前癌消对前列腺癌雄激素受体共调节因子的影响

【作者】 闫玥捷；

【导师】 郝钰；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2012， 硕士

【摘要】 研究背景前列腺癌是欧美国家老年男性最常见的恶性肿瘤,我国一直被认为是前列腺癌的低发地区,但近几十年来随着人们生活条件提高和生活方式的改变,我国前列腺癌发病率呈明显上升趋势。前列腺癌可分为雄激素依赖型和雄激素非依赖型两种类型。早期雄激素依赖型前列腺癌对雄激素阻断治疗比较敏感,但大多数患者在经历12-18个月雄激素阻断治疗后,雄激素依赖型前列腺癌会转变为雄激素非依赖型前列腺癌,此时对抗雄激素治疗不再有效,也无其他较好治疗方法,最终导致患者死亡。中医治疗癌症,源远流长,方法众多,疗效可靠,潜力巨大。具有补肾祛瘀功效的中药复方“前癌消”在临床治疗前列腺癌取得了较好的疗效。本课题组前期对该方的动物实验研究已证实前癌消可以延缓前列腺癌从雄激素依赖向雄激素非依赖的转化且对已进展到雄激素非依赖阶段的前列腺癌病变有较好的控制作用；同时也提示了在前列腺癌雄激素依赖阶段该方可能具有性激素样作用,应当慎用。前期体外细胞实验结果表明,前癌消对激素依赖性前列腺癌细胞可能有一定的促进生长作用,而与雄激素剥夺治疗联合使用,可加强雄激素剥夺治疗对于前列腺癌细胞生长的抑制作用。近年来的研究表明,雄激素受体(AR)活性的异常与雄激素非依赖性前列腺癌的发生关系密切。前列腺癌细胞中AR共调节因子的异常是导致AR异常活性的关键因素之一本实验目的：观察前癌消对裸鼠前列腺癌瘤组织及人雄激素依赖细胞系LNCaP细胞雄激素受体及其共调节因子的表达,以探讨前癌消治疗前列腺癌的相关分子机制。方法首先进行人雄激素依赖细胞系LNCaP细胞培养,培养成功后将该细胞与基质胶的1：1混合物接种到裸鼠皮下,制备前列腺癌动物模型。造模成功后,当裸鼠肿瘤长至450mm3左右时,随机分为模型组、单纯中药组、单纯去势组、去势加中药组四组。单纯去势组、去势加中药组行去势手术摘除睾丸,然后给予单纯去势组生理盐水灌胃,去势加中药组前癌消水煎剂灌胃,同时模型组、单纯中药组分别灌胃生理盐水和前癌消水煎剂；以上灌胃四周后处死动物,摘取瘤组织称重并做石蜡包埋切片,银染技术检测瘤细胞中核仁组成区嗜银蛋白AgNOR表达量的变化,免疫组织化学染色观察瘤组织AR共调节因子SRC-1、NCoR的表达情况。将40只雄性Wistar大鼠随机分为对照组、单纯给药组、单纯去势组和去势给药组,对去势组行去势手术摘除双侧睾丸。术后一周,给药组以临床等效剂量前癌消水煎剂灌胃,不给药组以生理盐水灌胃,共3天后,行腹主动脉采血,获得4组不同的含药血清：对照组、单纯给药组、单纯去势组和去势给药组。用各组大鼠血清对对数生长期人雄激素依赖细胞系LNCaP细胞实施干预,48小时后检测：细胞增殖和凋亡相关因子：K i67.Bax、 Bc12mRNA的表达(荧光定量RT-PCR); AR基因与蛋白表达(荧光定量RT-PCR, Westernblot);雄激素受体共调节因子SRC-l,NCoR的表达(荧光定量RT-PCR)。结果1.前癌消对裸鼠前列腺癌组织中瘤细胞核仁组成区嗜银蛋白AgNOR表达的影响核仁组成区嗜银蛋白AgNOR颗粒呈黑棕色颗粒,分布于核内,颗粒形态呈聚集型弥漫型；模型组AgNOR颗粒表达最多,单纯中药组AgNOR表达比模型组少(P<0.01),单纯去势组AgNOR表达比模型组少(P<0.01),去势加中药组AgNOR表达比单纯去势组少(P<0.01)。2.前癌消对裸鼠前列腺癌组织中AR共调节因子SRC-1、 NCoR表达的影响SRC-1在模型组、单纯给药组、去势给药组、单纯去势组均有表达。单纯去势组(雄激素非依赖性前列腺癌)和模型组(雄激素依赖性前列腺癌)相比,SRC-1表达明显增加(p<0.01)；单纯给药组和模型组相比,SRC-1表达明显减少(P<0.01)；去势给药组与单纯去势组相比SRC-1表达也明显减少(P<0.01),说明前癌消可能是通过降低前列腺癌细胞SRC-1蛋白的表达水平,从而降低AR的转录活性,延缓前列腺癌雄激素非依赖性的转变过程。NCoR在模型组、单纯给药组、去势给药组均有表达,而单纯去势组(雄激素非依赖性前列腺癌)无明显表达。单纯去势组和模型组相比,NCoR表达明显减少(p<0.01)；单纯给药组和模型组相比,NCoR表达明显增加(P<0.01)；去势给药组与单纯去势组相比NCoR表达也明显增加(P<0.01),提示前癌消可能通过升高NcoR蛋白表达水平从而延缓前列腺癌雄激素非依赖性的转变过程。3.前癌消对LNCaP细胞增殖和凋亡相关因子的影响四组细胞模型增殖因子Ki67mRNA以及凋亡相关因子Bax、 Bcl-2mRNA的表达水平的荧光定量PCR的结果显示：单纯给药组Ki67mRNA的表达大于对照组(P<0.05),单纯去势组Ki67mRNA的表达小于对照组(P<0.05),去势给药组Ki67mRNA的表达小于单纯去势组(P<0.05)。四组细胞模型BaxmRNA的表达水平不具有统计学意义(P>0.05)。单纯给药组Bcl-2mRNA的表达大于对照组(P<0.05),单纯去势组Bcl-2mRNA的表达小于对照组(P<0.05),去势给药组Bcl-2mRNA的表达小于单纯去势组(P<0.05)。4.前癌消对LNCaP细胞AR基因与蛋白表达的影响四组细胞模型AR表达western-blot的结果显示：单纯给药组AR的表达大于对照组(P<0.05),单纯去势组AR的表达小于对照组(P<0.05),去势给药组AR的表达小于单纯去势组(P<0.05)。四组细胞模型ARmRNA表达荧光定量PCR的结果显示：单纯给药组ARmRNA的表达大于对照组(P<0.05),单纯去势组ARmRNA的表达小于对照组(P<0.05),去势给药组ARmRNA的表达小于单纯去势组(P<0.05)。5.前癌消对LNCaP细胞AR共调节因子SRC-1、 NCORmRNA表达的影响单纯给药组SRC-lmRNA的表达大于对照组(P<0.05),单纯去势组SRC-lmRNA的表达小于对照组(P<0.05),去势给药组SRC-lmRNA的表达小于单纯去势组(P＜O.05)。单纯给药组NCoRmRNA的表达小于对照组(P<0.05),单纯去势组NCoRmRNA的表达大于对照组(P<0.05),去势给药组NCoRmRNA的表达大于单纯去势组(P<0.05)。结论：前癌消对未经去势治疗的前列腺癌细胞具有一定促进增殖作用,这可能与其含有补肾中药的性激素样作用有关,需要进一步研究；前癌消可抑制去势后前列腺癌细胞的AR表达,并可减少AR共刺激因子SRC-1的表达而增加AR共抑制因子NCoR的表达,从而降低AR的转录活性,抑制AR介导的前列腺癌细胞的增殖,且通过抑制Bcl-2的表达而影响前列腺癌细胞的凋亡。这可能是前癌消延缓前列腺癌从雄激素依赖到非依赖转化以治疗前列腺癌的机制之一。更多还原

