【作者】 袁明振；

【导师】 赵升田；

【作者基本信息】 山东大学 ， 外科学， 2007， 博士

【摘要】 研究背景与目的：前列腺是成年男性的重要器官，具有分泌前列腺液、协助射精、排尿等多种重要功能。前列腺生理功能的紊乱及病理变化可以导致许多疾病的发生，严重影响男性的身心健康。良性前列腺增生（BPH）是我国男性发病率最高的良性肿瘤，60岁以上老年人有50％发生良性前列腺增生症也是最常见的老年病致死原因，前列腺癌（PCa）一直是西方国家男性最常见的恶性肿瘤。在我国尽管前列腺癌的发病率相对较低，但近几年也有逐年上升的趋势。对前列腺癌进行早期发现和有效的治疗已使总体死亡率有了明显的降低。然而，晚期前列腺癌的治疗仍然是一个难题。慢性前列腺炎由于其不适症状重、治疗效果不满意，影响着患者的正常生活，因而也一直困扰着国内外泌尿外科医生。前列腺疾病的危害如此之大足以引起我们的重视，然而前列腺这些疾病的发病机理很多还不清楚，治疗效果还很不满意，还需要进一步深入研究。前列腺的生长与功能受多种神经系统的调节，包括胆碱能神经与去甲肾上腺素能神经，近年来对前列腺的组织结构与功能的研究已经越来越深入和广泛，特别是对调节前列腺功能的植物神经系统的研究显示前列腺疾病发生、发展与神经系统的调节关系密切。在良性前列腺增生症的治疗上α受体阻滞剂已经取得了巨大的成功，雄激素阻断也已经成功地用于前列腺癌的内分泌治疗。然而对于胆碱能M受体在前列腺生理功能与疾病发生发展中的作用还不是很清楚，也还没有这方面的药物应用于前列腺疾病的治疗。认识胆碱能M受体在前列腺中的作用，并把胆碱能药物用于前列腺疾病的治疗，首要的问题是弄清楚M受体在前列腺组织的分布特征，目前在良性前列腺增生组织及前列腺肿瘤细胞系已有这方面的研究，但是在正常前列腺各区带的分布规律目前还很少有研究报道，可查的资料也是本课题组2006年关于3种M受体亚型在正常前列腺表达规律的研究报道。最近几年，关于前列腺癌发生和进展中发生的分子生物学改变方面的知识增加迅速，对肿瘤分子生物学更好的理解对于建立新的对策来延缓或抑制肿瘤的进展具有重要的意义。研究表明在许多肿瘤细胞都有胆碱能受体表达，并且表达强度高于正常。对乳腺癌及其他肿瘤细胞系的研究发现，M₃受体与肿瘤的发生、分级密切相关，胆碱能受体的活动通过不同的信号途径可以刺激细胞的增殖。既往的研究发现M₁、M₂、M₃能刺激细胞的生长与分化，而M₄的这种功能则不明显。并且起调节作用的主要是M₃受体。这说明M₃受体表达的变化在肿瘤发生中具有重要的作用，了解M₃受体在正常前列腺、良性前列腺增生、前列腺癌组织中的表达变化规律对于探讨其在前列腺肿瘤发生、发展中的作用及其分子生物学机制具有重要的意义。在肿瘤的发生、发展过程中，肿瘤细胞的增殖与新生血管的形成是重要的环节，在乳腺癌的LMM3细胞系的研究发现，其主要受体亚型为M₃，且表达量是LM3细胞的40倍。胆碱能受体激动剂CARB能够刺激LMM3细胞的生长与新生血管生成，选择性的M₃受体拮抗剂pf-HHSiD能抑制这种作用，并且试验表明CARB能刺激VEGF-A的表达，选择性的M₃受体拮抗剂pf-HHSiD也能抑制这种作用。这说明M₃受体在LMM3细胞的生长与血管生成中起着十分重要的作用。目前关于M₃受体与良性前列腺增生、前列腺癌发生发展的关系方面的研究还很少，并且已有的研究也是在没有严格意义上的正常对照的基础上完成的。血管生成受到正负两方面因子的调控：血管生成刺激因子和血管生成抑制因子。血管内皮生长因子（vascular endothelial growth factor，VEGF）和内皮抑素（Endostatin ES）分别是目前已知的最强的血管生成促进因子和抑制因子，联合检测二者在肿瘤组织中的表达有助于更加深入地了解促血管生成因素和抑制血管生成因素在血管生成和肿瘤浸润转移中的作用。血管内皮生长因子是肿瘤血管新生的重要作用因子。在绝大多数恶性肿瘤中均呈现VEGF及其受体的高表达，以旁／自分泌作用机制，促进内皮细胞分裂、增殖、诱导血管新生，并可直接作用于肿瘤细胞，刺激肿瘤细胞生长。在前列腺内VEGF可由正常导管内皮细胞、腺癌细胞和浸润的淋巴细胞合成。VEGF的表达随着人类前列腺癌细胞的转移特性的增加而增加，而且恶性肿瘤细胞合成、分泌的VEGF刺激血管生成的效率更高。VEGF受体在前列腺肿瘤血管内皮细胞和肿瘤细胞上均有表达，这说明VEGF除了以旁分泌刺激血管内皮细胞增殖外，还能通过自分泌调节肿瘤细胞增殖。Endostatin是从血管内皮瘤培养液中分离出的18型胶原蛋白C端氨基酸片段。它可特异性地抑制内皮细胞增殖、迁移和肿瘤血管生成，诱导细胞凋亡，使肿瘤原发灶及转移灶处于休眠状态，停止生长。文献报道肿瘤内微血管密度（microvessel density MVD）反映了肿瘤间质血管生成的强度，随着MVD的增高，肿瘤的复发呈上升趋势。目前MVD已经成为评价肿瘤组织内新生血管形成程度的重要指标。研究前列腺组织中VEGF与内皮抑素、M₃受体三者在前列腺正常组织与肿瘤组织中的表达关系对于研究M₃受体在前列腺肿瘤发生发展中的作用，对于深入研究前列腺肿瘤的发生与发展机制以及对于晚期肿瘤治疗靶点的选择均具有重要的意义，此研究在人体前列腺肿瘤还未查见相关报道。材料与方法：本研究分两部分进行，第一部分，首先应用实时荧光定量RT-PCR方法对M受体及其各亚型在正常前列腺各区带的mRNA表达进行研究，探讨各亚型在各区带的基因表达差异，然后进一步对在前列腺组织表达较高并与肿瘤发生密切相关的三种受体亚型（M₁、M₂、M₃）通过Western blot方法检测其在正常前列腺、良性前列腺增生、前列腺癌组织中的表达差异，获得各亚型在不同前列腺组织中的蛋白表达特征，分析不同亚型在前列腺正常功能发挥与疾病发生、发展中的作用。第二部分，先通过RT-PCR方法研究M₃、VEGF、ES在正常前列腺、良性前列腺增生、前列腺癌三种组织的基因表达，再应用免疫组化及Western blot两种方法并对比CD34的表达综合对比VEGF与内皮抑素在正常前列腺、良性前列腺增生、前列腺癌三种组织中的蛋白表达差异，分析这两种物质的表达在前列腺肿瘤发生、发展中的作用及与前列腺癌临床分期、gleason评分的关系：最后通过与M₃受体在正常前列腺与良性前列腺增生、前列腺癌中的表达进行对比研究，进一步探讨M₃受体在前列腺肿瘤发生、发展中的作用。这对于以后前列腺疾病治疗药物靶点的开发具有重要的意义。取材：RT-PCR及Western blot检测采用新鲜标本，其中正常前列腺组织采用健康成人新鲜尸体前列腺标本，良性前列腺增生、前列腺癌标本为采用前列腺切除手术或穿刺活检取得的标本（取自山东大学第二医院及渮泽市立医院2003-2006年的住院病人），所有标本取出后均放于液氮中，转-80℃冰箱保存，实验前取出复温。检测M受体亚型在前列腺各区带的表达所用正常前列腺标本7例，检测M₃受体、VEGF、内皮抑素基因与蛋白表达所用标本分别为：正常前列腺36例、良性前列腺增生36例、前列腺癌8例。