摘要
研究背景与目的：
慢性前列腺炎(chronic prostatitis, CP)是一种涉及几乎所有年龄组的男性疾病,中青年居多,是50岁以下男性就诊泌尿外科的主要原因；其症状复杂,包括疼痛、排尿症状以及性功能障碍等,迁延难愈的临床症状能够导致患者心理精神的改变,严重干扰了患者的生活质量,美国国立卫生研究院已经将慢性前列腺炎归为四大影响患者生活质量的疾病之一。前列腺炎患病率大约10-14%,高达50%的男性在其一生中的某个阶段可能受到前列腺炎症状的困扰[3]。国内梁朝朝等基于人口数量的横断面研究,8.4%被确诊具有前列腺炎症状,4.5%根据NIH-CPSI以及前列腺炎症状学确诊为前列腺炎。前列腺炎的分类复杂,传统的分类方法以Meares-Stamey实验为依据将前列腺炎分为四类：急性细菌性前列腺炎(Acute bacterial prostatitis,ABP)、慢性细菌性前列腺炎(Chronic bacterial prostatitis, CBP)、慢性非细菌性前列腺炎(Non-bacterial prostatitis, NBP)、前列腺痛(Prostatodynia, PD)等,这一分类的依据是基于前列腺炎症状持续的时间以及严重程度、尿道不同节段是否存在脓细胞和病原菌等,尽管这一分类系统并未经过验证,但已经被几代泌尿外科医生所采用。1995年美国国家糖尿病、消化道疾病和肾病协会前列腺炎工作组(TheNational Institute of Diabetes and Digestive and Kidney Diseases, NIDDK) [6]召集慢性前列腺炎工作组会议,制定了新的前列腺炎定义和分类：Ⅰ型：急性细菌性前列腺炎(Acute bacterial prostatitis, ABP)、Ⅱ型：慢性细菌性前列腺炎(Chronic bacterial prostatitis, CBP)、Ⅲ型：慢性非细菌性前列腺炎／慢性骨盆疼痛综合症(Chronic nonbacterial prostatitis/chronic pelvic pain syndrome, CP/CPPS)和Ⅳ型：无症状性前列腺炎(Asymptomatic inflammatory prostatitis, AIP)等；根据前列腺按摩液(EPS)中是否存在白细胞将CP/CPPS又分为炎症型(Ⅲa)和非炎症型(Ⅲb)两类,前者EPS中含有较多的白细胞,而后者EPS中白细胞数量在正常范围。CP/CPPS是慢性前列腺炎中占有比例最高的一类,绝大部分CP(高达90%)属于此种类型。与Ⅰ、Ⅱ两型前列腺炎不同,Ⅲ型前列腺炎前列腺组织内应用传统细菌学技术检查无感染征象,慢性疼痛是其主要症状。以往分类依据是基于如下假设：1)EPS、精液、前列腺按摩后尿液等发现细菌是前列腺炎症状和精液异常的原因；2)精液或前列腺液中存在白细胞是炎症的标志；3)不能够发现白细胞或细菌,能够排除感染或炎症。但是,上述标准均未被确定地证实,因此,“非细菌性前列腺炎”和“前列腺痛”被代以“慢性骨盆疼痛综合症”。将两者归为一类是因为无论是否存在前列腺炎症,并不影响慢性前列腺炎临床症状,不能改变慢性前列腺炎预后以及对治疗效果的反应等。提出这一新的概念说明现在对前列腺炎病因及其发病机制了解甚少；而且慢性前列腺炎症状可能不仅与前列腺一个器官有关,极可能涉及到前列腺以外的器官。CP/CPPS的发病原因迄今尚未明确,目前认为其发病与多种因素有关,包括病原微生物的感染、神经性因素、外伤、自身免疫反应、尿流动力学因素、精神心理因素等。近几年自身免疫反应机制得到了较多的研究,并且已经具有自身免疫性前列腺炎的动物模型,即啮齿类动物实验性自身免疫性慢性前列腺炎动物模型(experimental autoimmune prostatitis, EAP)。Wistar大鼠以及不同品系的小鼠,包括C57BL／6小鼠、NOD小鼠等作为模型动物,采用前列腺匀浆提纯液作为免疫原,联合CFA免疫动物,成功制作了免疫性前列腺炎的模型,运用此种模型动物脾脏分离的淋巴细胞,经体外刀豆蛋白刺激后转输给未经免疫动物能够诱发慢性前列腺炎,并在前列腺组织内出现明显的炎症细胞浸润。研究证实,EAP前列腺组织内浸润的淋巴细胞以Th1类辅助性T淋巴细胞为主；运用此种免疫性前列腺炎动物模型研究发现,炎症能够导致小鼠出现类似于临床CP/CPPS症状,包括疼痛症状等；利多卡因前列腺内注射可以缓解疼痛,说明疼痛症状与前列腺炎有关：而且,应用Wistar大鼠制作的EAP模型,其精液质量受到明显的影响。这一实验结果说明,免疫性前列腺炎,作为一种非细菌性前列腺炎,能够引起常见的临床症状,且对患者造成生殖功能方面的影响。
