17β-雌二醇联合去势建立大鼠慢性前列腺炎模型评价及神经内分泌分化初步研究

发布时间：2018-05-03 07:56

本文选题：慢性前列腺炎 + 慢性非细菌前列腺炎　；参考：《安徽医科大学》2014年博士论文

【摘要】：目的采用17p-雌二醇联合去势建立SD大鼠慢性非细菌性前列腺炎模型(Chronic Nonbacterial Prostatitis,CNBP),评价造模结果。方法取2月龄SPF级雄性Sprague Dawley(SD)大鼠80只,随机分为8组,即A组：空白对照组(blank control)(n=10);B组：生理盐水组(saline)(n=10);C组：假手术组(sham operation)(n=10);D组：单纯去势组(Castration)(n=10);E组：单纯17β-雌二醇(17β-Estradiol)(0.25mg/2ml/kg)(n=10);F组：17p-雌二醇(0.15mg/2ml/kg)(低剂量组)联合去势(n=10)、G组：17β-雌二醇(0.20mg/2ml/kg)(中剂量组)联合去势(n=10)；H组：17β-雌二醇(0.25mg/2ml/kg)(高剂量组)联合去势(n=10)。按照国际前列腺炎组织学诊断与分度标准评估造模结果,并分析不同浓度17β-雌二醇联合去势建模效果。结果大体形态观察可见H组大鼠前列腺与周围组织粘连较紧密,充血水肿,前列腺包膜不完整。光镜下观察见前列腺组织结构破坏,伴有不均匀淋巴样组织增生,前列腺导管破坏或者部分基底膜破坏,炎性细胞多成群状分布,可见淋巴小结或滤泡形成。D组、E组、F组及G组光镜下可见散在淋巴细胞及单核细胞浸润,表现为轻度或中度炎症。A组、B组、C组大鼠前列腺大体形态观察可见包膜完整腺体表面较光滑、平整。光镜下观察未见明显组织炎症表现。在评价炎症的核心指标方面,H组大鼠慢性前列腺炎组织学炎症分级与D组、E组、F组及G组比有统计学差异(P0.05),表现出中、重度的炎症反应。而D组、E组、F组及G组两两相比无统计学差异(P0.05)。炎症范围而言,H组大鼠慢性前列腺炎炎症分级同D组、E组、F组及G组比有统计学差异(P0.05),表现出多灶性或弥漫性的炎症反应,而其他组表现为局灶性或多灶性表现。结论17β-雌二醇联合去势可以诱导建立SD大鼠慢性非细菌性前列腺炎(CNBP)模型,建模成功后组织病理学表现与临床非细菌慢性前列腺炎病理表现相似,其组织病理炎症与17β-雌二醇浓度有一定的正相关性。单一采用17p-雌二醇或者去势可以诱导大鼠前列腺产生轻度的、局灶性炎症,临床代表性较差。从评价炎症的核心指标来说,单一采用17β-雌二醇或者去势手术诱导的SD大鼠的慢性非细菌性前列腺炎效果不够稳定,不适合作为动物模型。背景及目的前列腺炎,特别是慢性前列腺炎(CP)一直是泌尿外科和男科的主要问题。该病发病机制尚不明确,而且治愈率低、复发率高,可以影响男性性功能及生殖功能,给患者身心健康及家庭带来不利影响,因此了解其发病机制对于慢性前列腺炎的治疗十分必要。目前有关慢性前列腺病因学研究多集中于病原学、尿液返流、上皮功能异常、神经内分泌异常等方面。近年相关研究表明,慢性前列腺炎的发生与神经内分泌之间有一定的联系。前列腺神经内分泌细胞(Neuroendocrine cell, NEC)又称胺前体摄取及脱羧(Amine Precursor Uptake And Decarboxylation,APUD)细胞,研究发现前列腺神经内分泌细胞存在分泌上皮细胞之间,可以通过分泌激素多肽及5-羟色胺发挥作用,调节前列腺细胞生长及分泌活动。本文主要通过采用17β-雌二醇联合去势手术(双侧睾丸切除)建立SD大鼠非细菌性慢性前列腺炎(CNBP)模型,研究神经内分泌细胞重要标志性蛋白：嗜铬粒蛋白A(Chromogranin A, CgA)、神经醇化酶(Neuronal specific enolase, NSE)、神经生长因子(Nerve growth factor, NGF)及P物质(Substance P, SP)在慢性前列腺炎组织中的改变及其相关性,以了解神经分泌分化在慢性前列腺炎发病中的作用。方法利用建立的SD大鼠的慢性非细菌性前列腺炎动物模型,分别利用免疫组化组织化学(Immunohistochemical,IHC)、免疫印迹(Western blot, WB)、酶联免疫吸附法(Enzyme-linked immunosorbent assay, ELLS A)及实时定量PCR(Real-time quantitative PCR, qPCR)从蛋白及基因表达水平研究A组、B组、C组、D组、E组、H组不同组别大鼠神经内分泌细胞重要标志蛋白：嗜铬粒蛋白A(CgA)、神经醇化酶(NSE)、神经生长因子(NGF)及炎症相关P物质(SP)的变化,并分析嗜铬粒蛋白A(CgA)、神经醇化酶(NSE)、神经生长因子(NGF)分别与P物质(SP)的相关性。结果 1.免疫组织化学结果显示CgA、CgA、NSE、SP在模型组H组表达较A组、B组、C组、D组、E组增强。 2.免疫印迹半定量研究发现神经内分泌细胞代表性蛋白CgA、NSE、NGF在慢性前列腺炎模型组(H组)表达上调,与其A组、B组、C组、D组、E组比较有统计学差异(P0.05)。 3.酶联免疫吸附法定量检测大鼠血清模型组(H组)CgA、NSE、NGF、SP物质含量在慢性前列腺炎模型组(H组)表达上调,与其它各组两者相比有统计学意义(P0.05)。相关性分析得出Cg A与SP的表达成正相关性(γ,=0.35,P0.01)；NSE与SP的表达成正相关(γ=0.29, P0.01); NGF与SP的表达正相关(γ,=0.50,P0.01)。 4.实时定量PCR定量研究发现CgA (F=109.4)、NSE (F=68.4)、NGF (F=65.4). SP (F=61.1)在慢性非细菌性前列腺炎模型组中表达上调,与其他各组相比有统计学意义(P0.05)。相关性分析得出CgA与SP的表达成正相关性(γ=0.45, P0.01); NSE与SP的表达成正相关(γ=0.53,P0.01)；NGF与SP的表达正相关(γ=0.37,P0.01) 结论 1.17β-雌二醇皮下注射联合去势手术建立慢性非细菌性前列腺炎模型组中神经内分泌细胞标志性蛋白CgA、NSE、NGF表达上调,提示慢性前列腺炎中存在神经内分泌分化现象,推测由17p-雌二醇和(或)去势诱导产生。 2. CgA、NSE、NGF分别与SP存在相关性,提示神经内分泌分化可能与炎症相关,SP是重要的致痛因子,推测神经内分泌细胞可能参与引起慢性非细菌性前列腺炎的神经原性疼痛。
[Abstract]:Objective to establish 17p- SD (Chronic) Nonbacterial Prostatitis (CNBP) model by using estradiol combined with castration, and to evaluate the results of modeling.
Methods 80 rats of 2 month old SPF grade Sprague Dawley (SD) were randomly divided into 8 groups, namely group A, blank control (n=10), B group: normal saline group (saline), C group: sham operation group; simple 17 beta estradiol (17 beta) ) (n=10); group F: 17p- estradiol (0.15mg/2ml/kg) (low dose group) combined castration (n=10), group G: 17 beta estradiol (0.20mg/2ml/kg) (medium dose group) combined castration (n=10); H group: 17 beta estradiol (0.25mg/2ml/kg) (0.25mg/2ml/kg) (high dose group) combined castration (n =10). Evaluation of model results according to the histological diagnosis and graduation criteria of international prostatitis and analysis Different concentrations of 17 beta estradiol combined with castration were used to model the effect.
