炎症对前列腺增生的影响及前列化浊方的干预效应研究

发布时间：2018-05-03 00:22

  本文选题：前列化浊方 + 前列腺炎　； 参考：《中国中医科学院》2014年硕士论文

【摘要】：慢性前列腺炎(chronic prostatitis,CP)是中青年男性的常见病和多发病,约占泌尿外科门诊患者的33%。该病缠绵难愈、反复发作,给患者造成很大的生理痛苦及心理负担。美国国立卫生研究院已将前列腺炎、心肌梗死、不稳定性心绞痛和活动性Crohn病等一起列为影响居民生活质量最为严重的慢性疾病。然而,目前其发病机制尚未完全阐明,大量研究表明多种细胞因子构成的网络和级联反应可能是导致CP的主要原因。细胞因子信号转导抑制蛋白(suppressor of cytokine signal,SOCS)能够调节细胞对细胞因子的反应。但是,SOCS在本病发生发展过程中发挥的作用至今尚未得到论证。 导师既往临床研究证实,前列化浊方(黄柏、茵陈、赤芍、红花、三七、柴胡、香附)在改善前列腺炎患者症状,提高患者生活质量方面具有满意的疗效和显著的优势。很多学者研究认为前列腺炎是一种自身免疫性疾病。基于此,本研究以实验性自身免疫性前列腺炎(experimental autoimmune prostatitis,EAP)大鼠模型为载体,通过前列化浊方的干预,采用RT-PCR和Western-blot方法检测大鼠前列腺组织相关细胞因子及SOCS的表达,揭示细胞因子与SOCS在CP发生发展中的共同作用,阐明前列化浊方治疗CP的作用机理,为临床应用前列化浊方提供实验依据。 前列腺炎和良性前列腺增生症(benign prostatic hyperplasia,BPH)常同时存在,相互影响。尽管有临床研究报道慢性炎症在BPH发病中发挥重要作用,与未患有前列腺炎男性相比,患有CP的患者更易发展为BPH。但是,目前CP和BPH的相关性研究大多以BPH患者为研究对象,研究者将BPH患者前列腺切除后获取的标本行病理学检查,以分析炎症和BPH的相互关系。有学者认为这类研究属回顾性研究,未能明确CP和BPH的发病顺序,也未能排除年龄在BPH发病中的影响。所以炎症和BPH之间的关系尚不明确,炎症在BPH进展中的作用机制尚缺乏强有力的证据。 Biochim Biophys Acta杂志2013年1月刊出Kim HJ等采用脂多糖注射大鼠前列腺方法建立前列腺炎大鼠模型,研究CP导致BPH的机制。鉴于此,本研究在国内率先以EAP大鼠模型为载体,采用前瞻性研究方法初步探索炎症在BPH进展中的作用机制,既可明确炎症与BPH的发病顺序,也能够排除年龄在BPH进展中的影响,有利于揭示炎症在BPH进展中的作用机制及阐明前列化浊方干预BPH进展的机理。 国内外均有学者认为有效防治前列腺炎,可以降低BPH的发生和减缓BPH的进展,慢性非细菌性前列腺炎为防治BPH提供了新靶点,为了延缓或阻断BPH发生发展,未来的研究应主要集中于细胞因子,它可能成为潜在的治疗靶点。故本研究以细胞因子、SOCS、Bcl-2和病理形态学等多角度多层次开展深入研究,以期揭示炎症在BPH进展中的作用机制,干预BPH的发生发展。 本研究为导师承担的国家自然基金面上项目(81072814),研究分为两部分：文献综述和动物实验研究。在导师指导下,我参与了课题前期准备、建立动物模型、灌胃给药、实验取材、统计分析等工作。 1.文献综述：本部分分别从NIH-Ⅲ型前列腺炎与相关细胞因子的研究进展,炎症在BPH进展中作用机制的研究进展两个方面进行综述。 2.动物实验研究：炎症对前列腺增生的影响及前列化浊方的干预效应研究 目的：观察前列化浊方对EAP大鼠的干预效应。 方法：取240-300gSD雄性大鼠66只,随机分为正常组12只,实验组54只。采用注射自身抗原的方法建立实验性自身免疫性前列腺炎(experimental autoimmune prostatitis,EAP)大鼠模型,实验组分别于0、7、14、21天腹腔注射百白破疫苗0.5ml,并同时皮内多点注射大鼠前列腺蛋白提纯液(浓度15g/L)和福式完全佐剂的混悬液(比例1：1)1.0ml。正常组分别行腹腔及皮内多点注射0.9%生理盐水注射液0.5、1.0ml。在第35天,实验组处死6只大鼠,将前列腺组织送西苑医院病理室检验,病理结果证实成功建立了前列腺炎大鼠模型。剩余48只大鼠随机分为模型组、前列化浊方低剂量组(以下简称低剂量组)、前列化浊方中剂量组(以下简称中剂量组)、前列化浊方高剂量组(以下简称高剂量组),每组12只大鼠。除模型组和正常组给予蒸馏水外,低剂量组、中剂量组、高剂量组分别给予低剂量前列化浊方(按5.25g/kg体重给药)、中剂量前列化浊方(按10.5g/kg体重给药)和高剂量前列化浊方(按21.0g/kg体重给药),每天灌胃给药一次,连续干预30天后,腹腔注射20%乌拉坦(用量按体重计算,0.4ml/100g)麻醉大鼠。局部皮肤消毒,在无菌条件下,下腹部正中切口,根据膀胱及精囊的位置找到前列腺,取出前列腺组织,剪除多余脂肪组织,用冷生理盐水洗净,摘取左侧叶前列腺分别置固定液中,用于常规HE染色,光镜下观察各组大鼠前列腺腺腔及间质的炎性细胞浸润和增生情况。