CD4+辅助性T淋巴细胞(Th)在适应性免疫应答中发挥着重要的调节作用,根据分泌的细胞因子的不同,经典分类方法将辅助性CD4+T淋巴细胞分为Th1与Th2细胞两大亚群,前者主要产生γ干扰素(Interferonγ, IFNγ),其效应与IFNγ及白细胞介素12(Interleukin 12, IL12)有关,调节细胞免疫应答,清除细胞内病原体；后者主要分泌白细胞介素4(interleukin 4, IL4)、白细胞介素5(Interleukin 5,IL5)、白细胞介素13(Interleukin 13, IL13)等,其效应主要与IL4有关,介导体液免疫反应和超敏反应,清除感染的寄生虫。Th17辅助性T淋巴细胞是以分泌白细胞介素-17(Interleukin 17, IL17)为主的一类辅助性T淋巴细胞,是近几年在小鼠自身免疫性疾病模型的研究中新发现的一类辅助性T淋巴细胞。Th17T淋巴细胞与Thl和Th2两类辅助性T淋巴细胞不同,以分泌IL17为主,研究发现此类CD4+辅助性T淋巴细胞与多种自身免疫性疾病有关,例如类风湿关节炎、多发性硬化、系统性红斑狼疮、血管炎以及其他免疫性疾病如银屑病、多发性硬化、肾病综合征、克罗恩病和溃疡性结肠炎等。IL17属于促炎症因子,其主要功能是作为前炎症介质在局部诱导白细胞介素6(Interleukin 6, IL6)、NO、前列腺素E2等放大局部炎症,通过诱导细胞集落刺激因子和白细胞介素8(Interleukin 8, IL8)的分泌参与中性粒细胞募集反应；IL17促进单核细胞分泌炎症因子包括肿瘤坏死因子a(Tumor necrosis factor a, TNFa)、白细胞介素1β(Interleukin 1β, IL-1β)等并与其发挥协同作用,进一步加强炎症反应；白细胞介素23(Interleukin 23, IL23)是由树突状细胞和其他抗原提呈细胞分泌的一个促炎症细胞因子,由一个独特的p19亚单位以及与白细胞介素12(Interleukin 12, IL12)共用的p40亚单位组成,是调节IL17产生的重要细胞因子,能够特异性地刺激记忆性CD4+T淋巴细胞,促进Th17细胞的表达,并在Th17细胞的扩增和稳定中发挥重要作用,从而在自身免疫性疾病中发挥十分重要的作用。
本实验运用文献介绍的EAP模型制作方法,应用C57BL／6小鼠制作免疫性前列腺炎动物模型,运用一步法实时定量RT-PCR方法检测EAP前列腺组织Th17辅助性T淋巴细胞相关的细胞因子包括IL17、IL23p19和IL23p40等的表达情况；并应用免疫组化方法,检测EAP小鼠前列腺组织中Th17淋巴细胞浸润状况,从而阐明Th17淋巴细胞与小鼠免疫性前列腺炎发病之间的关系,为慢性前列腺炎发病的免疫机制及其治疗措施的研究提供理论依据。
本研究共分为三部分进行：
一、C57BL／6小鼠实验性免疫性前列腺炎(EAP)模型的制作；
二、免疫性前列腺炎组织Th17辅助性T淋巴细胞浸润状况免疫组化研究；
三、免疫性前列腺炎组织Th17辅助性T淋巴细胞相关细胞因子表达研究。
第一部分C57BL／6小鼠EAP模型制作
方法：
C57BL／6小鼠作为模型动物,40只,清洁级,随机分为两组,各20只,一组为免疫性前列腺炎症组(EAP组),一组为对照组。
应用Wistar大鼠前列腺组织蛋白提纯夜作为免疫原,调节蛋白浓度至2mg／ml；小鼠水合氯醛腹腔注射麻醉后,每只小鼠多点皮下注射前列腺蛋白提纯夜0.5ml,同时腹腔注射CFA0.5ml与百白破疫苗0.1ml,分别在0,30天免疫注射两次；首次免疫后60天,再将炎症组和对照组小鼠分别随机各分为两组,即将炎症组小鼠随机分为两组,一组8只,用于RT-PCR检测；一组为12只,用于前列腺组织淋巴细胞浸润的免疫组化研究；对照组小鼠同样分为两组,各为8只和12只,分别用于RT-PCR与免疫组化检测。
小鼠10%水合氯醛腹腔注射麻醉(0.03ml／10g),手术切取前列腺,用于RT-PCR检测的炎症组和对照组各8只小鼠的前列腺分为两部分,一部分大约为前列腺体积的1／4,用4%多聚甲醛固定,待用于HE染色,检测前列腺组织内炎症细胞的浸润状况；余部分前列腺组织液氮冷冻后转于-80℃低温冰箱保存,留待行RT-PCR检测Th17淋巴细胞相关的细胞因子表达情况；用于免疫组化的炎症组和对照组各12只小鼠,前列腺组织4%多聚甲醛固定24小时,30%蔗糖沉糖后,转于-80℃低温冰箱保存,留待行冰冻切片免疫组化,检测CD4+、CD8+、Th17淋巴细胞在前列腺组织内的浸润状况,同时行HE染色检测炎症细胞的浸润情况。
结果：
1小鼠前列腺大体形态观察
首次免疫后60天,手术切取小鼠前列腺。炎症组小鼠前列腺与周围组织严重粘连,不易分离,前列腺组织可见充血、水肿；对照组小鼠前列腺与周围组织无粘连,无充血,手术分离容易。
两组小鼠体重比较：对照组小鼠体重22.7-26.1g,平均25.0±2.2g；炎症组小鼠体重21.7-28.3g,平均23.7±1.9g；两组比较t=1.6,p>0.05,差别无统计学意义；
两组小鼠前列腺重量比较：对照组小鼠前列腺重量23.2～33.5mg,平均28.5±3.9 mg；炎症组小鼠前列腺重量26.1-42.3mg,平均32.7±5.4mg；两组比较t=-1.5,p>0.05,差别无统计学意义。