免疫组化标本采用石蜡包埋标本，正常前列腺组织来自健康、新鲜尸体前列腺标本，良性前列腺增生、前列腺癌标本为采用前列腺切除手术或穿刺活检取得的标本（取自山东大学第二医院2002-2006年的住院病人），标本经10％福尔马林浸泡，石腊包埋。其中正常前列腺36例，良性前列腺增生36例，前列腺癌36例。操作在超净台进行，取新鲜标本时，每取一个部位均更换手术刀片，正常前列腺组织每个标本取相邻3块，分别做RT-PCR与Western blot及做石腊块用于免疫组化。实验方法：一、实时荧光定量RT-PCR方法检测正常成人前列腺各区带M受体各亚型（M₁～M₅）基因表达实验所用物品的去RNase处理，将组织低温下研碎，组织提取总mRNA，合成cDNA，进行PCR扩增反应，荧光定量RT-PCR反应条件：95℃10s，95℃5s，60℃45s，40个循环；得到每个部位、每个受体的Ct值，用△Ct法转换为其初始相对浓度。其换算公式为：△Ct=Ct_样本-Ct_β-actin，平均相对含量=2^-△Ct。统计学处理应用SAS9.0软件进行统计学处理，统计方法为配伍组设计资料的方差分析。并应用q检验进行组间两两比较，P＜0.05认为差异有统计学意义。二、RT-PCR方法检测M₃受体、VEGF、内皮抑素在正常前列腺、良性前列腺增生、前列腺癌组织中的基因表达实验所用物品的去RNase处理，组织低温下研碎，提取总RNA，进行逆转录（RT）反应合成cDNA。聚合酶链反应（PCR）。定量分析：将凝胶电泳图像输入美国Kodak凝胶分析系统，应用1 D Image Analysis Software进行表达强度分析，按下公式计算相对系数。相对系数=细胞因子表达强度／β-actin表达强度统计学处理应用SAS9.0软件进行统计学处理，统计方法为随机设计资料的方差分析。应用q检验进行组间两两比较，P＜0.05认为差异有统计学意义。M₃与VEGF采用pearson相关性分析。三、免疫组化法检测VEGF、内皮抑素、CD34在正常前列腺、良性前列腺增生、前列腺癌组织中的蛋白表达首先将蜡块连续切片4片，片厚4μm。取胎盘组织以PBS代替一抗做阴性对照，把每组标本的3片分别做VEGF、内皮抑素及CD34免疫组化，另一切片做HE染色。根据HE染色，对每个样本进行病理学审查（由病理科医生协助完成），排除病变样本。结果判定以染色阳性物质呈棕黄色。M₃、VEGF、内皮抑素染色结果判定：根据阳性细胞显色深浅及阳性细胞百分率分级：①显色深浅：不显色0分；浅黄色1分；棕黄色2分；深褐色3分。②阳性细胞百分率：无阳性细胞显色0分；阳性细胞显色＜25％为1分；25％～50％为2分；阳性细胞显色＞50％为3分。二者得分相加：0分—阴性（-）；1～2分—弱阳性（+）；3～4分—阳性（++）；5～6分—强阳性（+++）。结果判断在双盲下进行，每张切片由两位病理医师分别阅片记数。CD34染色阳性的单个内皮细胞或一簇内皮细胞作为一个微血管，在200×视野下计数3个血管密度最高区域（热点）的微血管数目，其平均值即为肿瘤的微血管密度（MVD）。统计学处理应用SAS9.0软件进行统计学处理，统计方法为随机设计资料的方差分析与x²检验，P＜0.05认为差异有统计学意义。M₃与VEGF采用pearson相关性分析。四、Western blot方法检测三种M受体亚型（M₁、M₂、M₃）、VEGF、内皮抑素的蛋白表达取新鲜组织块，称量后提取蛋白（M受体提取膜蛋白、VEGF、内皮抑素提取胞浆蛋白）、加上养液、煮沸、在Bio-Rad垂直电泳仪中灌注质量分数10％分离胶和质量分数7.5％浓缩胶，加入不同组样本，稳压120V电泳90min，然后在转膜仪上将凝胶上的蛋白转移到PVDF膜上，稳压100V转移60min，取出膜后用7％脱脂牛奶封闭60min，加入一抗，4℃过夜，次日加入抗兔二抗孵育2h，DAB显色，扫描图像，输入美国Kodak图像分析系统，应用1 D Image Analysis Software进行表达强度分析，按下公式计算相对系数。相对系数=细胞因子表达强度／β-actin表达强度统计学处理应用SAS9.0软件进行统计学处理，统计方法为随机设计资料的方差分析。应用q检验进行组间两两比较，P＜0.05认为差异有统计学意义。VEGF与ES采用pearson相关性分析。结果：第一部分1、荧光定量RT-PCR方法检测结果显示五种M受体亚型在正常成人前列腺均有不同程度的基因表达，各个区带各受体的表达存在差异。以M₂、M₃、M₁受体的表达为主。在前纤维肌区M₂的表达明显高于其他受体（P＜0.05），约是M₃表达量的3倍；M₃、M₁、M₅、M₄的表达差异无统计学意义（P＞0.05）。在移行带M₂的表达明显高于其他受体（P＜0.0001），约是M₃表达量的三倍。M₃的表达明显高于M₄（P＜0.05）。在中央带M₂的表达最高，但只与M₄之间比较差异有统计学意义（P＜0.05）；M₂、M₁、M₃、M₅四者间差异无统计学意义（P＞0.05）。在外周带各受体亚型的表达差异均无统计学意义（P＞0.1）。2、Western blot检测显示M₁、M₂、M₃在正常前列腺、良性前列腺增生、前列腺癌组织中的蛋白表达存在差异。在正常前列腺M₂的表达高于M₃，更高于M₁，三者比较差异均具有统计学意义（P＜0.01）。在良性前列腺增生与前列腺癌组织M₂、M₃受体的表达高于M₁，差异具有统计学意义（P＜0.01）。M₂、M₃受体之间的表达差异无统计学意义（P＞0.05）。在正常前列腺与病变前列腺组织中表达差异具有统计学意义。M₁受体在正常前列腺、良性前列腺增生、前列腺癌三种组织的表达差异无统计学意义（P＞0.05）。M₂受体在正常前列腺、良性前列腺增生、前列腺癌三种组织的表达差异无统计学意义（P＞0.05）。M₃受体在前列腺癌组织的表达高于在良性前列腺增生组织的表达，正常前列腺组织的表达最低，差异均具有统计学意义（P＜0.01）。第二部分1、RT-PCR结果显示M₃受体、VEGF、内皮抑素在正常前列腺、良性前列腺增生、前列腺癌组织的基因表达存在差异M₃受体在不同前列腺组织的基因表达存在差异，表达顺序为：前列腺癌＞良性前列腺增生＞正常前列腺。三者间差异均具有统计学意义（P＜0.01）。VEGF在不同前列腺组织的表达不同，表达顺序为前列腺癌＞良性前列腺增生＞正常前列腺。三者间差异均具有统计学意义（P＜0.01）。内皮抑素在不同前列腺组织的表达不同，其均值顺序为前列腺癌＞良性前列腺增生＞正常前列腺。三者间差异均不具有统计学意义（P＞0.05）。M₃受体的表达与VEGF的表达具有正相关关系（r=0.4999 P＜0.001）。2、免疫组化结果显示在正常前列腺、良性前列腺增生、前列腺癌组织VEGF、内皮抑素的蛋白表达存在差异CD34在不同组织的表达强度顺序为：前列腺癌＞良性前列腺增生＞正常前列腺（P＜0.05）。MVD与前列腺癌临床分期的关系比较，因为样本含量少，合并临床Ⅰ、Ⅱ期与临床Ⅲ、Ⅳ期相比，P＞0.05，差异无统计学意义。