【作者】 王文成；

【导师】 项琪；

【作者基本信息】 暨南大学 ， 生物工程（专业学位）， 2018， 硕士

【摘要】 目的:前列腺癌是全球男性常见的恶性肿瘤之一。由于人口老龄化、动物脂肪摄入量增加以及体育运动减少等诸多因素,前列腺癌发病率和死亡率逐年增加。以雄激素受体(AR)为核心的雄激素剥夺治疗长期使用会导致去势抵抗性前列腺癌(CRPC)的发生。17β-羟甾脱氢酶Ⅲ(17β-HSD3)是前列腺癌治疗独立于雄激素受体的新靶点,本研究对实验室自主合成的姜黄素系列衍生物进行了 17β-HSD3为靶点的抑制剂筛选,以期找到新的前列腺癌治疗小分子药物。方法:(1)采用放射免疫分析法检测给药后稳定转染并表达17β-HSD3的LC540(17β-HSD3)细胞上清中睾酮和孕酮的含量来筛选对17β-HSD3敏感的姜黄素衍生物;(2)实时荧光定量PCR法(RT-qPCR)检测不同化合物不同浓度作用后,LC540(17β-HSD3)细胞中17β-HSD3基因的表达情况;(3)四甲基偶氮唑盐微量酶反应(MTT)比色法检测化合物对LNCap、PC3和LC540(17β-HSD3)细胞增殖的影响;(4)蛋白免疫印迹法(Western Blot)检测LC540(17β-HSD3)细胞株中睾酮生物合成通路中五个关键酶:17β-HSD3、StAR、CYP11A1、CYP17以及3β-HSD的表达情况;(5)体外睾丸微粒体法检测目标化合物H10对17β-HSD3蛋白酶活性的影响;(6)SD大鼠连续七天给药,7天后取血检测体内激素含量;(7)构建LNcap荷瘤裸鼠模型,随机分成5组:生理盐水组(A);溶剂组(B);H10低(10mg/kg,C)、中(30mg/kg,D)、高(50mg/kg,E)三个剂量组,腹腔注射(i.p)给药。记录给药期间各组裸鼠荷瘤体积及体重变化,处死动物前取血清测定裸鼠体内激素水平;(8)小鼠急性毒性试验初步考察目标化合物H10的安全性,处死动物后取脏器进行组织形态学观察。结果:(1)利用LC540(17β-HSD3)细胞株筛选出3个有潜力的姜黄素衍生物:H1、H10和H11。其中H10显著降低了 LC540(17β-HSD3)细胞上清中睾酮水平,但对孕酮水平无影响;而H1和H11同时降低了睾酮和孕酮水平,其他化合物则对睾酮和孕酮水平无影响;(2)H10处理LC540(17β-HSD3)细胞株后可下调17β-HSD3基因的表达水平,且具有剂量依赖性;0.25、0.5和1 μmol/L三个浓度的H10均显著下调了 17β-HSD3蛋白的表达(P<0.05),但对睾酮合成通路上的StAR、CYP11A1、CYP17和3β-HSD的表达无显著影响;(3)0、10、20和40 μmol/L的H10处理睾丸微粒体后,体系中睾酮浓度分别为1.19±0.07ng/ml、0.90±0.10ng/ml、0.56±0.06ng/ml、0.49±0.09ng/ml;(4)SD 大鼠连续腹腔注射给药七天后(10、30和50 mg/kg/d),体内睾酮水平显著下降,且呈现剂量依赖性,而孕酮水平保持不变;(5)H10给药组裸鼠的肿瘤体积增长明显慢于生理盐水组和溶剂组(P<0.05)。生理盐水组、溶剂组、H10给药组(低、中、高)的肿瘤体积分别为510.80±99.08mm3、494.49±81.91 mm3、424.45±95.24mm3、243.67±58.49mm3、269.44±78.98 mm3;各组裸鼠血清睾酮水平分别为 1.53 ±0.56 ng/ml、1.41 ±0.65 ng/ml、0.41 ±0.30 ng/ml、0.22±0.07ng/ml、0.19±0.16 ng/ml(means±SD,n=6);(6)小鼠腹腔注射H10后(因溶解度原因最高剂量为100 mg/kg)在实验剂量范围内对KM小鼠无不良影响,病理切片显示心、肝、脾、肺、肾和睾丸组织结构正常。结论:本研究以17β-HSD3为靶点,从一系列姜黄素衍生物中筛选出了其选择性抑制剂H10,并进一步通过体内外实验验证了 H10对17β-HSD3基因和蛋白表达的抑制作用。在LNCap细胞株前列腺癌裸鼠模型中,H10能有效降低裸鼠体内睾酮水平并抑制肿瘤的生长。结果显示,姜黄素衍生物H10是一种潜在的以17β-HSD3为靶点的小分子抑制剂,可作为前列腺癌治疗药物进行进一步的研究。更多还原