迅速取右侧前列腺组织置液氮中,用于RT-PCR和Western-blot检测。RT-PCR法测定大鼠前列腺组织Interleukin-1(IL-1)、Interleukin-6(IL-6)、Interleukin-10(IL-10) Interleukin-17(IL-17)、Tumor necrosis factor-a(TNF-a)和B cell leukemia-2(Bcl-2)的mRNA表达水平,Western-blot法检测大鼠前列腺组织SOCS-1和SOCS-3表达水平。 结果： (1)组织病理学观察显示：模型组腺体排列紊乱,紧密,大量腺体呈乳头状增生,腺体上皮由立方状变为高柱状,腺腔内可见中性粒细胞；间质增生,血管扩张充血,组织水肿,大量中性粒细胞及少量淋巴细胞浸润。低剂量组、中剂量组和高剂量组炎症和腺体增生均呈不同程度减轻。组织病理学结果证实：成功建立了EAP大鼠模型,前列化浊方具有治疗前列腺炎和干预BPH进展的作用。 (2)炎症因子检测显示：与正常组比较,模型组大鼠前列腺组织IL-10表达下降(P0.01),IL-1、IL-6、IL-17和]NF-α表达均增高(P0.01)。表明EAP大鼠前列腺组织存在强烈的炎症反应。 与模型组比较,治疗组(低剂量组、中剂量组和高剂量组)大鼠前列腺组织IL-6、IL-17和TNF-α的表达均有下降(P0.01)；其中,中剂量组下降尤为显著(P0.01)。 与模型组比较,IL-1在中剂量组大鼠前列腺组织表达下降明显(P0.01),但高剂量组和低剂量组前列腺组织中IL-1水平与模型组比较无显著性差异(P0.05)。 与模型组比较,治疗组大鼠前列腺组织IL-10表达均上升(P0.01),中剂量组和低剂量组上升明显。提示前列化浊方对炎症因子的影响可能与剂量有关。 (3)凋亡抑制因子Bcl-2检测显示：与正常组比较,模型组大鼠前列腺组织Bcl-2的表达明显升高(P0.01)；与模型组比较,中剂量组大鼠前列腺组织Bcl-2的表达明显降低(P0.01)。 (4)SOCS-1和SOCS-3检测显示：与正常组比较,模型组大鼠前列腺组织SOCS-1和SOCS-3表达显著升高(P0.01)；与模型组比较,治疗组大鼠前列腺组织SOCS-1和SOCS-3表达均下降(P0.01),中剂量组下降最显著。 结论： 模型组大鼠病理形态学检测结果及前列腺组织炎症因子的表达水平均证实成功建立了EAP大鼠模型；治疗组大鼠前列腺组炎症和增生缓解,从形态学证实了前列化浊方治疗CP和干预BPH进展的疗效。 模型组大鼠前列腺组织前炎症因子表达显著高于正常组,治疗组大鼠前列腺组织前炎症因子表达显著低于模型组。提示前列化浊方可能通过抑制前炎症细胞因子表达发挥治疗作用。 模型组大鼠前列腺组织IL-10表达显著低于正常组,治疗组大鼠前列腺组织IL-10表达显著高于模型组。提示前列化浊方治疗CP的作用可能通过升高抗炎症因子表达和调节免疫功能实现的。 模型组大鼠前列腺组织前炎症因子、SOCS-1和SOCS-3表达显著高于正常组,证实炎症因子与SOCS可能共同影响着CP的发生发展,JAK/STAT信号通路在CP的发生发展中可能扮演了重要的角色,有待深入研究。 模型组大鼠前列腺组织Bcl-2表达显著高于正常组,中剂量组大鼠前列腺组织Bcl-2表达显著低于模型组。病理形态学检测显示模型组大鼠前列腺组织大量腺体呈乳头状增生,腺体上皮由立方状变为高柱状；治疗组大鼠前列腺极少量腺体乳头状增生,间质增生不明显。提示炎症在BPH进展中可能扮演重要角色,前列化浊方可能通过减轻前列腺组织炎症反应和下调Bcl-2表达干预BPH进展。
[Abstract]:Chronic prostatitis (chronic prostatitis, CP) is a common and frequently occurring disease of young and middle-aged men. It accounts for the 33%. of the outpatients in the Department of Urology, which is difficult to recover and relapse, causing great physical pain and psychological burden to the patients. The National Institutes of health of the United States has put forward the prostatitis, myocardial infarction, unstable angina and active C. Rohn disease, etc., is listed as the most serious chronic disease affecting the quality of life of the residents. However, its pathogenesis has not yet been fully elucidated. A large number of studies have