MVD与前列腺癌Gleason评分的关系比较，评分≥8分的与评分＜8份的比较，P＞0.05，差异无统计学意义。VEGF在不同组织的表达强度顺序为：前列腺癌＞良性前列腺增生＞正常前列腺，差异均具有统计学意义（P＜0.01）。ES在不同组织的表达强度顺序为：前列腺癌＞良性前列腺增生＞正常前列腺，差异均具有统计学意义（P＜0.01）。VEGF与前列腺癌临床分期的关系比较，因为样本例数含量少，合并临床Ⅰ、Ⅱ期与临床Ⅲ、Ⅳ期相比，差异有统计学意义（P＜0.01）。VEGF与前列腺癌Gleason评分的关系比较，评分≥8分的与评分＜8分的比较，差异有统计学意义（P＜0.05）。ES与前列腺癌临床分期的关系比较，因为样本含量少，合并临床Ⅰ、Ⅱ期与临床Ⅲ、Ⅳ期相比，差异具有统计学意义（P＜0.01）。ES与前列腺癌Gleason评分的关系比较，评分≥8分的与评分＜8分的比较，差异具有统计学意义。（P＜0.05）3、Western blot检测结果显示VEGF、内皮抑素的蛋白表达存在差异VEGF在不同前列腺组织的表达不同，表达顺序为前列腺癌＞良性前列腺增生＞正常前列腺。三者间差异均具有统计学意义。（P＜0.05）内皮抑素在不同前列腺组织的表达不同，其均值顺序为前列腺癌＞良性前列腺增生＞正常前列腺。三者间差异均具有统计学意义（P＜0.05）。VEGF的表达与内皮抑素的表达具有正相相关（r=0.5582 P＜0.001）。结论：1、前列腺各个区带均有不同程度的5种M受体亚型的基因表达，主要以M₂、M₃、M₁为主。2、M₂、M₃、M₁受体在正常前列腺的蛋白表达与基因表达具有相同的趋势，M₃受体在前列腺肿瘤组织的蛋白表达高于在正常前列腺的表达，与前列腺肿瘤的发生、发展关系密切。3、VEGF、ES与前列腺肿瘤的发生及前列腺肿瘤的临床分期、gleason评分有密切关系。4、M₃、VEGF、ES在前列腺组织的表达具有正相直线相关关系。5、M₃、VEGF、ES、CD34可进一步研究作为前列腺肿瘤诊断指标及治疗新靶点的选择。更多还原

【Abstract】 Research background and objects:Prostate which is an important organ of male adult has many important functions, such as prostatic fluid secretion, ejaculation assisting, urination and so on. The physiological and functional disorder of the prostate and its pathological changes can cause many diseases, even affect the physical and mental health of men. Benign prostatic hyperplasia is,of the highest incidence disease among tumors of men in our country. Prostate cancer is one of the most common malignant tumors for men in western country. Although the incidence of prostate cancer is low in our country, it has the tendency to increase in recent years. Chronic prostatitis affects patients’ life because of severe symptoms and unsatisfied treatment, so that it continuously harasses urologists all over the world. Prostatic diseases’ disadvantages are serious enough to call our attention, but the mechanism of these diseases is still unknown and the treatment is not so satisfied.Detective and aggressive treatment of early prostate cancer resulted in a significant decline of overall mortality rates. However, the treatment of advanced tumor stages is still a crucial problem. Knowledge on molecular alterations taking place in the development and progression of prostate cancer increased rapidly in the past few years, thanks also to the use of novel techniques that enabled to perform broad gene expression profiling of tumors. This better understanding of the molecular biology of prostate cancer led to the establishment of novel strategies to delay/inhibit tumor progression.The growth and function of prostate is regulated by many nervous systems, including cholinergic and epinephrine nerves systems. In the past few years the construction and function of prostate have been deeply researched. The researches of autonomic nerve system’s regulation for function of the prostate demonstrated that the regulation of nerve system was intimately related to the genesis and development of prostatic diseases. Great success has been obtained in the research of alpha receptor inhibitor used in