【Abstract】 Objective:Prostate cancer is one of the common malignancies in men worldwide.Due to many factors such as the aging of the population,the increase of animal fat intake and the decrease of physical activity,the incidence and mortality of prostate cancer increase year by year.Long-term use of androgen deprivation therapy centered on the androgen receptor(AR)leads to the development of castration-resistant prostate cancer(CRPC).17β-hydroxysteroid dehydrogenase III(17β-HSD3)is a new target for prostate cancer treatment independent of androgen receptor.In this study,17β-HSD3 was targeted for the curcuminoid derivatives synthesized in the lab.Screening for inhibitors to find new small molecule drugs for the treatment of prostate cancer.Method:(1)A radioimmunoassay was used to screen 17β-HSD3-sensitive curcumin derivatives by measuring the content of testosterone and progesterone in the supernatant of LC540(17β-HSD3)cells after administration;(2)Real time qPCR was used to detect the expression of 17β-HSD3 gene in LC540(17β-HSD3)cells after treatment with different concentrations of compounds;(3)MTT colorimetric assay was used to detect the effect of compound on the proliferation of LNCap,PC3 and LC540(17β-HSD3)cells;(4)Western Blot was used to detect the expression of 17β-HSD3,StAR,CYP11A1,CYP17 and 3β-HSD in the testosterone biosynthetic pathway of LC540(17β-HSD3)cell line;(5)Testis microsomes were extracted and subjected to enzyme kinetics reaction to detect the effect of compound H10 on 17β-HSD3 protease activity;(6)SD rats were given for seven consecutive days,and blood samples were taken after 7 days to detect the hormone content;(7)LNcap tumor-bearing nude mice models were constructed and randomly divided into 5 groups:normal saline group(A);solvent group(B);H10 low(10 mg/kg,C),medium(30 mg/kg,D),high(50 mg/kg,E)Three dose groups were administered intraperitoneally(ip).The tumor volume and body weight of each group of nude mice during the administration period were recorded,and serum levels of nude mice were measured before sacrifice;(8)The acute toxicity test of mice was used to preliminarily examine the safety of the target compound H10.After the animals were sacrificed,the organs were taken for histomorphological observation.Result:(1)Three potential curcumin derivatives were selected using the LC540(17β-HSD3)cell line:H1,H10,and H11.H10 significantly reduced testosterone levels in the supernatant of LC540(17β-HSD3)cells,but had no effect on progesterone levels;while H1 and H11 also reduced testosterone and progesterone levels,while other compounds had no effect on testosterone and progesterone levels.;(2)H10 treatment of the LC540(17β-HSD3)cell line down-regulated the expression of the 17β-HSD3 gene in a dose-dependent manner.H1 concentrations of 0.25,0.5,and 1 μM significantly down-regulated the expression of 17β-HSD3 protein(P<0.05),but had no significant effect on the expression of StAR,CYP11A1,CYP17 and 3β-HSD on the testosterone synthesis pathway;(3)After 0,10,20 and 40 μmol/L H10 treatment of testicular microsomes,the testosterone concentrations in the system were 1.19±0.07 ng/ml,0.90±0.10 ng/ml,0.56±0.06 ng/ml,and 0.49±0.09 ng/ml,respectively;(4)After 7 days(10,30,and 50 mg/kg/d)of continuous administration of SD rats,the level of testosterone decreased significantly in a dose-dependent manner,while the level of progesterone remained unchanged;(5)The tumor volume growth of nude mice in the H10 group was significantly slower than that in the saline group and the solvent group(P<0.05).The tumor volumes in the saline group,solvent group,and H10-administered group(low,medium,and high)were 510.80±99.08 mm3,494.49±81.91 mm3,424.45±95.24 mm3,243.67±58.49 mm3,and 269.44±78.98 mm3,respectively;The serum testosterone levels in nude mice were 1.53±0.56 ng/ml,1.41 ±0.65 ng/ml,0.41 ±0.30 ng/ml,0.22±0.07 ng/ml,and 0.19±0.16 ng/ml(means±SD,n=6),respectively;(6)The mice were intraperitoneally injected with H10(the highest dose for solubility reasons was 100 mg/kg),no adverse effects on KM mice in the experimental dose range,histological sections showed normal heart,liver,spleen,lung,kidney and testicular tissue.Conclusion:In this study,17β-HSD3 was used as a target,and its selective inhibitor H10 was screened from a series of curcumin derivatives,and the inhibitory effect of H10 on the expression of 17β-HSD3 gene and protein was further verified by in vitro and in vivo experiments.In the nude mouse model of LNCap cell line prostate cancer,H10 can effectively reduce testosterone levels in nude mice and inhibit tumor growth.The result shows that the curcumin derivative H10 is a potential small molecule inhibitor targeting 17β-HSD3 and can be used as a therapeutic drug for prostate cancer for further study.更多还原