shown that the network and cascade reaction of multiple cytokines may be the main causes of CP. The cytokine signal transduction inhibitor (suppressor of cytokine signal, SOCS) can be the main cause of the disease. Regulation of cell response to cytokines. However, the role of SOCS in the pathogenesis of this disease has not yet been demonstrated.
Previous clinical studies have confirmed that prostoriate turbid recipe (cypress, Artemisia, red peony, red flower, 37, bupleurum, and fragrant) has a satisfactory effect and significant advantage in improving the symptoms of prostatitis and improving the quality of life of the patients. Many scholars have studied that prostatic inflammation is a autoimmune disease. Based on this, this study is experimental The rat model of experimental autoimmune prostatitis (EAP) was used as a carrier. Through the intervention of Prostant turbid recipe, the expression of cytokines and SOCS in the prostate tissue of rats was detected by RT-PCR and Western-blot methods. The common effect of cytokines and SOCS in the development of CP was revealed, and the prostad turbid recipe was clarified. The therapeutic mechanism of CP is to provide experimental evidence for clinical application of prostaglandin turbid prescription.
Prostatitis and benign prostatic hyperplasia (benign prostatic hyperplasia, BPH) often exist and interact with each other. Although there are clinical reports that chronic inflammation plays an important role in the pathogenesis of BPH, patients with CP are more likely to develop into BPH. than those with no prostatitis, but the current correlation of CP and BPH is mostly BPH. The patients were studied. The researchers examined the pathological examination of specimens obtained after the prostatectomy in BPH patients to analyze the relationship between inflammation and BPH. Some scholars believe that this kind of study is a retrospective study that does not define the sequence of CP and BPH, nor does it exclude the influence of age in the pathogenesis of BPH. So there is no relationship between inflammation and BPH. It is clear that there is no strong evidence for the role of inflammation in the progression of BPH.