the treatment of benign prostatic hyperplasia, and androgen-blocking has been successfully used in the endocrine treatment of prostate cancer. But cholinergic muscarinic receptor’s functions in regulating the prostatic physiologic function, disease genesis and development is still unclear. And also there has been no such kind of drugs used in the treatment of prostatic diseases.In order to better understand the roles of cholinergic muscarinic receptors in prostate and to apply cholinergic drugs in the treatment of prostatic diseases, we should make out the distribution characteristics of muscarinic receptor in normal human prostatic tissue. Up to now there are only researches about muscarinic receptor on benign hyperplastic prostate tissue and prostatic tumor cell lines, but the report of distribution regularity in different zones of normal human prostate is only fewer. The only report is done by our research team about the distribution regularity of the three kinds of muscarinic receptor subtypes in normal human prostate in 2006The cholinergic receptor expression was prevalent in many tumors, in which mostly higher than in the normal tissue. The research about breast carcinoma and other tumors revealed that M₃ receptor was intimately related with the carcinogenesis and classification of tumor. The activity of cholinergic receptor could provoke the cell proliferate through different signal pathway. The previous research found out that M₁, M₂. M₃ receptors could stimulate cell growth and differentiation, but M₄ was not found having such funetions. Furthermore M₃ receptor is the main subtype to play the role of regulator, and be changed much in tumor generation. To understand the expression rule of M₃ receptor in healthy, hyperplastic, cancerous prostate tissue is very important for making clear the role of M₃ receptor and its molecular biologic mechanism in genesis and development of prostatic tumor.The proliferation of tumor cells and the formation of new blood vessels were important steps in the process of the genesis and development of tumor. In the research of breast cancer LMM3 cell line, it was found that the main subtype was M3 and its expression quantity was 40 folds of LM3 cell. The cholinergic receptor agonist CARB could provoke the growth and neovascular genesis of LMM3 cell, but this function could be suppressed by the selective M3 receptor antagonist. It was also demonstrated that CARB could promote the expression of VEGF-A, which could be inhibited by selective M3 receptor antagonist pf-HHSiD. All these findings told us that M3 receptor played an important role in LMM3 cell growth and neovascular genesis. But the researches about M 3 receptor in prostatic hyperplasia and cancer was much fewer, and the fewer finished study was done based on not strict normal control.Vascular endothelial growth factor（VEGF） is an important factor in neogenesis of tumor vessels. VEGF and its receptor are highly