两组小鼠前列腺指数(前列腺重／小鼠体重)比较：对照组小鼠前列腺指数0.0008-0.0014,平均0.0011±0.0002；炎症组小鼠前列腺指数0.0012-0.0018,平均0.0014±0.0001；两组比较t=-2.6,p<0.05,差异具有统计学意义。
2小鼠前列腺组织病理学观察
小鼠前列腺组织冰冻切片,厚度5μm,HE染色,光学显微镜观察组织病理学改变。炎症组小鼠前列腺可见轻重不同的炎症细胞浸润,浸润炎症细胞包括淋巴细胞、中性粒细胞、浆细胞等,以间质、血管周围浸润明显；部分腺泡可见扩张、腺体结构破坏等。对照组小鼠前列腺组织结构正常,未见明显炎症细胞浸润。
第二部分免疫性前列腺炎组织Th17辅助性T淋巴细胞浸润状况免疫组化研究
方法：
取冰冻保存(已经4%多聚甲醛固定)的小鼠前列腺组织,冰冻切片机连续切片,厚度5μm,贴片、吹干、4%多聚甲醛固定,按照SABC试剂盒说明书步骤,行免疫组化检测。结果判断以淋巴细胞胞浆和／或胞膜棕黄色着色并高于背景色为阳性,每张载玻片随机选取5个高倍视野,计数阳性细胞数量,取其均值作为阳性细胞数。
结果：
免疫组化检测结果：CD4、CD8、IL17三种标记在对照组和炎症组前列腺组织淋巴细胞均有表达,炎症组明显高于对照组。
CD4阳性细胞数量：对照组小鼠前列腺组织切片每高倍视野1.0-4.4个,平均2.4±1.1个；炎症组小鼠前列腺组织切片每高倍视野5.6-12.7个,平均9.1±2.1个；两组比较,t=9.7,p<0.05,差别具有统计学意义；
CD8阳性细胞数量：对照组小鼠前列腺组织切片每高倍视野0.2-1.4个,平均0.8±0.3个；炎症组小鼠前列腺组织切片每高倍视野2.7-6.8个,平均4.8±1.3个；两组比较,t=10.4,p<0.05,差别具有统计学意义；
IL17阳性细胞数量：对照组小鼠前列腺组织切片每高倍视野0.3-5.1个,平均2.3±1.8个；炎症组小鼠前列腺组织切片每高倍视野3.2-8.2个,平均5.7±1.8个；两组比较,t=4.7,p<0.05,差别具有统计学意义。
第三部分免疫性前列腺炎组织Th17T淋巴细胞相关细胞因子表达研究
方法：
取低温保存的小鼠前列腺组织约20mg,运用RNAprep pure动物组织总RNA提取试剂盒(离心柱型)说明书所列实验步骤进行操作,提取前列腺组织总RNA；提取的总RNA浓度为118ng／μl,纯度为1.95,符合RT-PCR检测要求；运用SYBR Green I一步嵌合荧光法实时定量RT-PCR检测前列腺组织Th17淋巴细胞相关细胞因子IL17、IL23p19、IL23p40等mRNA的表达；应用2-ΔCT表示各个细胞因子mRNA相对表达量。
结果：
IL17、IL23p19、IL23p40等mRNA的表达相对含量结果：
对照组小鼠前列腺组织IL17相对含量为0.0005-0.0081,平均0.0036±0.0029；炎症组小鼠前列腺组织IL17相对含量为0.0221-0.0980,平均0.0530±0.0265；组间比较t=-4.5,p<0.05,差别具有统计学意义；
对照组小鼠前列腺组织IL23p19相对含量为0.0008-0.0092,平均0.0051±0.0025；炎症组小鼠前列腺组织IL23p19相对含量为0.0107-0.0258,平均0.0177±0.0065；组间比较t=-4.5,p<0.05,差别具有统计学意义；
对照组小鼠前列腺组织IL23p40相对含量为0.0002～0.0072,平均0.0041±0.0028；炎症组小鼠前列腺组织IL23p40相对含量0.0092-0.0861,平均为0.0289±0.0283；组间比较t=-2.1,p<0.05,差异有统计学意义。
结论：
运用前列腺组织蛋白提纯液作为免疫原免疫C57BL／6小鼠成功制作免疫性前列腺炎模型；
1、小鼠免疫性前列腺炎组织Th17相关的细胞因子mRNA表达增高,前列腺组织内浸润的IL17+细胞明显增多,说明Th17淋巴细胞参与了免疫性前列腺炎的发病过程；
2、研究与Th17淋巴细胞有关的治疗方法可能有助于慢性前列腺炎治疗研究。
主要创新点：
1.运用免疫组化方法,首次检测了C57BL／6小鼠EAP模型前列腺组织内Th17辅助性T淋巴细胞浸润状况,研究免疫性前列腺炎发病与Th17T淋巴细胞之间的关系。
2.运用实时定量PCR法首次检测了EAP模型前列腺组织内Th17T淋巴细胞相关细胞因子IL17、IL23p19、IL23p40表达情况,研究免疫性前列腺炎发病与IL17等细胞因子之间的关系。
