【摘要】： 目的：研究聯(lián)合應(yīng)用WT1-siRNA和人參皂甙Rg3(genseno sideRg3,Rg3)在逆轉(zhuǎn)體外培養(yǎng)惡性黑素瘤細(xì)胞WM451對(duì)化療藥物順鉑(DDP)、氮烯咪胺(DTIC)耐藥的作用。 方法：構(gòu)建針對(duì)WTl基因的siRNA真核表達(dá)載體,利用脂質(zhì)體轉(zhuǎn)染W(wǎng)M451細(xì)胞,以G418篩選,獲得穩(wěn)定轉(zhuǎn)染細(xì)胞株,分別以逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(RT-PCR).蛋白質(zhì)印跡法(Western-Blot).免疫細(xì)胞化學(xué)染色方法檢測(cè)轉(zhuǎn)染細(xì)胞中WT1 mRNA及蛋白表達(dá)水平的改變,MTT法檢測(cè)WT1-siRNA.人參皂甙Rg3對(duì)WM451細(xì)胞增殖的影響及對(duì)細(xì)胞耐藥的逆轉(zhuǎn)作用,用電鏡和tunel法觀察WT1-siRNA.人參皂甙Rg3對(duì)WM451細(xì)胞凋亡的影響。 結(jié)果：成功構(gòu)建靶向人WT1基因的siRNA真核表達(dá)載體,獲得穩(wěn)定轉(zhuǎn)染細(xì)胞株,轉(zhuǎn)染細(xì)胞WT1 mRNA及蛋白水平明顯下調(diào)；WT1-siRNA干擾組細(xì)胞的凋亡率(51%)較對(duì)照組明顯增加(9%),干擾組細(xì)胞生長(zhǎng)受抑制；人參皂甙Rg3對(duì)黑素瘤細(xì)胞生長(zhǎng)同樣有抑制作用且與濃度呈正相關(guān),80ug/ml的人參皂甙Rg3細(xì)胞凋亡率(84%)較對(duì)照組明顯增加(3%)；MTT顯示W(wǎng)T1-siRNA分別逆轉(zhuǎn)細(xì)胞對(duì)順鉑、氮烯咪胺的耐藥性2.1、1.5倍,人參皂甙Rg3(5ug/ml)分別逆轉(zhuǎn)細(xì)胞對(duì)順鉑、氮烯咪胺的耐藥性2.34、2.01倍,聯(lián)合應(yīng)用WT1-siRNA和人參皂甙Rg3分別逆轉(zhuǎn)細(xì)胞對(duì)順鉑、氮烯咪胺的耐藥性3.81、3.42倍。 結(jié)論： 1.脂質(zhì)體轉(zhuǎn)染siRNA可特異性下調(diào)WT1 mRNA及蛋白水平。 2.下調(diào)WM451細(xì)胞WT1基因表達(dá)可促進(jìn)細(xì)胞凋亡,抑制細(xì)胞增殖。 3.人參皂甙Rg3可以誘導(dǎo)細(xì)胞凋亡,抑制細(xì)胞增殖,其抑制作用與濃度呈正相關(guān)。 4.單獨(dú)、聯(lián)合應(yīng)用WTl-siRNA和人參皂甙Rg3均可增加順鉑、氮烯咪胺對(duì)WM451細(xì)胞的抑制作用,逆轉(zhuǎn)WM451細(xì)胞對(duì)化療藥物的耐藥性,且以聯(lián)合的作用最強(qiáng),為臨床中西醫(yī)聯(lián)合治療提供了實(shí)驗(yàn)依據(jù)。
[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.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R739.5

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