【作者】 王强；

【导师】 宋波；

【作者基本信息】 第三军医大学 ， 外科学， 2009， 博士

【摘要】 背景慢性前列腺炎(chronic prostatitis,CP)是泌尿外科最常见疾病之一,该病病因、病理改变、临床症状复杂多样,其中疼痛是慢性前列腺炎患者最主要的症状。尽管前列腺炎一般不会对生命造成威胁,但可严重影响患者生活质量,尤其是对患者精神健康的影响比严重的糖尿病和慢性心力衰竭更为明显。由于慢性前列腺炎病因复杂,治疗困难,虽已有大量的基础和临床研究,但至今尚无突破性进展。因此,从疼痛机理研究入手,有望开创新的慢性前列腺炎治疗方法。由于前列腺疼痛呈现内脏牵涉痛特点,目前认为前列腺疼痛的持续与泛化与支配前列腺的L5~S2脊髓段的继发性病变有关。在慢性前列腺炎L5~S2脊髓段的继发性病变研究中,发现除神经元外,神经胶质细胞具有较大作用。神经胶质细胞是神经系统中除神经元以外的第二大类细胞,广泛分布于脑和脊髓。近年来发现,痛觉的传导和调制不仅仅是神经元的功能,活化的星形胶质细胞也通过和神经细胞的相互作用及炎性因子的分泌而参与病理性疼痛的发生。星形胶质细胞功能受许多神经体液因子的调节,目前引起广泛重视的有p物质(substance P, SP)、谷氨酸和各种炎性因子等,周围慢性疼痛可通过这些递质和生物因子的作用影响脊髓星形胶质细胞功能,从而参与慢性疼痛在脊髓初级中枢的传导和调制。SP不仅作为神经递质向中枢传递来自外界的物理、化学和温度等伤害性刺激,具有导致疼痛的作用,同时还可在接受刺激信号后由神经末梢在局部释放出来,参与损伤局部的免疫、炎症反应及损伤的修复。L5~S2脊髓段神经胶质细胞参与前列腺疼痛调节的可能机制研究中,信号转导通路的研究尤其重要。p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase, p38MAPK)属于哺乳动物应激与炎症激活的丝裂原活化蛋白激酶家族重要成员。p38MAPK信号通路参与了细胞的生长发育及细胞间功能同步等多种生理过程,并与炎症、应激反应的调控密切相关,被认为是细胞信息传递的交汇点和共同通路。脊髓cAMP反应元件结合蛋白(cAMP responseelement binding protein,CREB)转录因子属于碱性氨基酸亮氨酸拉链(bZIP)转录因子家族,参与多种生物分子基因表达的调控。核转录因子kB (Nuclear factor-kappa b,NF-kB)是能调节多种炎症和免疫基因表达的一种重要的转录调节因子。有证据表明,这些因子在疼痛机制中发挥一定的作用,研究这些因子的生物学特性及其在慢性前列腺疼痛脊髓胶质细胞活化中的调节机理,有助于进一步了解慢性前列腺疼痛的发病机制,及促进抗慢性前列腺疼痛药物的有效开发。目的1、观察L5-S2脊髓段背根神经节(dorsal root ganglion,DRG)和体外培养星形胶质细胞活化中p38MAPK的变化,观察炎性因子水平的变化及与p38MAPK的关系;2、探讨CREB和NF-KB在p38MAPK所致脊髓星形胶质细胞活化中的作用,明确脊髓星形胶质细胞活化中p38MAPK细胞信号转导途径及在大鼠慢性前列腺炎疼痛中的作用。方法第一部分:p38MAPK在P物质刺激脊髓星形胶质细胞体外活化中的作用分离培养SPF大鼠脊髓星形胶质细胞,设正常对照组(正常组)、SP刺激组(SP组,10-7mol/L)、SB203580阻断p38MAPK组(SB组,10μmol/L)和SP刺激+SB203580阻断p38MAPK组(SP+SB组)。WB法、RT-PCR法、ELISA法检测1h,3h,12h,24h和36h时p38MAPK、p-p38(磷酸化p38MAPK)含量及GFAP mRNA、TNF-α、NO水平和NOS活性的变化。第二部分:p38MAPK在慢性前列腺炎大鼠脊髓背根神经节内星形胶质细胞活化中的作用制作大鼠慢性前列腺炎疼痛模型,观察L5-S2脊髓段背根神经节中p38MAPK和磷酸化p38MAPK含量变化,测定培养上清液TNF-α、NO、NOS含量,同时应用p38MAPK抑制剂作用于动物模型(骶管内注射模型),观察抑制剂SB203580对p38MAPK活化与痛性物质表达的影响及关系。第三部分:CREB和NF-KB在p38MAPK所致脊髓星形胶质细胞活化中的作用观察体外胶质细胞在SP刺激后p-P38、p-CREB、NF-KBp65的变化及关系,观察抑制剂SB203580(p38MAPK inhibitor)、PD98059(CREB inhibitor)和SN50(NF-KB inhibitor)对各信号转导通路蛋白活化与痛性物质表达的影响及关系。分离培养SPF大鼠脊髓星形胶质细胞,设正常组、SP刺激组(SP组,10-7mol/L)、SP刺激+SB203580(10μmol/L)阻断p38MAPK组(SP+SB组)、SP刺激+ PD98059(10μmol/L)阻断CREB组(SP+PD组)、SP刺激+ SN50(10μmol/L)阻断NF-KB (SP+SN组)。WB法、免疫荧光法、ELISA法检测12h和24h时p-p38、p-CREB、NF-KBp65水平及GFAP、TNF-α、IL-1β水平变化。结果与结论第一部分:脊髓星形胶质细胞GFAP阳性表达率大于95%。SP组脊髓星形胶质细胞总p38MAPK水平无显著变化,1h后p-p38开始升高,3h后GFAP mRNA水平显著增高,同时NO、NOS和TNF-α水平显著增高。用SB203580阻断p38MAPK通路后,SP+SB组较SP组p-p38、GFAP mRNA、NO、NOS、TNF-a水平显著降低。SB组总p38MAPK、p-p38、GFAP mRNA、NO、NOS、TNF-α水平无显著变化。提示p38MAPK信号通路参与了体外培养脊髓星形胶质细胞P物质刺激活化过程,阻断p38MAPK信号通路可有效降低脊髓星形胶质细胞炎性因子水平。第二部分:前列腺炎组大鼠L5~S2脊髓背角中p-p38、GFAP、TNF-α、NO水平和iNOS活性在5d和10d均明显高于正常对照组并随时间增高。镇痛组大鼠L5~S2脊髓背角中p-p38、GFAP、TNF-α、NO水平和iNOS活性在5d和10d均低于前列腺炎组。提示脊髓p38MAPK信号通路参与了大鼠前列腺炎发生后脊髓痛觉传导及SB203580镇痛机制,阻断p38MAPK信号通路可有效降低脊髓炎性因子水平。第三部分:SP组脊髓星形胶质细胞p-p38、p-CREB、NF-KBp65显著升高,GFAP水平显著增高,同时TNF-α和IL-1β水平显著增高。与SP组比较,用SB203580阻断p38MAPK通路后,SP+SB组p-p38、p-CREB、NF-kBp65显著降低,GFAP、TNF-α和IL-1β水平显著降低。用PD98059阻断CREB通路后,SP+PD组p-p38、NF-kBp65无显著变化,p-CREB显著降低,GFAP水平降低,同时TNF-α和IL-1β水平降低。用SN50阻断NF-kB通路后,SP+SN组p-p38、p-CREB无显著变化,NF-kBp65显著降低,GFAP水平降低,同时TNF-α和IL-1β水平降低。提示体外培养中,SP刺激后脊髓星形胶质细胞显著活化,p38MAPK活化后通过CREB及NF-kB信号途径导致胶质细胞炎性因子水平显著升高。更多还原