expressed in most malignant tumors, through para/autoendocrine reaction, promoted endothelial cell division, proliferation and induced vascular neogenesis, directly affecting tumor cell and provoking it to grow. In prostate, VEGF could be synthesized by normal duct endothelial cell, adenoma cell and infiltrative lymphocyte.The stronger the metastatic characteristic of human prostatic cancer cell was, the higher VEGF expression would be, and neovascular genesis would be more effectively promoted by VEGF which was synthesized and secreted by malignant cell. The VEGF receptor expressed on both the vessel endothelium of prostatic tumor and the tumor cell showed that VEGF not only stimulated vascular endothelial proliferation by paraendocrine way but also regulated tumor cell proliferation by autoendocrine way.Endostatin was XVIII collagen C-terminal amino segment which was isolated from culture solution of vascular endothelioma. It could specifically inhibit endothelial cell proliferation, metastasis and tumorous angiogenesis, and could induce cell apoptosis, so that it made the primary and metastatic site of tumor stay in dormant state and stop growing.To study VEGF, endostatin and M₃ receptor’s relation in normal and tumor prostate is very important for further understanding the mechanism of tumorigenesis,development, and selecting the target of treatment in late stage tumor. At present the research on this aspect about human prostatic tumor has not been reported.For the above reason my research firstly studied the mRNA expression of muscarinic receptor subtypes in each zone of normal prostate and discussed the expression difference of each subtype among different zones. Then we detected the expression of those three kinds of receptors, among the normal, hyperplastic and cancerous prostate, using Western blot, which were highly expressed in the prostatic tissue and had intimate relationship with the tumor genesis. We gained the protein expression difference of each subtype in different zones of prostate tissue and analyzed the role of different subtype in normal human prostate,and in the genesis.developmeiit of diseases. At last using RT-PCR, immunohistochemistry, Westsern blot, we comparatively studied the expression of VEGF and endostatin in prostatic tissue at genetic and protein levels, together with the expression of M₃ receptor in normal, hyperplastic and cancerous prostate, and discussed their roles in genesis and development of prostatic tumor.These researches were important for understanding M receptor, VEGF and endostatin’s roles in prostate’s function educing and disease generation, also it is more important for developing new drugs for prostatic diseases .Material and method:Materials:The fresh normal prostates were obtained from healthy adult donators who were died of accident. The samples of hyperplastic, cancerous prostates were taken from the hospitalized patients in The Second Hospital of SDU