ml、50μg/ml、100μg/ml、200μg/ml、400μg/ml分别作用于人乳腺癌细胞株MCF-7。24h后抑制率分别为13.36%、26.41%、35.46%、44.19%、48.92%、53.65%。Rg3处理组与空白对照组比较,浓度为50μg/ml以上均明显抑制肿瘤细胞增殖(P<0.05);48h后抑制率分别为25.06%、37.84%、48.40%、54.79%、70.76%、75.68%,25μg/ml以上均有统计学意义(P<0.05)。作用时间上比较,24h和48h人参皂甙Rg3对肿瘤细胞的抑制率无统计学差异(P=0.051)。形态学分析,在倒置显微镜下可见,经药物作用后MCF-7细胞数目减少,细胞形态由原来的贴壁良好、饱满、多角形、梭形变为类圆形、悬浮、颜色变暗、无折光性,且随药物浓度的增加细胞形态变化越大。2.RT-PCR检测Rg3对基因FUT1、FUT4的表达水平:各实验组和空白对照组分别作用MCF-7细胞24h后,提取各组细胞mRNA,然后进行PCR反应。在凝胶成像系统下观察:对照组和药物组均有FUT1、FUT4基因、内参基因β-action的表达。经Lab-work软件分析对照组FUT1、FUT4表达较药物组强,在药物组随浓度增加(12.5μg/ml、25μg/ml、50μg/ml、100μg/ml)FUT1、FUT4表达减弱。对照组与药物组浓度为12.5μg/ml、25μg/ml、50μg/ml相比,无统计学差异(P>0.05);与浓度100μg/ml相比有显著的统计学差异(P=0.01)。药物组各个浓度之间比较:浓度100μg/ml分别与浓度12.5μg/ml、25μg/ml、50μg/ml相比,均有统计学差异(P<0.05),但是12.5μg/ml、25μg/ml、50μg/ml这三个浓度组相比无显著差异(P>0.05)。3.细胞免疫荧光检测LeY表达情况显示:对照组荧光最强,且随药物浓度的增加荧光强度逐渐减弱。药物浓度为100μg/ml组荧光强度最弱。结论:1.人参皂甙Rg3对MCF-7肿瘤细胞增殖有一定的抑制作用,并且随药物浓度的增加,对肿瘤细胞的增殖抑制能力增强。2.合成LeY的关键酶基因FUT1、FUT4随人参皂甙Rg3的作用,表达逐渐减弱,并呈剂量依赖性,有显著的统计学差异。3.MCF-7肿瘤细胞经人参皂甙Rg3作用后,免疫荧光检测显示LeY随药物浓度增加,表达下降。有统计学差异。4.人参皂甙Rg3具有抑制岩藻糖基转移酶(FUT1和FUT4),降低LeY在MCF-7细胞的合成,抑制肿瘤细胞的生长,提示Rg3抗肿瘤作用机制之一。更多还原

【Abstract】 Objective: To study the effect of ginsenoside Rg3 on Ley oligosaccharide antigen and its key enzyme (FUT1/4)in human breast cancer cell line MCF-7, cultured in vitro. It will be helpful to discover the mechanism of ginsenoside Rg3 in anti-tumor and lay a clue for treating cancer.Methods: Human breast cancer cell line MCF-7 was exposed to ginsenoside Rg3 at differential concentrations, respectively. The cell proliferation inhibition was measured by 3-〔4,5-dimethylthiazo-2-yl〕-2,5 diphenyl tetrazolium bromide(MTT) assay. The effect of ginsenoside Rg3 on FUT1 ane FUT4 genes was measured by reverse transcriptase-polymerase chain reaction (RT-PCR) 24 hours after ginsenoside Rg3 exposure. The change of the LeY antigen was examined with immunofluorescence 24 hours after ginsenoside Rg3 treatment.Results:1. Inhibitory effect of ginsenoside Rg3 on proliferation of MCF-7 cells. Human breast cancer cell line MCF-7 was exposed to ginsenoside Rg3 at the concentration of 12.5μg/ml、25μg/ml、50μg/ml、100μg/ml、200μg/ml、400μg/ml respectively. The inhibition ratio was 13.36%、26.41%、35.46%、44.19%、48.92%、53.65%, respectively after 24 hours and statistical significance can be found from the groups 50μg/ml to 400μg/ml Rg3 treatment compared with the control group (p<0.05). The inhibition ratio was 25.06%、37.84%、48.40%、54.79%、70.76%、75.68%, respectively, after 48 hours, statistical significance can be found from Rg groups of 25μg/ml to 400μg/ml (p<0.05). There was no statistical significance between 24 hours and 48 hours. Under an inverted microscope, we observed the cells were adherent, fullness, polygon in control group, the cells amout sparse, suspended, quasi-round, and no refractional nature in experiment groups;the highter the concentration, the greater the morphological change of cells became.2. The expression of FUT1 and FUT4 genes. Human breast cancer cell line MCF-7 cells were cultured between control group and experiment groups, mRNA of all groups cells was extracted after 24 hours. Then RT-PCR reaction was carried on. The genes FUT1 and FUT4 and internal referenceβ- action were expressed in the cells of all groups. The expression of FUT1 and FUT4 genes was high in control group compared with the experiment groups, with the highter the concentration was (12.5μg/ml、25μg/ml、50μg/ml、100μg/ml), the weaker the expression in experiment groups by Lab-work analysis. There was no statistical significance between control group and experiment groups (12.5μg/ml、25μg/ml、50μg/ml) (p>0.05), but markedly statistical significance can be found compare with 100μg/ml (p=0.01). There was markedly statistical significance between 100μg/ml and 12.5μg/ml,25ug/ml or 50μg/ml (p<0.01), but no statistical significance between 12.5μg/ml,25ug/ml and 50μg/ml (p>0.05).3. Immunofluorescence method under the fluorescent microscope, we observed that the strongest fluorescence expression in control group and with increase of the concentration of Rg3, the strength of fluorescence was gradually decreased. The weakeast fluorescence strength is 100ug/ml in experiment group.Conclusion:1. Ginsenoside Rg3 has anti-tumor effect on human breast cancer cell line MCF-7, the highter the concentration is, the greater the inhibitory effect on tumor cells proliferation will come. No statistical significance was found between Rg3 treatment in 24 hours and 48 hours.2. The expression of fucosyltransferase (FUT1/4) gene was higher in control group compared with that of Rg3 groups, ginsenoside Rg3 can down regulate the expression of FUT1/4gene. There was markedly statistical significance with the increased concentration of ginsenoside Rg3.3. Immunofluorescence show that the expression of LeY antigen decrease with increase of the concentration of Rg3. There was markedly statistical significance.4. Ginsenoside Rg3 can down regulate the expression of Fut1/4 gene and decrease the synthesis of Ley antigen, the resore inhibit the growth of tumor cells. This may supply an evidence of the important mechanisms related ginsenoside Rg3 in anti-tumor .更多还原