【作者】 韩勇军；

【导师】 吴新荣；

【作者基本信息】 华南理工大学 ， 制糖工程， 2011， 硕士

【摘要】 研究目的：鱼藤素(deguelin)是从豆科毛鱼藤植物中分离的一种黄酮类化合物,可抑制肺癌、胃癌、乳腺癌细胞等多种肿瘤细胞增殖并诱导其凋亡,体外实验表明鱼藤素可以抑制PI3K活性,降低pAkt磷酸化水平,下调环氧合酶-2(cyclooxygenase-2,COX-2)的蛋白表达,但是有关鱼藤素在体内的作用机制尚不清楚。本实验室利用模式生物斑马鱼胚胎研究鱼藤素初步发现：0.6μM浓度鱼藤素处理2细胞期胚胎,可以使胚胎在受精后4小时(4 hpf,4 hours post fertilization,又称sphere时期)停止发育,不能进行原肠胚运动,该表型与干扰斑马鱼胚胎COX/PGE2信号通路或PI3K/Akt信号通路的表型相似,推断鱼藤素可能抑制了斑马鱼胚胎的COX/PGE2信号通路,进而影响了下游PI3K/Akt信号通路,具体机制还有待探究。本课题希望利用荧光定量PCR,整胚原位杂交生物技术来检测斑马鱼上COX/PGE2信号通路和其下游相关基因的变化,而后选取与COX信号通路密切相关的肿瘤细胞,检测鱼藤素对其增殖的抑制作用,利用荧光定量PCR法检测这些肿瘤细胞中COX信号通路相关基因的变化,以验证鱼藤素的抗肿瘤作用与抑制COX信号通路相关性,为预测鱼藤素在体内抗肿瘤的作用机理提供基础依据。研究结果：(1)利用荧光定量PCR检测0.6μM鱼藤素作用前后的sphere期胚胎COX/PGE2通路相关基因,发现coxl, cox2b在转录水平并未发生明显变化,PGE2合成酶ptges、ptges3a的mRNA的表达下降水平与空白对照组相比有统计学差别(p<0.05),PGE2的受体ep2,ep4均发生显著下降,下游基因egrl和cyclin D1基因表达均发生了显著下调(p<0.01)。(2)0.16μM鱼藤素处理6 hpf时期胚胎至21体节期,用原位杂交的方法研究scl和gatal表达变化,发现在10体节期和21体节期,鱼藤素均抑制了scl和gatal的表达。5μM dmPGE2、0.16μM鱼藤素、5μM dmPGE2和0.16μM鱼藤素混合物处理10体节时期胚胎至30 hpf,用原位杂交的方法研究runxl表达,发现在30 hpf时期,与空白对照组相比,5μM dmPGE2提高了runxl表达,HSCs的数量明显增多,0.16μM鱼藤素抑制了runxl的表达,而用5μM dmPGE2和0.16μM鱼藤素混合物处理,与dmPGE2单独处理相比,runxl表达明显下降。(3)利用荧光定量PCR检测了21体节期胚胎COX/PGE2信号相关基因,另外scl, gatal, runxl, cmyb基因在这一时期对血细胞系前体均有特异性表达,对这四个标记性基因也进行了相对定量分析,其结果发现：coxl, ptges3a, ep4a, ep4b, egrl, cyclinDl, scl, gatal, runxl, cmyb基因均发生了显著下降(p<0.01), cox2b, ptges基因表达下降水平与空白对照组相比有统计学差别(p<0.05)。另外,利用荧光定量PCR法检测了30 hpf时期COX/PGE2信号通路相关基因,研究发现在30 hpf这一时期,COX/PGE2信号通路上cox2b基因发生了显著下降,其下游基因egrl也发生了显著下降(p<0.01), ep4b受体基因表达下降水平与空白对照组相比有统计学差别(p<0.05)。(4)鱼藤素作用于MCF-7, Mia PaCa-2, PC-3细胞48小时后,均可以抑制其细胞增殖,随着浓度的增加,抑制效果越为明显。荧光定量PCR检测鱼藤素作用48小时后这三个细胞的COX/PGE2信号通路相关基因,发现鱼藤素处理后,MCF-7细胞中COX1和EP2基因的表达发生了明显下降(p<0.01); Mia PaCa-2细胞中PTGES基因的表达水平发生了显著降低(p<0.01), EP4和CCND1基因的表达下降水平与空白对照组相比有统计学差别(p<0.05); PC-3细胞中COX1基因的表达水平发生了显著下降(p<0.01)。结论：(1)鱼藤素可以抑制斑马鱼胚胎体内COX/PGE2信号通路相关基因的表达。(2)鱼藤素可以抑制MCF-7, Mia PaCa-2, PC-3细胞增殖。(3)鱼藤素可以抑制MCF-7, Mia PaCa-2, PC-3细胞中COX/PGE2信号通路的相关基因的表达。更多还原