【Abstract】 BackgroundChronic prostatitis (chronic prostatitis, CP) is the most common urological diseases. The causes, pathological changes and clinical symptoms of this disease were complex and diverse. Pain is the most important symptoms in patients with chronic prostatitis. Although prostatitis is generally not life-threatening, but it can seriously affect the life quality of patients. The impact of prostatitis on mental health of patients is more serious than diabetes and chronic heart failure. Due to the etiology complexity and treatment difficulties of chronic prostatitis, although a large number of basic and clinical research have been done, but so far no breakthrough has been gained. Therefore, study of the mechanism of pain is expected to open up new treatment for chronic prostatitis. Prostate pain has the characteristics of visceral referred pain, for this reason, At present, it is recognized that the continuation and generalization of prostate pain is correlated with the secondary lesions of the L5 ~ S2 spinal cord innervating prostate.It was found that the glial cells have a role in the secondary lesions of the L5 ~ S2 spinal cord in the chronic Prostatitis as well as neurons, Glial cells, widely distributed in the brain and spinal cord, were the second largest category of cells of the nervous system in addition to neurons. In recent years, it was found that transmission and modulation of pain is not only the function of neurons, but also of activated astrocytes realized by the interaction with nerve cells and the secretion of inflammatory factors.Astrocyte function is regulated by many neurohumoral factors. At present, substance P, glutamic acid and a variety of inflammatory factors has caused widespread attention. Chronic pain can affect the function of spinal cord astrocytes through the role of these transmitters and biological factors and participate in the conduction and modulation of primary spinal cord center of chronic pain. SP, as a neurotransmitter, not only transmit physical, chemical noxious stimulation from the outside to the center leading to pain, but also can be released from nerve endings to participate in immunity, inflammation and repair of local injury after receiving the stimulating signal. In the possible mechanisms study of glial cells of L5~S2 spinal cord involved in the regulation of prostate pain, signal transduction pathway study is particularly important. p38 mitogen-activated protein kinase (p38MAPK) is an important member of the family of mitogen-activated protein kinases involved in mammalian stress and