染料木素对人胰腺癌细胞株PANC-1的作用研究

发布时间：2018-01-15 21:26

本文关键词：染料木素对人胰腺癌细胞株PANC-1的作用研究　出处：《山西医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:观察不同浓度染料木素对人胰腺癌细胞株PANC-1作用及其抑癌基因表达的的研究,从而为临床治疗胰腺癌提供新的思路方法。方法:本实验共分实验组(80,120,160,200μmol/L)的染料木素、空白对照组(PBS)和标准对照(吉西他滨)。各组培养基处理细胞12h、24h、36h、48h、60h后,细胞计数试剂盒(CCK-8)法检测人胰腺癌细胞株PANC-1在染料木素作用下的增殖活性;流式细胞术检测人胰腺癌细胞株PANC-1在染料木素木素作用下的凋亡细胞百分率;用逆转录-聚合酶连反应(RT-PCR)方法测定P53及P21抑癌基因的表达;Western blot法检测抑癌基因P53和P21的蛋白表达水平。实验结果统计学分析:细胞凋亡与细胞基因m RNA采用的是LSD法,细胞增殖数据采用的是配对t检验,P0.05比较差异有统计学意思。结果:1.CCK-8法检测染料木素对胰腺癌细胞(PANC-1)的生长抑制作用分别对160μmol/L与200μmol/L、120μmol/L与200μmol/L、160μmol/L与GEM组、200μmol/L与GEM组通过SPSS 13.0进行配对样本t检验,结果显示160μmol/L对GEM组无统计学差异(P=0.067),而其余三组P值均小于0.05;针对36h以内的数据,统计发现120μmol/L对GEM组P值亦小于0.05,而200μmol/L与GEM组无统计学差异(P=0.051)。根据以上结果,我们认为染料木素总体呈浓度依赖性和时间依赖性,其中36h内200μmol/L效果与GEM组较为接近,但160μmol/L总体效果最好,且与GEM组最为接近。2.流式细胞仪(FCM)各组凋亡指数比较FCM结果可以看出,4个染料木素分组中,80μmol/L对细胞增殖抑制效果并不明显,而120μmol/L和160μmol/L最接近GEM组的效果,200μmol/L晚期凋亡细胞占大部分。可见染料木素的作用有明显的浓度依赖性。3.Western bloting检测P53、P21等相关蛋白表达Western-blot检测蛋白水平显示160μmol/L和200μmol/L染料木素相对于空白对照能够明显的增强抑癌基因P21和P53的蛋白水平的表达,相对于标准组没有明显的差异。结论:1.通过FCM和CCK-8法表明染料木素作用具有浓度依赖性和时间依赖性,但是在低浓度时,染料木素的作用并不理想,而当浓度达到160μmol/L以上时其FCM结果与标准对照组吉西他滨非常接近,浓度达到200μmol/L时晚期凋亡细胞已占60%以上2.CCK-8法细胞增殖的结果显示160μmol/L的抑制效果在48h达到最佳,整体效果最好且曲线与标准对照相近,而Western-blot结果显示染料木素对P21、P53蛋白均有明显上调作用,浓度依赖性小。3.我们推断浓度为160μmol/L染料木素对胰腺癌细胞株PANC-1的抑制作用最佳,其可能通过下调细胞增殖、细胞周期相关基因以及上调P21、P53蛋白表达,使染料木素对胰腺癌细胞株PANC-1体外增殖的抑制作用表现出浓度及时间依赖性,从而发挥抑制癌细胞作用。
[Abstract]:Objective: To investigate the effects of genistein on the function of PANC-1 in human pancreatic cancer cell lines and expression of tumor suppressor genes, so as to provide new ideas for clinical treatment of pancreatic cancer. Methods: the experiment was divided into experimental group (80120160200 mol/L) of genistein, blank control group (PBS) and control (standard gemcitabine) and cultured 12h cells. Each treatment, 36h, 48h, 24h, 60H, cell counting Kit (CCK-8) method for the detection of human pancreatic cancer cell line PANC-1 in genistein under the effect of proliferation; flow cytometry of human pancreatic cancer cell line PANC-1 in dye lignin under the action of apoptosis percentage; reverse transcriptase polymerase chain reaction (RT-PCR) method for the determination of the expression of P53 and P21 tumor suppressor gene; Western blot method to detect the expression of tumor suppressor gene P53 and P21 protein levels. Statistical analysis results: cell apoptosis and expression of M gene RNA Using the LSD method. The cell proliferation data using the paired t test, P0.05 difference was statistically. Results: 1.CCK-8 assay of genistein on pancreatic cancer cells (PANC-1) growth inhibition of 160 mol/L and 200 mol/L, 120 mol/L and 200 mol/L, 160 mol/L and GEM group 200 mol/L GEM group and SPSS 13 paired samples t test, the results showed that 160 mol/L had no significant difference in GEM group (P=0.067), while the remaining three group P values were less than 0.05; for 36h within the data statistics found that 120 mol/L is less than 0.05 of the GEM group P, and 200 mol/L and GEM group had no statistical difference (P=0.051). According to the above results, we believe that genistein showed a dose-dependent and time-dependent manner, the effect of mol/L 200 in 36h and GEM group is close to 160 mol/L, but the overall effect is the best, and the GEM group is most close to the.2. flow cytometry (FCM) The apoptotic index compared with FCM results, 4 genistein group, 80 mol/L on cell proliferation inhibition effect is not obvious, while the 120 mol/L and 160 mol/L closest to the effect of group GEM, 200 mol/L of late apoptotic cells accounted for the majority. The effect of concentration of genistein showed significant dependence on.3.Western bloting detection of P53, P21 and other related protein expression level of Western-blot was detected 160 mol/L and 200 mol/L genistein compared with blank control can enhance the protein expression of tumor suppressor gene P21 and P53 significantly, compared with no significant differences between the standard group. Conclusion: 1. through FCM and CCK-8 method showed that genistein had concentration and time dependent manner, but at low concentrations, genistein's effect is not ideal, and when the concentration reaches above 160 mol/L the results of FCM and gemcitabine non standard group Very close to, the concentration reached 200 mol/L in late apoptotic cells accounted for more than 60% 2.CCK-8 cell proliferation showed inhibitory effect of 160 mol/L in 48h to achieve the best overall effect, and the best curve with the standard of photography, and Western-blot results showed that genistein on P21, P53 protein were significantly up-regulated, concentration dependent we conclude that the small.3. concentration of 160 mol/L inhibitory effects of genistein on human pancreatic cancer cell line PANC-1, it may down regulate cell proliferation, cell cycle related genes and upregulation of P21, P53 protein expression, so that genistein inhibits proliferation of human pancreatic cancer cell line PANC-1 in vitro showed a concentration and time dependent, so the inhibition of cancer cells.

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.9

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