【Abstract】 Objective:Deguelin, a flavonoid derived from Derris elliptica within the Leguminosae family, has been shown to inhibit Tumor cell proliferation and induce several types of cancers apoptosis, including lung, stomach and breast cancer cells. Deguelin inhibited PI3K activity and reduced pAkt levels and decreased expression of cyclooxygenase 2 in vitro studies on cancer cells. However, the mechanism action of deguelin in vivo is still not clear. By using the externally developing zebrafish embryos as a model, we found that embryos treated with 0.6μM deguelin at two-cell stage exhibited gastrulation arrest at 4 hours post fertilization stage(4 hpf, it is also called sphere stage) micking phenotypes of Cox-1, Ptges, EP4-deficient and inhibition PI3K/Akt signaling embryos. Therefore, we hypothesized that deguelin may disrupt the COX/PGE2 pathway in vivo. The functional role for deguelin in vivo remains virtually unknown. We hope to analysis the alterations of COX/PGE2 signaling genes transcript level in embryos treated with deguelin compared to control using qRT-PCR and whole-mount in situ hybridization. Then we selected tumor cells associated with COX pathway to observe the proliferative inhibition process by deguelin using MTT assay. QRT-PCR assay was performed to detect the alterations of COX/PGE2 signaling genes transcript level in these tumor cells to investigate the interaction between the effect of anti-tumor of deguelin and the inhibition of COX/PGE2 signal pathway.Results:(1) Using qRT-PCR to analysis the alterations of COX/PGE2 signaling genes transcript levels in embryos treated with 0.6μM deguelin compared to control, we found that PGE2 synthetases, ptges and ptges3a, were significantly reduced, else PGE2 receptors, ep2 and ep4, were significantly reduced. Consistently the downstream genes egrl and cyclin D1 were significantly decreased.(2) Using whole-mount in situ hybridization to observe the expression of scl and gatal in embryos treated with 0.16μM deguelin from 6 hpf to 21-somite stage compared to control, we found that deguelin decreased scl and gatal expression at 10-somite and 21-somite stages. At 30 hpf stage,0.16μM deguelin inhibited the expression of runxl compared to control embryos,5μM dmPGE2 enhanced runxl expression in control embryos, while the enhancing effect of dmPGE2 on HSCs was reduced significantly by deguelin.(3) QRT-PCR was used to examine the alterations of target genes transcript levels in the 21-somite stage of embryos treated with 0.16μM deguelin from 6 hpf to 21-somite stage compared to control embryos, we found that coxl, ptges3a, ep4a, ep4b, egrl, cyclinDl, scl, gatal, runxl, cmyb were downregulated in deguelin-treated embryos. At 30 hpf stage, ep4b was found to been downregulated by deguelin, cox2b and the downstream gene egrl were significantly decreased.(4) MCF-7, Mia PaCa-2 and PC-3 cells were treated with different concentrations of deguelin for 48 hours, resulted in the inhibition of cell proliferation in a dose-dependent manner. Using qRT-PCR to analysis the alterations of COX/PGE2 signaling genes transcript levels in these three tumor cells treatment with deguelin for 48 hours, the data showed that COX1 and EP2 were downregulated at the mRNA levels in MCF-7 cell. In Mia PaCa-2 cell, PTGES was downregulated significantly by deguelin, also EP4 and CCND1 transcript levels were decreased compared to control. In PC-3 cell, COX1 was downregulated significantly by deguelin.Conclusion:(1) Deguelin downregulated the expression of COX/PGE2 signaling genes in zebrafish embryos.(2) Deguelin inhibited the proliferation of MCF-7, Mia PaCa-2 and PC-3 cells.(3) Deguelin downregulated the expression of COX/PGE2 signaling genes in MCF-7, Mia PaCa-2 and PC-3 cells.更多还原