inflammatory activation.p38MAPK signaling pathway, considered as the intersection point and common pathway of the cell information transmission, involved in a variety of physiological processes such as cell growth and cellular function closely related to the regulation and control of inflammation and stress response. cAMP response element binding protein (CREB), involved in the regulation of gene expression of a variety of biological molecules, belongs to basic amino acid leucine zipper (bZIP) transcription factor family. Nuclear factor-kappa b ( NF-kB), regulating variety of inflammatory and immune gene expression, is an important factor in transcriptional regulation. There is evidence that these factors play a role in the pain mechanisms. Research on the biological characteristics of these factors and the regulation mechanism of glial cell activation of spinal cord in the chronic prostate pain contributes to a better understanding of the pathogenesis of chronic prostate pain and the promotion of effective development of anti-chronic prostate pain drug.Aim1. To observe the changes of p38MAPK of dorsal root ganglia (DRG) in L5-S2 of spinal cord and during the activation of cultured astrocytes in vitro. To observed the relationship between the changes of inflammatory factors and p38MAPK.2、To investigate the role of CREB and NF-KB in the p38MAPK-induced activation of spinal cord astrocytes, p38MAPK signal transduction pathway in the activation of spinal cord astrocytes cell and the role in the chronic prostate pain of rats.MethodsThe first part: The role of p38MAPK in the P material stimulation induced activation of spinal cord astrocytes in vitroAastrocytes, cultured from spinal cord of SPF rat and identified by immunofluorescence method with GFAP antibody, were grouped into the control group, the SP stimulus group(SP group) and the SB203580 interrupt p38MAPK group ( interrupt group)in which SP (10-7mol/L) and SB203580 (10μmol/L) were added to the supernatant for 12h. The WB method, RT-PCR method and ELISA method were used to determine the changes of p38MAPK, phosphorylated p38MAPK, GFAP mRNA, TNF-α, NO and NOS of astrocytes or supernatant at 1h,3h,12h,24h and 36h respectively.The second part: The role of p38MAPK in the activation of astrocytes of dorsal root ganglion cells in the spinal cord of rats with chronic prostatitisForty-five male SD rats were divided into the normal group (5 rats), the prostatitis group (10 rats ) and the chronic prostate pain treated with SB203580 group (the SB203580 group, 30 rats). In the SB203580 group, complete Freund’s adjuvant prostate was injected and intrathecal administration of SB203580 every 5 rats with 0.5μg/10μl、2.5μg/10μl and 12.5μg/10μl were applied respectively. After 5 and 10 days, Western blot and ELISA method were used to determine the changes of p-p38 (phosphorylated p38MAPK) , GFAP, TNF-α, NO and iNOS in the posterior horn of the L5~S2 spinal cord.The third part: The role of CREB and NF-KB in the p38MAPK-induced activation of spinal cord astrocytesAastrocytes, cultured from spinal cord of SPF rat, were grouped into the normal group, the SP stimulus group(SP group), the SP stimulus + SB203580 interrupt group(SP+SB group), the SP stimulus + PD98059 interrupt group(SP+PD group) and the SP stimulus + SN50 interrupt group (SP+SN group)in which SP (10-7mol/L), SB203580 (10μmol/L), PD98059 (10μmol/L) and SN50(10μmol/L) were added to the supernatant for 12h. The WB method, immunofluorescence method and ELISA method were used to determine the changes of p-p38, p-CREB, NF-KBp65, GFAP, TNF-α, IL-1βof astrocytes or supernatant at 12h and 24h.Main results and conclusoionsThe first part: The GFAP positive rate of the cultured cells was higher than 95%. p38MAPK level in the SP group did not have any change, whereas p-p38 level increased significantly after 1h and GFAP mRNA, NO, NOS and TNF-a level increased after 3h. When p38MAPK pathway was inhibited by SB203580 in the SP+SB group, GFAP mRNA, NO, NOS and TNF-a was significantly reduced compared with those in the SP group. The level of p38MAPK, p-p38, GFAP mRNA, NO, NOS and TNF-a in the SB group did not have changes compared with those in the normal group. These results indicate that p38MAPK signal pathway contributes to P substance induced activaton of spinal cord astrocytes according to inflammatory factors attenuation after p38MAPK signal pathway interruption by SB203580.The second part: p-p38, GFAP, TNF-α, NO and iNOS of the prostatitis group increased significantly at the 5th days and 10th day. while these index were significantly reduced compared with those in the SB203580 group. The chronic prostatitis can result in the activation of p38MAPK and more expression of GFAP, TNF-αand iNOS in L5~S2 spinal cord, suggesting the secondary inflammation of L5~S2 spinal cord which is probably correlated with the maintenance and enlargement of prostate pain.The third part: p-p38, p-CREB, NF-KBp65 level in the SP group increased significantly. In the same time, GFAP, TNF-a and IL-1βlevel increased significantly too. When p38MAPK pathway was inhibited by SB203580 in the SP+SB group, p-p38, p-CREB, NF-KBp65 level and GFAP, TNF-a and IL-1βwas significantly reduced compared with those in the SP group. In the SP+PD group, p-CREB level was significantly reduced compared with those in the SP group. In the SP+SN group, NF-KBp65 level was significantly reduced compared with those in the SP group. Astrocytes from spinal cord were significant activated after stimulated by SP in vitro , Inflammatory factors levels from glial cells were significantly increased through CREB and NF-KB signaling pathways after p38MAPK activation.更多还原