Results the general morphological observation showed that the prostate and surrounding tissue were closely adhered to the prostate in the H group, and the hyperemia and edema were not complete. The damage of the prostate tissue structure was observed under the light microscope, accompanied by the uneven lymphoid tissue hyperplasia, the destruction of the prostatic catheter or the broken part of the basement membrane, and the distribution of inflammatory cells in groups, and the lymph nodes were visible. Or follicular formation in group.D, group E, group F and group G showed infiltration of lymphocytes and mononuclear cells under light microscope, which showed mild or moderate inflammation in group.A, B group, group B and group C, the whole glandular surface of the prostate gland was smooth and smooth. No obvious tissue inflammation was observed under the light microscope. The core index of inflammation was evaluated. The histologic grade of chronic prostatitis in group H was significantly different from group D, group E, group F and G group (P0.05), showing a moderate and severe inflammatory response. There was no statistical difference between group D, E, F and G group 22 (P0.05). Differences (P0.05) showed multifocal or diffuse inflammatory response, while the others showed focal or multifocal manifestations.
Conclusion the combined castration of 17 beta estradiol can induce the establishment of a chronic non bacterial prostatitis (CNBP) model in SD rats. The histopathological performance of the model is similar to that of the clinical non bacterial chronic prostatitis after successful modeling. The histopathological inflammation is positively correlated with the concentration of 17 beta estradiol. A single 17p- estradiol or castration is used alone. It can induce mild, focal inflammation and poor clinical representation in the prostate of rats. From the core indicators of inflammation, the effect of a single 17 beta estradiol or castrated SD rat chronic non bacterial prostatitis is not stable and is not suitable as an animal model.
Background and objective prostatitis, especially chronic prostatitis (CP), has always been the main problem in the Department of Urology and the male family. The pathogenesis of the disease is not clear, the cure rate is low, the recurrence rate is high, the male sexual function and reproductive function can be affected, and the patients' physical and mental health and family are adversely affected. Therefore, the pathogenesis of the disease is understood to be chronic. The treatment of prostatitis is very necessary. At present, the study of chronic prostatic etiology focuses on etiology, urine reflux, abnormal epithelial function, and neuroendocrine abnormalities. In recent years, related studies have shown that there is a certain relationship between the occurrence of chronic prostatitis and neuroendocrine cells. Neuroendocri NE cell, NEC), also known as amine precursor uptake and decarboxylation (Amine Precursor Uptake And Decarboxylation, APUD) cells, found that the prostate neuroendocrine cells exist