Background:Chronic prostatitis(CP) is a multifactorial problem, which affects men of all ages and in all demographics, and it is the main reason for the patients under 50 years old to go to hospital. Chronic prostatitis symptoms usually include pain, voiding and sexual dysfunction, even some patients usually experience psycological problems. Men with prostatitis tend to experience serious impairments in health-related quality of life (QOL). The QOL of patients with prostatitis is comparable with that of a patient suffering from recent myocardial infarction, unstable angina, or Crohn's disease. The morbidity of CP is about 10-14%, and more than 50% of the whole male suffered this disease in their life. A native population-based cross-sectional study reported by Liang-Zhaozhao indicated that 8.4% of them in china had symptoms of CP,4.5% of them were diagnosed as CP by NIH-CPSI and CP symptomatology. The classification of prostatitis is very complicated, and it was classify into 4 kinds by the traditional classification, they were (1) Acute bacterial prostatitis (ABP);(2) Chronic bacterial prostatitis (CBP);(3) Non-bacterial prostatitis (NBP); and (4) Prostatodynia (PD). This classification is based on the duration of the symptoms of CP and the serious of the symptoms of it. Though it was never validated but it was used by the urological doctors for some generations. In 1999, the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)) proposed a new classification:(1) Acute bacterial prostatitis is an acute infection of the prostate(ABP); (2) Chronic bacterial prostatitis is a recurrent infection of the prostate(CBP); (3) Chronic nonbacterial prostatitis/chronic pelvic pain syndrome (CP/CPPS), where there is no demonstrable infection. Subgroup of this class are:(3.1) Inflammatory CPPS, where white cells are found in the semen, EPS, or VB3. (3.2) Non-inflammatory CPPS, where white cells are not found in the semen, EPS, or VB3. (4) Asymptomatic inflammatory prostatitis (AIP), where there are no subjective symptoms, but white blood cells are found in prostate secretions or in prostate tissue during an evaluation for other disorders.