from 2000 to 2006, and from prostatectomy or core biopsy. All the tissues were put in nitrogen liquid and kept in -80℃refrigerator for further using. Seven samples of normal prostate were used in detecting M receptor subtype in each zone of prostate. The specimens used in detecting M₃ receptor, VEGF, endostatin at genetic and protein levels included 36 normal prostates, 36 hyperplastic ones, and 8 cancerous ones.The samples detected with immunohistochemistry were paraffin-embedded ones. The normal prostatic tissue was from fresh corpses of healthy adults. The hyperplastic, cancerous samples were from hospitalized patients of The Second Hospital of SDU from 2000 to 2006, taken with prostatectomy or care biopsy. The samples were soaked in 10% formalin and paraffin-embedded. There were 36 normal prostate, 36 hyperplastic ones and 36 cancerous ones. All the operations were taken on super clean bench. During taking fresh samples, the operating knife blade should be changed for different site. Every sample of normal prostate was taken out 3 pieces, respectively used in RT-PCR,western blot and made into paraffin-embedded block which would be used in immunohistochemistry.Methods:I .Detect muscarinic receptor subtypes’genetic expression in each zone of normal human prostate with real-time fluorescent quantitative RT-PCRProcess the experiment material with RNase. Levigate the tissue at low temperature. Extract the total mRNA. Synthesize cDNA. Carry out PCR amplification reaction. Real Time PCR reaction condition: 95℃10s, 95℃5s, 60℃45s, 40 cycles; Every receptor’s threshold cycle（Ct） value of each site which we got was transformed into its initial relative concentration with△Ct way. The transformation formula:△Ct=Ct_sample-Ct_β-actin. The mean relative quantity = 2^-△Ct . Apply SAS9.0 software in statistical treatment. The statistical method was variance analysis to pair-matched data. And q test was used to compare every two groups. The difference was considered as statistically significant when P<0.05.II .Using RT-PCR detect M₃ receptor, VEGF, endostatin’s expression in normal,hyperplastic, cancerous prostate at genetic level:The experiment material was processed with RNase. Levigate the tissue at low tempreture. Extract the total RNA.The RNA was then reverse transcribed into cDNA. Input gel electrophoresis picture to the American Kodak gel analystic system.Use 1 D Image Analysis Software to analyse the expression intensity.Calculate the relative coefficient with the formula below.The relative coefficient=cytokine expression intensity/β-actin expression intensityIII. Detect VEGF, endostatin, CD34’s expression in normal, hyperplastic, and cancerous prostate with immunohistochemical methodFirstly we cut 4 serial sections with thickness 4 micrometres from the paraffin block. Fetal tissue with PBS instead of first Ab serves as negative control. The 3 slices of every group were respectively used in VEGF , endostatin and