【作者】 宁晨；

【导师】 齐琳； Yang Xia；

【作者基本信息】 中南大学 ， 外科学， 2012， 博士

【摘要】 研究背景阴茎异常勃起(Priapism)是指与性欲无关的阴茎异常持续勃起状态,是泌尿男科的急症,如不及时有效治疗,将引起阴茎组织损伤坏死、海绵体纤维化,最终导致阴茎勃起功能障碍(Erectile dysfunction, ED)。该疾病虽在普通人群中不常见,但高发于某些特殊人群,如镰状红细胞贫血症(Sickle cell disease)、粒细胞性白血病、多发性骨髓瘤、阴茎勃起功能障碍药物治疗中的并发症等。该疾病发病机制仍然不十分清楚,临床上缺乏针对性有效治疗手段。因此,深入探究阴茎异常勃起发病机理,寻找其有效治疗方法,尤显必要。一氧化氮(Nitric oxide, NO)-环磷酸鸟苷(Cyclic guanosine monophosphate, cGMP)信号通路在阴茎勃起中的重要调节作用已得到公认,但也有研究结果表明,在阴茎勃起中尚存在其它重要的因子与信号通路。近年来人们再度认识了腺苷信号通路在阴茎异常勃起中的作用。新近本课题组前期试验研究意外地发现腺苷脱氨酶(Adenosine deaminase, ADA)基因敲除小鼠表现出类似于人的阴茎异常勃起症状(自发的持续阴茎勃起伴有阴茎纤维化),阴茎组织中腺苷水平明显增高,并可通过腺苷脱氨酶注射治疗得到缓解。这一重要的发现强烈提示腺苷信号通路在阴茎异常勃起的发生发展中起着十分重要作用,是一个很有前景的研究领域。腺苷(Adenosine),由腺嘌呤与核糖组成,腺苷特异性受体包括4种类型：A1,A2A,A2B和A3。腺苷的代谢主要受ADA调节,ADA催化腺苷不可逆地转变为次黄嘌呤腺苷。本课题组前期研究结果表明,腺苷A2B受体是阴茎正常和异常勃起过程中的关键环节。特异性阻断腺苷A2B受体,有可能成为阴茎异常勃起及纤维化防治新靶点。阴茎异常勃起伴随着缺血缺氧过程,而同时缺血缺氧又可加重阴茎异常勃起。病理生理研究表明,机体组织的缺血缺氧与腺苷水平密切相关。缺氧诱导因子-1(Hypoxia-inducible factor-1, HIF-1)是一个参与调节与缺氧反应相关基因表达的重要转录因子,其α亚单位(HIF-1α)为HIF-1的活性亚基和调节亚基,近年来备受关注。HIF-1α与阴茎异常勃起发病是否有联系?是否也参与其腺苷信号通路?值得进一步探究。基于上述,本研究拟在前期研究基础上,以ADA基因缺陷小鼠和SCD转基因小鼠(SCD transgenic mice, SCD Tg小鼠)两种阴茎异常勃起的动物模型为研究对象,采用新近研制出的腺苷A2B受体抑制剂—PSB1115,特异性阻断腺苷A2B受体,观察其对阴茎异常勃起及纤维化的作用影响,并试从腺苷-腺苷A2B受体-HIF-1α-内皮型一氧化氮合成酶(Endothelial NO synthase, eNOS)信号通路,深入探讨其机制,为寻找临床防治阴茎异常勃起及纤维化的新靶点、新方法提供直接的实验依据。实验方法本研究包括四个部分：一、两种阴茎异常勃起小鼠模型的维持及基因型鉴定1、采用本实验室前期使用基因敲除技术制作的ADA基因缺陷小鼠模型。出生后即给予聚乙二醇腺苷脱氨酶(Polyethylene glycol ADA, PEG-ADA)替代治疗,维持其性发育至成熟,在该模型鼠出生后第7天,行酶谱分析鉴定基因型。取ADA-／-纯合子鼠,继续维持饲养。2、采用转基因技术制作的SCD Tg小鼠模型。于模型鼠出生后第6周进行基因型鉴定,分别使用①剪尾取血,涂片染色,寻找镰状红细胞。②取尾组织,提取RNA, PCR检测二种方法。取SCD Tg纯合子鼠,继续维持饲养。二、腺苷A2B受体抑制剂对阴茎异常勃起的影响分别在ADA基因缺陷小鼠和SCD Tg小鼠,使用特异性腺苷A2B受体抑制剂—PSB1115作干预治疗药物,实验用药从小鼠12周龄开始,PSB1115以10mg.kg-1.d-1剂量腹腔注射,持续用药2周。实验共分6组,每组5只：(1)空白对照组：周龄相符的野生型C57BL/6雄性小鼠+等量生理盐水；(2)阴性对照组：周龄相符的野生型C57BL/6雄性小鼠+PSB1115治疗；(3)ADA对照组：ADA基因缺陷雄性小鼠+等量生理盐水；(4)ADA治疗组：ADA基因缺陷雄性小鼠+PSB1115治疗；(5)SCD对照组：SCDTg雄性小鼠+等量生理盐水；(6)SCD治疗组：SCDTg雄性小鼠+PSB1115治疗；电刺激小鼠阴茎海绵体神经模拟生理性阴茎勃起。采用最大阴茎海绵体内压、总阴茎海绵体内压、阴茎勃起初始期速率三个指标,检测、评估小鼠阴茎勃起功能状态。采用HLPC方法检测各组小鼠阴茎组织腺苷浓度。三、腺苷A2B受体抑制剂治疗阴茎异常勃起的分子机制分别于模型鼠(体内实验)和细胞培养(体外实验)二个研究层面进行实验。实验小鼠分组及实验条件同第二部分,取阴茎组织抽提总RNA；采用人微血管内皮细胞-1(Human microvascular endothelial cells-1,HMEC-1),分别以腺苷类似物5’-N乙烯碳氨基腺苷(5’-N-ethylcarboxamidoadenosine, NEC A,10μM)、腺苷A2B受体抑制剂(PSB1115,10μM)干预处理,于Oh、3h、6h、2h、24h分别收取细胞抽提总RNA；采用Real-time PCR方法检测HIF-1α及eNOS的基因表达；并进一步在HMEC-1细胞实验中采用基因沉默方法沉默HIF-1α基因,以明确HIF-1α在腺苷-腺苷A2B受体-eNOS信号通路中的作用。四、腺苷A2B受体抑制剂对阴茎纤维化的作用及其机制实验对象与分组同第二部分,小鼠用药时间延至6周。取阴茎组织病理切片作HE染色、Masson’s Trichrome染色和免疫组化染色,分别检测阴茎胶原纤维/平滑肌面积比率,α平滑肌肌动蛋白(α-smooth muscle actin, α-SMA)阳性面积/海绵体总面积百分比,评估纤维化程度。另取阴茎组织抽提总RNA,用Real-time PCR方法检测纤维化相关基因表达：α2(Ⅰ)胶原、转化生长因子β1(Transforming growth factor β1,TGF-β1),纤溶酶原活化抑制物-1(Plasminogenactivator inhibitor-1,PAI-1)。结果1、通过酶谱分析与血涂片及基因型鉴定,成功筛选出ADA基因缺陷小鼠纯合子(ADA-/-)和SCD Tg小鼠纯合子。2、与野生型小鼠相比,14周龄的ADA基因缺陷小鼠与SCD Tg小鼠阴茎内腺苷水平显著增高(P<0.05)；电刺激后阴茎勃起状态明显增强,存在阴茎异常勃起倾向性,表现为最大阴茎海绵体内压、总阴茎海绵体内压、阴茎勃起初始期速率显著增加(P<0.05); PSB1115治疗可有效预防和缓解ADA基因缺陷小鼠与SCD Tg小鼠阴茎异常勃起倾向性,表现为最大阴茎海绵体内压、总阴茎海绵体内压、阴茎勃起初始期速率较ADA对照组显著下降(P<0.05)。