The constituent ratio of CP/CPPS is the highest one in the 4 kinds of prostatitis, about 90% percent of the whole patients with prostatitis belong to CP/CPPS, different from other 3 kinds, there is not evidence which can prove that there is bacteria in the prostatic tissue or EPS in CPPS patients by the traditional bacterial technology, and pain is the most common symptom of this kind of prostatitis. The early classification is based on this assumption that 1) the finding of bacteria in EPS, semen or the urine after prostate message is the reason of the symptoms of prostatitis and the abnormity of semen; 2)the exist of white blood cells in semen or EPS is the marker of the inflammation of prostate; 3)the inflammation or infection can be excluded if the white blood cells or bacteria can not be found in EPS or semen. But all above were not validated up to now, so the term "nonbacterial chronic prostatitis" and "Prostatodynia" were taken the place by the term "CPPS". The reason is whether there is inflammation in prostate tissue or not, the symptoms of the prostatitis were not influenced, and the prognosis and the curative effects of it were not influenced too. On the other hand, the new concept indicate that we know little about the etiology of chronic prostatitis, and maybe the symptoms of chronic prostatitis were involved more than one organ. The etiology of CPPS is not clear up to now, and many factors were thought to be related to its oncoming, include infection, injury, nerves factors, autoimmune, urine follow-metry, and psychological problems,et al.
The rats or mice were injected with The purified prostate tissue protein combined with complete Freund's adjuvant(CFA) and diphtheria pertussis tetanus (DPT) vaccine at 0 and 30 days, The model of animal chronic prostatitis was made successfully in rodent animals. And the adoptive transfer of the lymphocytes suspended from the immuned mice spleens can also induce the prostate inflammation of mice and the obvious infiltration of inflammatory cells in the prostate can be found. It was proved that the main kind of lymphocytes infiltrate in the prostate was Th1 lymphocyte, and the mice immuned with this method can be found the similar symptoms of clinical prostatitis. Applying the rats model of autoimmune prostatitis to study the influence on the reproduction by this disease, the quality of semen of the rats was injured obviously.