【Abstract】 ObjectiveProstate cancer is the most common malignant tumor of the older men in the United States and Europe, our country has always been considered a low-incidence area of prostate cancer, however, as the living conditions and lifestyle changes, the incidence of prostate cancer showed a increasing trend year after year in China and the patient is also gradually getting younger and younger. Prostate cancer can be divided into two typesof androgen-dependent prostate cancer and androgen-independent prostate cancer. Early androgen-dependent prostate cancer is more sensitive to androgen deprivation therapy, However,after a period of12to18months,nearly all patients under the treatment of hormone deprivation therapies are progressing into androgen-dependent prostate cancer.Hormone therapy introduce no therapeutic actions in this stage, neither other therapies. A rapid progression can be observed in this stage and most patients stride to the end of the life.Chinese medicine performs well in the clinical practice in treating different Kinds of cancers and it is more and more employed in cancer treatment these years for the advantages of little side effect, multi-targets and multi functioning levels, focusing on comprehensive regulation of the body compared to the modern therapies.The QAX created by prof. Pei Wang,who is a Chinese oncologists,on the basis of his experiences in treating cancers, which is called the QAX with the function of nourishing the kidney and promoting blood circulation, The previous research in our laboratory indicated that the QAX should be used with cautions during the stage of androgen-dependent prostate cancer and it will give good performance on treating the disease during the androgen-independent stage.Besides, it can delay the disease progression from androgen-dependent stage into the androgen-independent.LNCaP cells, the classic androgen-dependent prostate cancer cells, are used in our program to test the effects of the QAX above in vitro, and We will further study on the basis of preliminary in vivo experiments to discuss the molecular mechanism of prostate cancer and to provide more theoretical basis for further clinical application.MethodsFirst, human androgen dependent cell line LNCaP cell was cultured.The cells with the1:1mixture of matrigel was inoculated in nude mice, preparing animal models of prostate cancer. After the modeling, nude mice bearing prostate cancer is the study objective and the QAX is intervening factors.When the tumor in nude mice grew to about45Omm3, randomly divided into the normal control group, the QAX group, the castration group and the castration+QAX group, the normal control group and the castration group were given saline after the castration, the QAX group and the castration+QAX group were given the QAX.Four weeks after the QAX intervention, all animals were sacrificed and removal of the tumor.Immunohistochemistry staining tumor for SRC-1and NCOR expression in tumor tissue. Using factors related to proliferation label AgNOR,examine it expressing characteristic in four groups.The methods of intragastric administration of equivalent dose decoction and surgical removal of the testicles