3、与野生型小鼠相比,ADA基因缺陷小鼠与SCDTg小鼠阴茎内HIF-1α、eNOS基因表达水平显著增高(P<0.05),PSB1115治疗可使上述基因表达水平显著下降；在HMEC-1细胞实验中进一步证实,腺苷类似物(NECA)可上调HIF-1α、eNOS基因表达(P<0.05),腺苷A2B受体抑制剂可下调HIF-1α、eNOS基因表达(P<0.05),提示腺苷-腺苷A2B受体-HIF-1a-eNOS信号通路可能参与了ADA基因缺陷小鼠及SCDTg小鼠阴茎异常勃起。4、与野生型小鼠相比,18周龄ADA基因缺陷小鼠及与SCDTg小鼠表现出明显的阴茎纤维化,其纤维化指标(纤维面积/海绵体平滑肌面积比、抗α平滑肌肌动蛋白)及纤维化相关的基因表达(α2(Ⅰ)胶原、TGF-β1、PAI-1)显著增加；PSB1115治疗可有效预防和缓解阴茎纤维化的发生。结论1、在ADA基因缺陷小鼠和SCD Tg小鼠两种阴茎异常勃起模型鼠,阴茎组织腺苷浓度显著增高,并通过腺苷A2B受体导致阴茎异常勃起；2、特异性腺苷A2B受体抑制剂-PSB1115可有效预防和缓解ADA基因缺陷小鼠与SCD Tg小鼠的阴茎异常勃起及纤维化；3、腺苷-腺苷A2B受体-HIF-1α-eNOS信号通路是阴茎异常勃起的重要分子机制之一；4、特异性阻断腺苷A2B受体有望成为防治阴茎异常勃起和纤维化的新靶点。更多还原

【Abstract】 BackgroundPriapism is defined as abnormally prolonged and persistent penile erection in absence of sexual provocation or desire. This disorder is considered as a urological emergency which need early treatment to avoid the risks of structural damage of the penis and erectile dysfunction (ED), a known complication. Priapism is rarely been seen in general population, but it is associated with a number of different medical conditions, such as sickle cell disease (SCD), ED pharmacotherapeutic complications, neurological disease, and solid malignancies. Due to lack of fundamental understanding of the etiology and pathophysiology of priapism, effective approaches to manage the disorder are limited. Thus, mechanism-based approaches which aim at preventing and curing priapism are needed.Nitric oxide (NO) is the best characterized molecule to regulate penile erection, but that does not exclude a role of other signaling molecules, either released by neruons, and/or endothelial and smooth muscle cells in the regulation of this process. Recently, our lab unexpectedly found that mice deficient in the purine catabolic enzyme adenosine deaminase (ADA) exhibited priapism seen in human, which suggest adenosine may play a key role in priapism and normal penile erection and indicated ADA-deficient mice could be employed as a novel and important model to study priapism like the other well-established animal model of priapism, SCD transgenic (Tg) mice. We further demonstrated that excess adenosine in penile tissue contributed to priapism through adenosine A2B receptor and decreasing adenosine level in penis by ADA enzyme therapy could prevent and reverse the occurrence of priapism. These results all suggested that adenosine signaling pathway plays an important role in the regulation of normal and abnormal penile erection.Adenosine is a well-known potent vasodilator that elicits its physiological effects on target cells by engaging its four specific G-protein-coupled receptors:A1, A2A, A2B, and A3. ADA is the key enzyme in adenosine metabolism, which catalyses irreversible conversion of adenosine to inosine. Based on our previous studies, adenosine A2B receptor is the main adenosine receptor contributing to priapism,which indicate that adenosine A2B receptor signaling pathway may be a novel therapeutic target for erectile disorders, especially