According to the types of cytokines secreted by the CD4+ T helper lymphocytes, it was divided into two kinds, include Thl and Th2 helper T cells. The main cytokines secreted by the former cells include IFNy and IL12, and the function of it was related with IFNy and IL12 to regulates the cellular immunity, wiping out the pathogen in the cells; the main cytokines secreted by the later T cells were IL4、IL5、IL13 et al, and the function of it was related with IL4, regulating the humoral immunity to wipe out the parasite. In recent years a new kind of assistant T lymphocyte was found which secret IL17 mainly, and it has many to do with lots of autoimmune diseases such as rheumatoid arthritis(RA), multiple sclerosis, systemic lupus erythematosus (SLE), Crohn's disease, ulcerative colitis et al. IL17 is belong to pro-inflammatory cytokines and it can induce the secretion of IL6, NO, PGE2 so as to amplify the local inflammation; it can induce the recruiting response of neutrophile granulocyte by stimulating the secretion of colony stimulating factor and IL8; IL17 can promote the secretion of Tumor necrosis factor a(TNFa), Interleukin 1B(IL-1B), and they have synergistic reaction and thereby strengthen the inflammation reaction. IL23 is one kind of pro-inflammatory cytokine secreted by DCs and other antigen presenting cells, it was made of two subunits include p19 and p40 which is shared with Interleukin 12(IL12), and it is a important factor regulates the production of IL17. IL23 can promote the expression of Th17 cell, and has important funtion in the amplification of Th17 cells.
We use the method introduced by papers to make the C57BL/6 mice model of experimental autoimmune prostatitis, and measure the expression of Th17 related cytokines include IL17, IL23p19 and IL23p40 and study the infiltration of Th17 lymphocytes in the prostate tissue by real time RT-PCR and immunol histochemistry respectively as well.
Part one The making of experimental autoimmune prostatitis(EAP) with C57BL/6 mouse
Methods:
40 C57BL/6 mice were used to make the model of autoimmune prostatitis. They were divided into 2 groups randomly, each group had 20 mice. One group was as EAP group and the other was as control group. The prostates of Wistar rats were used to make purified extract, which would be used as antigen. The concentration of the extract was diluted to 2mg/ml. after the mouse were anesthetized by chloral hydrate by intraperitoneal injection,0.5ml prostate extract was injected subcutaneously as the same time each mouse was injected with CFA intra-peritonealy (0.5ml) combined with DPT vaccine(0.1ml),30 days after the first injection, the second injection was performed.60 days after the first injection, the mice were operated by open surgery to harvest the prostates. And the EAP group and the control group was divided in to two groups individually randomly, in the other words, the EAP group was divided into 2 groups and as the same time the control group was divided into 2 groups too, one group has 8 mice and the other group has 12 mice. The group of 12 mice was used to do the study of the CD4+、CD8+ and IL17+ lymphocytes infiltration in the prostate tissue, and the other group of 8 mice was used to study the expression of Thl7cell related cytokines in the prostate tissue.
The mice were anesthetized by chloral hydrate, the abdominal wall of the mice were opened and the prostate were harvest. The prostate which would be used to check the cytokines were divided in to two parts, one part was about 1/4 volume of the whole prostate was fixed into 4% paraformldehyde for about 24 hours for the use to observe the infiltration of inflammatory corpuscle in the prostate tissue, and the other part was snapped in to the liquid nitrogen and then shift to-80℃low temperature refrigerator so as to be used to do the RT-PCR.
The prostates of the other 12 mice whose prostates would be used to do immunol histochemistry were fixed into paraformldehyde for about 24 hours and then shift to the-80℃low temperature refrigerator for the use of immuno histochemistry later.
Results:
1 The observation of the gross appearance of the prostate
60 days after the first immune injection, the prostates of the C57BL/6 mice were harvest. In the operation we could find a serious adhension between the prostate and the adjacent tissues and organs, include the pelvic wall and the rectum in the EAP group, the prostate was congestion with blood and has obvious oedema, and the operation was difficult to isolate the prostate.but in the control group there was not adhenssion and it was easily to isolate the prostate.
