脂多糖通过降低下丘脑Kisspeptin-GPR54系统表达抑制性腺轴功能和GnRH脉冲治疗男性IHH生精疗效分析

发布时间：2018-03-10 07:36

  本文选题：Kisspeptin　切入点：GPR54　出处：《北京协和医学院》2015年博士论文　论文类型：学位论文

【摘要】：近十年的研究阐明：下丘脑Kisspeptin神经元分泌Kisspeptin多肽,作用于促性腺激素释放激素(Gonadotropin-releasing hormone, GnRH)神经元上的Kisspeptin受体-GPR54,刺激GnRH分泌,进而调控垂体-性腺轴功能。下丘脑Kisspeptin-GPR54系统成为生殖内分泌疾病病理生理机制研究以及临床治疗药物开发应用的热门靶点。本文第一部分,研究脂多糖(Lipopolysaccharide, LPS) 对 Kisspeptin-GPR54系统表达的影响,进而阐述LPS抑制性腺轴功能的病理生理机制；第二部分,比较GnRH脉冲输注与人绒毛膜促性腺激素/人绝经期促性腺激素(Human Chorionic Gonadotropin/Human Menopausal Gonadotropin, HCG/HMG)联合肌注治疗男性特发性低促性腺激素性性腺功能减退症(Idiopathic hypogonadotropic hypogonadism,IHH)生精的疗效,并分析影响生精疗效的因素。第一部分：脂多糖通过降低下丘脑Kisspeptin-GPR54系统表达抑制性腺轴功能研究背景：LPS可抑制性腺轴功能；但其病理生理学机制尚不清楚。LPS导致的应激状态(伴糖皮质激素升高),也参与LPS抑制性腺轴功能。下丘脑Kisspeptin-GPR54系统统领调控下丘脑-垂体-性腺轴功能,可能介导LPS及其所致应激状态对性腺轴功能的抑制。研究目的：观察LPS和地塞米松(Dexamethasone, Dex)对Kisspeptin-GPR54系统表达的影响。研究方法：1.体外培养MCF7细胞系,用LPS(10μg/ml和20 gg/m1)和Dex(10-6 mol/L和10-7 mol/L)干预72 h,评价干预6 h、24 h、48h和72 h后Kisspeptin和GPR54 mRNA (Real-Time PC R)和蛋白表达量(Western印迹)的变化；2.成年雄性小鼠去势4周后,检测去势前后LH水平和下丘脑视前区Kisspeptin和GPR54免疫组化表达；用LPS (100μg/Kg)和Dex (1mg/Kg)干预去势成年雄性小鼠4周,检测干预前后LH水平和下丘脑视前区Kisspeptin和GPR54免疫组织化学染色变化。研究结果：1.在MCF7细胞系中,和对照组相比,LPS (10μg/ml和20μg/ml)和Dex(10-6mol/L和10-7mol/L)均显著降低Kisspeptin mRNA(P0.05)和蛋白表达；LPS(10μg/ml和20μg/ml)和Dex(10-6mol/L口10-7 mol/L)均显著增加GPR54 mRNA(P均0.05)和蛋白表达；2.与正常成年雄性小鼠比较,去势4周后成年雄性小鼠LH水平显著升高(P0.01),下丘脑Kisspeptin表达显著增加(P0.05),下丘脑GPR54表达无变化(P0.05)。在去势成年雄性小鼠中,与对照组比较,LPS和Dex均显著降低去势成年雄性小鼠LH水平(均P0.01)；LPS降低(P0.05)而Dex不改变(P0.05)去势成年雄性小鼠下丘脑视前区Kisspeptin和GPR54免疫组织化学染色表达。研究结论：LPS可以通过降低下丘脑Kisspeptin-GPR54系统表达抑制性腺轴功能。地塞米松不通过改变下丘脑Kisspeptin-GPR54系统表达发挥其抑制性腺轴功能。进一步支持Kisspeptin神经元是成年雄性小鼠睾酮负反馈调节作用部位。和Dex(10-6mol/L和10-7mol/L)均显著降低Kisspeptin mRNA(P0.05)和蛋白表达；LPS(10μg/ml和20μg/ml)和Dex(10-6mol/L口10-7 mol/L)均显著增加GPR54 mRNA(P均0.05)和蛋白表达；2.与正常成年雄性小鼠比较,去势4周后成年雄性小鼠LH水平显著升高(P0.01),下丘脑Kisspeptin表达显著增加(P0.05),下丘脑GPR54表达无变化(P0.05)。在去势成年雄性小鼠中,与对照组比较,LPS和Dex均显著降低去势成年雄性小鼠LH水平(均P0.01)；LPS降低(P0.05)而Dex不改变(P0.05)去势成年雄性小鼠下丘脑视前区Kisspeptin和GPR54免疫组织化学染色表达。研究结论：LPS可以通过降低下丘脑Kisspeptin-GPR54系统表达抑制性腺轴功能。地塞米松不通过改变下丘脑Kisspeptin-GPR54系统表达发挥其抑制性腺轴功能。进一步支持Kisspeptin神经元是成年雄性小鼠睾酮负反馈调节作用部位。