priapism and penile fibrosis.In the penis of patients with ischemic priapism, the corpus cavernosum and vascular endothelial cells are exposed to intermittent hypoxic episodes. Hypoxia-inducible factor-1(HIF-1) is a homodimeric transcriptional complex which promotes expression of numerous genes which involve in oxygen and energy-providing substrates supplements under hypoxia condition. And adenosine is also highly induced under ischemic and hypoxia condition, which could regulate HIF-1α expression. It would be quite interesting to determine whether HIF-1α involves in the pathogenesis of priapism.To extend our findings to therapeutic possibility for priapism and penile fibrosis, we choose adenosine A2B receptor specific antagonist PSB1115, which could be used in vivo research, and took the advantages of two independent priapic animal model, ADA-deficient mice and SCD Tg mice to evaluate the efficiency of PSB1115treatment to priapism and penile fibrosis in vivo and to determine the role of adenosine-HIF-1α-eNOS signaling pathway in priapism.MethodOur study is generally consisted of four parts.1. Genotyping of ADA-deficient mice and SCD Tg mice, two well-accepted animal models of priapism.1) We took advantage of ADA-deficient mice which generated from our lab as an animal models of priapism. We routinely treat the ADA-deficient mice with PEG-ADA to make them survive and reproductive system mature. In this study, zymogram analysis was employed to detect the ADA activity at7th days after birth to determine the newborn mice’s genotyping.2) SCD Tg mice express exclusively human sickle hemoglobin and have the major features found in humans with sickle cell disease. For genotyping of these mice, two methods including blood smear test and PCR are used at6weeks after birth.2. Therapeutic effects of adenosine A2B receptor specific antagonist PSB1115on priapism in mice were observed.ADA-deficient mice and SCD Tg mice were treated with PSB1115(10mg.kg-1.d-1) by intraperitoneal injection for2weeks. These mice were divided into6groups (n=5):1) Control:age-matched wild-type (WT) C57BL/6mice+saline2) Control+PSB1115:age-matched wild-type (WT) C57BL/6mice+PSB11153) ADA-/-:ADA-deficient mice+saline4) ADA-/-+PSB1115:ADA-deficient mice+PSB11155) SCD:SCD Tg mice+saline6) SCD+PSB1115SCD Tg mice+PSB1115A well-established technique was used in vivo to mimic normal penile erection by measuring the intracavernosal pressure (ICP) to assess the erectile response to cavernous nerve stimulation. Maximum ICP, total ICP (area under curve) and slope were detected as the main parameters for evaluating the erectile function. HPLC was used to measure the adenosine level in penile tissue.3. To determine the underlying mechanism of adenosine mediated priapism via adenosine A2B receptorStudies of in vivo and in vitro were employed. Penile tissues were collected from6groups. HIF-1α and eNOS gene expressions were measured. Human microvascular endothelial