were employed to prepare4groups of different rat serums, the Normal Control group, the QAX group, the Castration group and the Castration+QAX group. The serums described above are used to interfere the human androgen-dependent prostate cancer LNCaP cells to examine ARmRNA and protein (quantitative real time RT-PCR, Western blot), factors related to proliferation label Ki67and factors related to apoptosis label BCL-2、 Bax (quantitative real time RT-PCR), steroid receptor coactivator-l(SRC-l),nuclear receptor corepressors(NCOR)(quantitative real time RT-PCR).Results1. Using immunohistochemistry technique labeling SRC-1、 NCOR,study expressing characteristic of them in four groups of tumor tissue.the Normal Control group, the QAX group, the Castration group and the Castration+QAX group.Regarding expression of AR, it shows that high expression in tumor cells of the Castration group, AR in the Normal Control group is less than the Castration group:AR expression in the QAX group was less than the Normal Control group, AR in the Castration+QAX group was less than the QAX group.AR in the Castration group is less than the Castration+QAX group; AR expression in the Normal Control group was less than the QAX group.2. Using factors related to proliferation label AgNOR,examine it expressing characteristic in four groupshigh expression in tumor cells of the Normal Control group, AR in the QAX group is less than the Normal Control group the Castration+QAX group:AR expression in the Castration group was less than the Castration+QAX group.3. ARmRNA and proteinARmRNA and protein in the Normal Control group was less than the QAX group; AR expression in than the Castration+QAX group was less the Castration group.4. factors related to proliferation label Ki67and factors related to apoptosis label BCL-2、 BaxKi67and BCL-2in the QAX group is less than the Normal Control group the Castration+QAX group:Ki67and BCL-2expression in the Castration group was less than the Castration+QAX group.5. Steroid receptor coactivator-1(SRC-1), nuclear receptor corepressors(NCOR)SRC-1in the QAX group was less than the Normal Control group the Castration+QAX group:SRC-1expression in the Castration group was less than the Castration+QAX group.SRC-1in the QAX group was more than the Normal Control group the Castration+QAX group:SRC-1expression in the Castration group was more than the Castration+QAX group.Conclusion1. The QAX with the function of nourishing the kidnedy and Promoting blood circulation can reduce the SRC-1. AgNOR expression of the tumor tissue of the nude mice.2. The QAX with the function of nourishing the kidnedy and Promoting blood circulation can promote the AR、Ki67mRNA、 SRC-1mRNA、 Bcl-2mRNA,and reduce expression of NCORmRNA expression of LNCaP cells.3. Castration can suppress the AR mRNA and protein、 Ki67mRNA SRC-lmRNA、 Bcl-2mRNA expression of LNCaP cells, and promote the expression of NCOR mRNA.4. AR mRNA and protein、 Ki67mRNA、 SRC-lmRNA、 Bcl-2mRNA expression in the Castration and the QAX group was less than in the Castration group.更多还原