The weight of the mice body of the control group was 22.7～26.1g, and the mean weight of it was 25.0±2.2g; the weight of the mice body of the EAP group was 21.7～28.3g, and the mean weight was 23.7±1.9g, t=1.6, p>0.05, and the differentce between the two groups has not statistics significance.
The weight of the prostate of the control group was 23.2～33.5mg, and the mean weight was 28.5±3.9 mg; the weight of the prostate of the EAP group was 26.1～42.3mg, and the mean weight was 32.7±5.4mg, and the difference between the two group has not statistics too(t=-1.5, p>0.05).
The index of the prostate of the two groups were 0.0011±0.0002(0.0008～0.0014)and 0.0014±0.0001 (0.0012～0.0018) respectively and the difference was statistics (t=-2.6, p<0.05).
2 The histopathology
The frozen section was made with 5μm. And the histo pathology was observed by microscope. In the EAP group the inflammation was founded and there were a lot of infiltration of inflammation cells in the proastate, the cells infiltrate in the prostate was lymphocytes, granulocyte cells and plasmacytoid lymphocyte.
The most seriously places infiltrated with inflammation cells was in the interstitial, and the surrounding of the vessils. It could be found oedema, dillation of the gland, and the destruction in the gland.
On the other hand, there was not infiltration of inflammation cells in the control group.
Part 2 The study of the infiltration of Thl7 cells in the prostate tissue
Methods:
The prostate stored in the low temperature ice box was made frozen section in 5μm, mounted on the slide glass, dry by airing, fixed in 4% paraformaldehyde for about 30 minutes, according to the instruction of SABC kit to do the immuno histochemistry test. The diagnosis of the result was according to the stain of the cells. if the membrane and the plasm was stained brown madder it was looked as possive.
Five high power field was selected and count the number of possive cells in each field, then make the average number as the result of each cells.
Results:
The result of immuno histochemistry test:
The number of CD4 possive cells was 1.0～4.4 in each high field and the average was 2.4±1.1 in the control group; The number of CD4 possive cells was 5.6～12.7 in each high field and the average was 9.1±2.1 in the EAP group; there is statistic significance between the two groups (t=9.7,p<0.05).
The number of CD8 possive cells in the control group was 0.2-1.4 each high field and the average was 0.79±0.32; The number of CD8 possive cells was 2.7～6.8 in each high field and average was 4.82±1.31 in the EAP group; there is statistic significance between the two groups (t=10.35,p<0.05).
The number of IL17 possive cells in the control group was 0.3～5.1 in each high field and the average was 2.28±1.77 in the control group; The number of IL17 possive cells was 3.2～8.2 in each high field and the average number of IL17 positive cells was 5.67±1.76 in the EAP group; there is statistic significance between the two groups (t=4.70,p<0.05).
Part 3 The study of the expression of the Th17 cell related cytokines in the prostate tissue
Methods:
About 20mg tissue of the prostate was used to extract RNA. The method was according to the illustrate of the kit.
The concentration of the RNA was 118ng/μl, and the rate of purity was 1.95, it was fit for the RT-PCR.
One step real time RT-PCR was used to check the mRNA expression of Th17 lymphocytes related cytokines including IL17 and IL23 (p19/p40) in the protate tissue of EAP mice, and compared the difference of the cytokines between the EAP and control group.
2"-CT was used to express the content of mRNA of each cytokine.
Results:
The mRNA relative amount of IL17, IL23p19 and IL23p40 in the mice prostate tissue of control and EAP group were 0.0036±0.0029 vs 0.0530±0.0265 (t=-4.5,p<0.05), 0.0051±0.0025 vs 0.0177±0.0065 (t=-4.5,p<0.05),0.0041±0.0028 vs 0.0289±0.0283 (t=-2.1,p<0.05) respectively, and the difference between the two groups were statistically.
Conclussion:
The EAP model was made successfully by immuned with prostate extract.
1) The expression of Th17 cell related cytokines raised and the infiltration of CD4+、CD8+ and IL17 positive cells was increased, we can conclude that the Th17 cell was related with the ongoing of the EAP prostatitis.
2)The therapeutic method related with Th17 cells would benefit to the treatment of CPPS.
引文
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