cell-1(HMCE-1) was treated with NECA (adenosine analogy) and/or PSB1115and observed the variation of HIF-1α and eNOS gene expression. HMCE-1with HDF-1α knocked down was used to determine the regulation effect of HIF-1α on eNOS.4. Therapeutic effects of adenosine A2B receptor specific antagonist PSB1115on penile fibrosisMice with PSB1115were treated for6weeks and penile tissues were collected for histological examination (H&E staining, Masson’s Trichrome staining and a-Smooth muscle actin (a-SMA) immuno-histochemistry staining) to evaluate penile fibrosis. Real-time PCR was used to measure fibrotic gene expression profile (α2(Ⅰ) procollagen, TGF-β1, and plasminogen activator inhibitor-1),Results1. ADA-deficient mice homozygote were identified by zymogram analysis and SCD Tg mice homozygote were genotyped by blood smear test and PCR.2. Compared with WT mice, levels of adenosine in penile tissue of ADA-deficient mice and SCD Tg mice were significantly increased. ADA-deficient mice and SCD Tg mice displayed an obvious elevated maximum ICP as well as total ICP (area under curve) and the initial rate of ICP (Slope), compared with control mice. However,2weeks PSB1115treatment for ADA-deficient mice and SCD Tg mice dramatically reduced the maximum ICP, total ICP and slope compared to ADA-deficient mice and SCD Tg mice,which were also similar to control mice.3. Compared with WT mice, ADA-deficient mice and SCD Tg mice had a higher level of HIF-la and eNOS gene expression which were reduced after2weeks PSB1115treatment. In vitro, the results showed that in HMCE-1cells treated with NECA (adenosine analog), the mRNA levels of HIF-1α and eNOS were significantly elevated. However, these elevations were inhibited by PSB1115treatment. In HIF-1a knockdown HMEC-1cells, NECA treatment couldn’t increase the eNOS mRNA level. These results suggested that adenosine mediated eNOS gene expression by modulating HIF-1α via adenosine A2B receptor in endothelial cells.4. We found that PSB1115treatment significantly reduced penile injury and fibrosis measured by H&E staining, immunohistochemistry and Trichrome staining in both mouse models. Consistently, we found that PSB1115significantly reduced fibrotic gene expression including procollagen I, TGF-β1, and plasminogen activator inhibitor-1mRNA. Conclusion1. Elevated levels of adenosine in penile tissue of ADA deficient mice and SCD Tg mice, two well-accepted priapism animal models, contribute to priapism via adenosine A2B receptor.2. Adenosine A2B receptor specific antagonist PSB1115prevents and relieves priapic feature and penile fibrosis both in ADA deficient mice and SCD Tg mice.3. Adenosine-HDF-1α-eNOS signaling pathway contributes to priapism in ADA-deficient mice and SCD Tg mice via adenosine A2B receptor.4. Interfering adenosine A2B receptor signaling is likely an effective therapy to treat and prevent priapism and penile fibrosis and which offers a novel therapy for human with priapism.更多还原