between secretory epithelial cells, and can play a role by secreting hormone peptides and 5- serotonin to regulate the growth and secretion of anterior gland cells. This article mainly adopts 17 The non bacterial chronic prostatitis (CNBP) model of SD rats was established with beta estradiol combined with castration (bilateral testicle excision) to study the important markers of neuroendocrine cells: chromogranin A (Chromogranin A, CgA), neuroglycolase (Neuronal specific enolase, NSE), nerve growth factor (Nerve growth) and substances ( Substance P (SP) changes in chronic prostatitis tissues and their correlation, in order to understand the role of neurosecretory differentiation in the pathogenesis of chronic prostatitis.
Methods using the established model of chronic non bacterial prostatitis in SD rats, Immunohistochemical, IHC, Western blot, WB, enzyme linked immunosorbent assay (Enzyme-linked immunosorbent assay, ELLS A) and real-time quantitative PCR were used. The expression level of A group, group B, group C, group D, group E, group H, and group H were important markers of neuroendocrine cells: chromaffin A (CgA), neuroalkinase (NSE), nerve growth factor (NGF) and P substance in inflammation (SP), and the analysis of chromaffin protein, neurotrophic factor and nerve growth factor respectively The correlation of substance (SP).
Result
1. immunohistochemical results showed that CgA, CgA, NSE and SP in the model group H group were higher than those in A group, B group, C group, D group and E group.
2. the semi quantitative study of immunoblotting found that the expression of representative protein CgA, NSE, NGF in the chronic prostatitis model group (group H) was up regulated, and there was a statistically significant difference between the group A, the B group, the C group, the D group and the E group (P0.05).
The expression of CgA, NSE, NGF, SP in the model group (group H) of chronic prostatitis (group H) was up regulated by 3. enzyme linked immunosorbent assay (group H), compared with other groups (P0.05). The correlation analysis showed that the expression of Cg A and SP was positive correlation (gamma, =0.35, P0.01), and positive correlation (gamma, =0.35, P0.01). 9, P0.01); NGF is positively correlated with the expression of SP (gamma, =0.50, P0.01).
4. quantitative study of real-time quantitative PCR found that CgA (F=109.4), NSE (F=68.4), NGF (F=65.4). SP (F=61.1) was up regulated in the chronic non bacterial prostatitis model group (P0.05). The correlation analysis showed that CgA was positively correlated with the SP table. 3, P0.01); NGF is positively correlated with the expression of SP (gamma =0.37, P0.01).
conclusion
1.17 beta estradiol subcutaneous injection combined with castration to establish a chronic nonbacterial prostatitis model group, the expression of neuroendocrine cell marker protein CgA, NSE, NGF expression was up-regulated, suggesting the existence of neuroendocrine differentiation in chronic prostatitis, which is presumed to be induced by 17p- estradiol and (or) castration.
2. CgA, NSE, and NGF are associated with SP, suggesting that neuroendocrine differentiation may be associated with inflammation. SP is an important pain causing factor. It is suggested that neuroendocrine cells may be involved in neurogenic pain causing chronic abacterial prostatitis.

【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R697.33;R-332

【参考文献】