染料木黄酮抑制雄激素受体信号通路抗去势抵抗前列腺癌的体外研究

发布时间：2018-04-01 02:24

本文选题：大豆异黄酮　切入点：染料木黄酮　出处：《成都医学院》2017年硕士论文

【摘要】：研究表明,大豆异黄酮(soy isoflavones)具有抗前列腺癌的作用,但其对去势抵抗前列腺癌(castrate-resistant prostate cancer,CRPC)的作用及机制尚未明确。前列腺癌经去势治疗后,前列腺癌细胞仍可通过从头合成途径(de novo)合成雄激素,从而激活雄激素受体(androgen receptor,AR)信号通路,继而调节基因转录,诱导前列腺特异性抗原(prostate-specific antigen,PSA)分泌及促进增殖,产生去势抵抗。因此,阻断癌细胞内部雄激素从头合成,抑制AR激活是CRPC防治的靶点。目的探讨染料木黄酮(genistein)对去势抵抗前列腺增殖的影响,以及对雄激素信号途径的抑制作用及分子机制,为植物化学物防治CRPC提供实验数据。方法以22RV1、VCa P细胞为CRPC细胞模型,人正常前列腺上皮细胞RWPE-1为对照。(1)CCK-8法检测细胞活力,流式细胞术检测细胞周期分布,细胞免疫化学法检测Ki-67;(2)酶联免疫法检测PSA,二氢睾酮(DHT),睾酮(T);(3)Western Blot检测P53、Cyclin D1、PCNA及de novo中各种催化酶表达,包括类固醇生成快速调节蛋白(St AR)、胆固醇调节元件结合蛋白(SREBP)、胆固醇侧链裂解酶(CYP11A1)、由17α羟化酶/20-裂解酶(CYP17A1)、3-羟基类固醇合成酶(3βHSD)、醛酮还原酶(AKR1C3)、5α还原酶2(SRD5A2)及胞浆蛋白、核蛋白中AR的表达;(4)实时荧光PCR检测AR下游基因FKBP5、STEAP1、TMPRSS2表达;(5)激光共聚焦检测AR的表达及胞内定位;(6)免疫共沉淀法检测HSP90与AR的相互作用。结果(1)genistein能有效抑制22RV1、VCa P细胞的增殖能力,且这种作用具有剂量时间效应;(2)genistein抑制CRPC细胞Ki-67、PCNA的表达,抑制CRPC细胞PSA的分泌,阻滞细胞周期于G2/M期;(3)genistein下调CRPC细胞AR的表达,抑制AR的核易位;(4)genistein下调CRPC细胞中AR下游基因STEAP1、TMPRSS2的表达;(5)genistein降低CRPC细胞中雄激素水平,下调从头合成途径关键酶AKR1C3、SRD5A2、CYP11A1及3βHSD的表达。结论Genistein通过下调CRPC细胞从头合成途径中的AKR1C3、SRD5A2、CYP11A1及3βHSD的表达,阻断雄激素的从头合成,抑制AR信号通路的激活及核易位,发挥抗去势抵抗前列腺癌增殖的作用。
[Abstract]:Studies have shown that soy isoflavones has an anti-prostate cancer effect, but its role and mechanism in castration against prostate cancer is not clear. Prostate cancer cells can still synthesize androgen via de novoa pathway, which activates androgen receptor AR-signaling pathway, regulates gene transcription, induces prostate-specific antigen-PSAs secretion and promotes proliferation. Therefore, blocking androgen biosynthesis and inhibiting AR activation in cancer cells are the targets of CRPC. Objective to investigate the effect of genistein on ovariectomized prostate proliferation. The inhibitory effect on androgen signaling pathway and its molecular mechanism provide experimental data for phytochemical prevention and treatment of CRPC. Methods 22RV1VCAP cells were used as CRPC cell model, and normal prostatic epithelial cells (RWPE-1) were used as control. The cell cycle distribution was detected by flow cytometry, the expression of P53Cyclin D1PCNA and various catalytic enzymes in de novo were detected by enzyme-linked immunosorbent assay (Elisa). These proteins include steroid fast regulatory protein St ARN, cholesterol regulatory element binding protein SREBP, cholesterol side chain cleavage enzyme CYP11A1, CYP17A1 / 3-hydroxysteroid synthase 3 尾 HSDN, aldehydes and ketone reductase AKR1C3A5A2) and cytosolic protein. The expression of AR in nuclear protein was detected by real-time fluorescence PCR. The expression of FKBP5 / STEAP1 / TMPRSS2 was detected by laser scanning. Laser confocal method was used to detect the expression of AR and the immunoprecipitation method was used to detect the interaction between HSP90 and AR. Results Genistein could effectively inhibit the proliferation of 22RV1VCAP cells. This effect has a dose-time effect, which inhibits the expression of Ki-67 CRPC, inhibits the secretion of PSA in CRPC cells, and inhibits the down-regulation of AR expression in CRPC cells at G _ 2 / M phase by genistein. Inhibition of AR nuclear translocation 4 genistein down-regulates the expression of AR downstream gene STEAP1 and TMPRSS2 in CRPC cells and reduces the level of androgen in CRPC cells. The expression of CYP11A1 and 3 尾 HSD in ab initio synthesis pathway was down-regulated by Genistein. Conclusion Genistein can inhibit the activation and nuclear translocation of AR signal pathway by down-regulating the expression of AKR1C3A5A2CYP11A1 and 3 尾 HSD in the ab initio synthesis pathway of CRPC cells. Play the role of anti-castration resistance to prostate cancer proliferation.
【学位授予单位】：成都医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.25

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