CD34’s immunohistochemical detection. The other one was HE stained. Every sample was inspected by the pathologist. In the result evaluation the positive stained should be brown. M₃, VEGF, endostatin’s staining result assessment: to classify them according to the extent of positive cell coloration and positive cell percentage: i according to the coloration:no color score 0;buff 1;buffy 2;sable 3. ii according to the positive cell percentage: no positive cell score 0; positive cell<25% 1; positive cell 25%-50% 2; positive cell>50% 3;Then add the two score : 0-negative（-）;1-2-weakly positive（+）; 3-4-positive（++）;5 -6-strongly positive（+++）.The result assessment was carried out with double blind method. Every slice was inspected and counted by two pathologists respectively. We considered one single endothelium or one cluster of endothelium, which was CD34 positive stained as one microvessel. We counted the microvessel number of 3 different visual field where the vessel density was the highest in 200X view field. The mean value was treated as tumor’s microvessel density.iv. Detect M₁, M₂, M₃, VEGF and endostatin’s protein expression in normal, hyperplastic, cancerous prostate with western-blot methodFresh tissue were taken and weighted.Then the protein（we extracted membrane protein for M receptor, extracted the cytoplasmic protein for VEGF and endostatin） was extracted and denatured.Different samples were added in Bio-Rad vertical electrophoresis apparatus which had been perfused with weight percentage 10% separating gel and weight percentage 7.5% stacking gel, and ran for 90 min at stable voltage 120 volt. Then buffer transferred the protein in the gel onto PVDF membrane with transferring membrane, at stable voltage 100 volt for 60 min. The membrane was taken out and blocked by 7% skimmed milk for 60 min. Then it was incubated with first Ab overnight at 4℃, washed thoroughly and with the second Ab for 2 hours. Stained the membrane with DAB and scanned the picture. The picture was inputted into the American Kodak Image Analystc System. Using 1 D Image Analysis Software analyzed the expression density. The relative coefficient was calculated with the formula below:The Relative Coefficient=Cytokine expression density/β-actin expression densityResults:I .The fluorescent quantitation RT-PCR detection demonstrated that five muscarinic receptor subtypes were all expressed to different extent at genetic level in normal human prostate.There was difference among these receptors’ expression in different zones. It was mainly M₂, M₃, and M₁ receptors expressed.In prefibromuscular zone, M₂ was obviously higher than other receptors（P<0.05）,it is about three times of M₃’s expression; The difference among M₃, M₁, M₅, M₄’s expression was not statistically significant（P>0.05）.In transitional zone, M₂’s expression was the highest （P<0.0001） in all the 5 receptors ,it is about 3 times of M₃’s.M₃’s was obviously higher than M4’s（P<0.05）.In