Biochim Biophys Acta magazine published Kim HJ and Kim HJ in January 2013 to establish prostatitis model in rats by injecting lipopolysaccharide into the prostatitis rat model and study the mechanism of BPH caused by CP. In view of this, this study took the lead in the domestic EAP rat model as the carrier, and explored the mechanism of inflammation in the progression of BPH by prospective study methods. It is true that inflammation and the sequence of BPH can also exclude the influence of age on the progress of BPH, and to reveal the mechanism of inflammation in the progress of BPH and to clarify the mechanism of the intervention of prostaglandin turbid prescription in the progress of BPH.
Both domestic and foreign scholars believe that effective prevention and control of prostatitis can reduce the occurrence of BPH and slow down the progress of BPH. Chronic non bacterial prostatitis provides a new target for the prevention and control of BPH. In order to delay or block the development of BPH, the future research should focus mainly on the cytokine, and it may be a potential target for treatment. In order to reveal the mechanism of inflammation in the progress of BPH and to interfere with the development of BPH, many angles and multiple levels, such as cytokine, SOCS, Bcl-2 and Pathomorphology, are carried out in depth.
This study is the national natural fund project (81072814) undertaken by the tutor. The study is divided into two parts: literature review and animal experiment. Under the guidance of the tutor, I participated in the preparation of the project, the animal model, the gavage, the experimental materials, the statistical analysis and so on.
1. literature review: this part is a review of the progress of NIH- III prostatitis and related cytokines, and the progress of the research on the mechanism of inflammation in the progress of BPH.
2. animal experimental study: the effect of inflammation on benign prostatic hyperplasia and the intervention effect of prostaglandin turbid recipe
Objective: To observe the intervention effect of Qian lie Hua Zhuo Fang on EAP rats.
Methods: 66 male 240-300gSD rats were randomly divided into 12 rats in the normal group and 54 in the experimental group. The experimental autoimmune prostatitis (experimental autoimmune prostatitis, EAP) rat model was established by injection of self antigen. The experimental group was injected with a pertussis vaccine 0.5ml in the abdominal cavity on 0,7,14,21 days, and the Pinedo point was given at the same time. The rat prostatic protein purification fluid (concentration 15g/L) and the mixed suspension of full adjuvant (1:1) in the normal group were injected into the abdominal cavity and the intraperitoneal injection of 0.9% saline injection of 0.9% physiological saline injection 0.5,1.0ml. for thirty-fifth days. The experimental group died of 6 rats. The prostate tissue was sent to the Xiyuan Hospital pathology laboratory, and the pathological results confirmed the successful establishment of the test. The remaining 48 rats were randomly divided into model group, low dose group (hereinafter referred to as low dose group), low dose group of prostad turbid prescription (hereinafter referred to as low dose group), middle dose group (hereinafter referred to as middle dose group), high dose group (hereinafter referred to as high dose group), each group of high dose group (hereinafter referred to as high dose group), each group was given the low dose group except the model group and the normal group. In the middle dose group, the high dose group was given a low dose of prostapido turbid (5.25g/kg weight), the middle dose of prostapido turbid prescription (according to 10.5g/kg weight) and the high dose of prostachiated turbid prescription (according to 21.0g/kg weight) was administered every day, and after 30 days of continuous intervention, the intraperitoneal injection of 20% urantan (the dosage according to weight, 0.4ml/100g) anesthesia Intoxicated rats, local skin disinfection, under the aseptic condition, the lower abdomen is median incision, according to the location of the bladder and seminal vesicle to find the prostate, remove the prostate tissue, remove the excess fat tissue, clean the prostate with cold physiological saline, and take the left lobe prostate respectively in the fixed solution, which is used for routine HE staining, and the prostate gland cavity of each group is observed under light microscope. RT-PCR and Western-blot were used to detect Interleukin-1 (IL-1) in the prostate tissue of rats, Interleukin-6 (IL-6), Interleukin-10 (IL-10) Interleukin-17 (IL-17). The expression level of mRNA and Western-blot were used to detect the expression level of SOCS-1 and SOCS-3 in rat prostate tissue.
Result锛，

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