联合应用WT1-siRNA和人参皂甙Rg3对恶性黑素瘤细胞WM451耐药性的逆转

发布时间：2019-02-13 13:13

【摘要】： 目的：研究联合应用WT1-siRNA和人参皂甙Rg3(genseno sideRg3,Rg3)在逆转体外培养恶性黑素瘤细胞WM451对化疗药物顺铂(DDP)、氮烯咪胺(DTIC)耐药的作用。 方法：构建针对WTl基因的siRNA真核表达载体,利用脂质体转染WM451细胞,以G418筛选,获得稳定转染细胞株,分别以逆转录聚合酶链反应(RT-PCR).蛋白质印迹法(Western-Blot).免疫细胞化学染色方法检测转染细胞中WT1 mRNA及蛋白表达水平的改变,MTT法检测WT1-siRNA.人参皂甙Rg3对WM451细胞增殖的影响及对细胞耐药的逆转作用,用电镜和tunel法观察WT1-siRNA.人参皂甙Rg3对WM451细胞凋亡的影响。 结果：成功构建靶向人WT1基因的siRNA真核表达载体,获得稳定转染细胞株,转染细胞WT1 mRNA及蛋白水平明显下调；WT1-siRNA干扰组细胞的凋亡率(51%)较对照组明显增加(9%),干扰组细胞生长受抑制；人参皂甙Rg3对黑素瘤细胞生长同样有抑制作用且与浓度呈正相关,80ug/ml的人参皂甙Rg3细胞凋亡率(84%)较对照组明显增加(3%)；MTT显示WT1-siRNA分别逆转细胞对顺铂、氮烯咪胺的耐药性2.1、1.5倍,人参皂甙Rg3(5ug/ml)分别逆转细胞对顺铂、氮烯咪胺的耐药性2.34、2.01倍,联合应用WT1-siRNA和人参皂甙Rg3分别逆转细胞对顺铂、氮烯咪胺的耐药性3.81、3.42倍。 结论： 1.脂质体转染siRNA可特异性下调WT1 mRNA及蛋白水平。 2.下调WM451细胞WT1基因表达可促进细胞凋亡,抑制细胞增殖。 3.人参皂甙Rg3可以诱导细胞凋亡,抑制细胞增殖,其抑制作用与浓度呈正相关。 4.单独、联合应用WTl-siRNA和人参皂甙Rg3均可增加顺铂、氮烯咪胺对WM451细胞的抑制作用,逆转WM451细胞对化疗药物的耐药性,且以联合的作用最强,为临床中西医联合治疗提供了实验依据。
[Abstract]:Aim: to study the effect of combined use of WT1-siRNA and ginsenoside Rg3 (genseno sideRg3,Rg3) on the reversal of the chemotherapeutic drug resistance to cisplatin (DDP), zomidomide (DTIC) in cultured malignant melanoma cells WM451 in vitro. Methods: the eukaryotic expression vector of siRNA targeting WTl gene was constructed. WM451 cells were transfected with liposome and screened by G418. The stable transfected cells were obtained by reverse transcriptase polymerase chain reaction (RT-PCR). Western blotting (Western-Blot). Expression of WT1 mRNA and protein in transfected cells was detected by immunocytochemical staining and WT1-siRNA. was detected by MTT Effects of ginsenoside Rg3 on proliferation and reversal of drug resistance of WM451 cells were studied by electron microscope and tunel. Effects of ginsenoside Rg3 on apoptosis of WM451 cells. Results: the eukaryotic expression vector of siRNA targeting human WT1 gene was successfully constructed and stable transfected cell lines were obtained. The WT1 mRNA and protein levels of transfected cells were down-regulated significantly. The apoptosis rate of WT1-siRNA interference group (51%) was significantly higher than that of control group (9%). Ginsenoside Rg3 also inhibited the growth of melanoma cells and had a positive correlation with the concentration. The apoptosis rate of Rg3 cells in 80ug/ml (84%) was significantly higher than that in the control group (3%). MTT showed that WT1-siRNA reversed the resistance of cells to cisplatin and azomidomidine by 2.1 times, and ginsenoside Rg3 (5ug/ml) reversed the resistance of cells to cisplatin and azomidine by 2.34 times, respectively. The combination of WT1-siRNA and ginsenoside Rg3 reversed the resistance of the cells to cisplatin and azomidomidine by 3.81 ~ 3.42 times respectively. Conclusion: 1. SiRNA transfection with liposome can specifically down-regulate the level of WT1 mRNA and protein. 2. Down-regulation of WT1 gene expression in WM451 cells can promote cell apoptosis and inhibit cell proliferation. 3. Ginsenoside Rg3 can induce cell apoptosis and inhibit cell proliferation. 4. WTl-siRNA and ginsenoside Rg3 alone could increase the inhibitory effect of cisplatin and azenomidine on WM451 cells and reverse the resistance of WM451 cells to chemotherapeutic drugs. It provides experimental basis for clinical combined treatment of traditional Chinese and western medicine.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R739.5

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