central zone, M₂’s expression was the highest, but it was only statistically significant when it was compared with M4（P<0.05）;the differences among M₂, M₁, M₃ and M₅ were not statistically significant（P>0.05）.In peripheral zone there was no significant difference among those receptor subtypes （P>0.1）.II .Western blot showed that there was protein expression difference among M₁, M2, M3 in normal, hyperplastic and cancerous prostatic tissue. In normal human prostate M₁ receptor expressed the highest（P<0.01）. The differences of M3 receptor’s expression in normal and pathological prostate tissue were statistically significance （P<0.05）,while M₂ and M₁’s wasn’t（P>0.05）.The highest expression receptor in cancerous prostate tissue was M3 receptor（P<0.01）.III. RT-PCR demonstrated that there was gene expression difference among M3 receptor, VEGF, endostatin in normal, hyperplastic and cancerous prostate tissue.M3 and VEGF’s expression was higher in cancerous prostate than in normal prostate with significant difference （P<0.01）.But endostatin’s expression difference among the three different kinds of tissue wasn’t statistically significant（P>0.05）.It has positive correlation between M₃ and VEGF’s expression （P<0.001） .IV .The immunohistochemistry revealed that there were protein expression differences of VEGF and endostatin in normal, hyperplastic and cancerous prostatic tissue. VEGF and endostatin’s expression were higher in prostate cancer tissue than in BPH tissue, while their expression were lowest in normal prostate tissue with the significant differences （P<0.01）. In prostate cancer the more the Gleason score was,the more higher VEGF and endostatin were expressed （P<0.05） .Furthermore the expression in the late clinical stage wre higher than in the early stage （P<0.05） .V .The Western blot indicated that VEGF and endostatin expressed highest in prostate cancer tissue ,moderate in BPH tissue, lowest in normal prostatic tissue with the significant differences （P<0.05 ） . It has positive correlation between VEGF and endostatin’s expression （P<0.001） .Conclusions:I .There are genetic expressions of all the five muscarinic receptor subtypes in each zone of prostate, mainly are M₂, M₃, M₁.II .In normal human prostate, the genetic expression of M₂, M₃, M₁ is similar to their protein expression,M3 receptor’s protein expression is higher in cancerous than in normal prostatic tissue, which means it concerned with tumorigenesis of prostate .III.M₃, VEGF and endostatin’s expression in prostatic cancer is related with the tumor’s clinical stage and pathological Gleason grading.IV. It has positive correlation between M₃, VEGF and endostatin’s expression in human prostate tissue.M₂, M₃, VEGF and endostatin’s expression is high in prostatic cancer tissue.V.M₃, VEGF, ES, and CD34 are related with genesis and development of prostatic tumor. They can be used as new targets of diagnosis and treatment for prostastic tumor.更多还原