第二部分：GnRH脉冲输注与HCG/HMG联合肌注治疗男性IHH患者生精疗效比较和影响因素分析研究目的：比较GnRH脉冲式皮下输注和HCG/HM G联合肌注治疗男性IHH的生精疗效,并分析影响生精疗效的因素。研究方法：回顾性分析2010年5月至2014年10月在北京协和医院内分泌科门诊就诊的男性IHH患者92例。患者自愿选择一种治疗方案,并据此将92例IHH患者分成两组：GnRH脉冲式治疗组(GnRH组,n=40);HCG/HMG联合治疗组(HCG/HMG组,n=52)。观察GnRH组在治疗后第1天、第3天、第5天、第7天和每月的LH及FSH水平；每月随诊观察两组患者血总睾酮(Serum total testosterone, TT)水平、睾丸体积(Testicular volume, TV)和精子生成例数的变化情况。并比较GnRH组和HCG/HMG组在治疗过程中TT水平、TV、精子生成率和精子初现时间的差异；并分析影响生精疗效的因素。研究结果：1.所有患者均治疗3个月以上。GnRH组和HCG/HMG组随访中位时间分别为8.2(3.0-18.4)个月和9.2(3.0-18.6)个月,差别无统计学意义(P=0.413)。2.GnRH组治疗3天时,LH(0.5±0.6vs.1.6±1.1 IU/L,P0.05)和FSH(1.3±1.1vs.4.3±3.9IU/L,P0.05)水平较治疗前显著升高。GnRH组治疗2个月时与治疗前比较,TT(1.0±1.0 vs.8.2±7.3 nmol/L, P0.01)水平显著升高,TV(2.3±1.5 vs.6.0±2.5ml,P=0.001)显著增大。GnRH组患者末次随诊TT水平(7.4±5.2)nmol/L和TV(8.1±4.0)m1分别较治疗前TT水平(1.0±1.0) nmol/L和TV (2.3±1.5) ml显著升高和增大,均P0.01。HCG/HMG组末次随诊TT水平(14.4±48.0)nmol/L和TV(7.6±4.2)m1分别较治疗前TT水平(0.8±0.6)nmol/L口TV (2.3±2.1) ml显著升高和增大,均P0.01。GnRH组和HCG/HMG组分别有20人(20/40,50.0%)和15人(15/52,28.8%)生成精子,组间差异有统计学意义(P=0.038)。精子初现时间方面,GnRH组(6.5±3.1)月短于HCG/HMG组(10.8±3.7)月(P=0.001)。精子初现时,GnRH组和HCG/HMG组的TT水平分别为(9.0±5.1)nmol/L和(14.8±8.8)nmol/L(P=0.034)。3.在GnRH组和HCG/HMG组,各自生成精子和未生成精子IHH患者基础TV比较,差异无统计学意义(均P0.05)；在GnRH组和HCG/HMG组,各自IHH患者Logistic回归分析基础TV对生精疗效影响,差异无统计学意义(均P0.05)。研究结论：GnRH脉冲式皮下输注治疗男性IHH 比 HCG/HMG联合肌注更早产生精子。基础睾丸体积不影响GnRH脉冲输注和HCG/HMG联合肌注生精疗效。
[Abstract]:Nearly ten years of research demonstrates that hypothalamic Kisspeptin neurons secrete Kisspeptin polypeptide on gonadotropin releasing hormone (Gonadotropin-releasing, hormone, GnRH) neuronal Kisspeptin receptor -GPR54 stimulated GnRH secretion, and regulation of hypothalamic pituitary gonadal axis function. The Kisspeptin-GPR54 system has become a research pathophysiological mechanism of reproductive endocrine diseases as well as the popular target for clinical treatment of drug the development and application. In the first part of this paper, the research of lipopolysaccharide (Lipopolysaccharide, LPS) on expression of Kisspeptin-GPR54 system, and then discusses the pathophysiological mechanism of LPS inhibition of gonadal function; the second part, GnRH pulse infusion and human chorionic gonadotropin / human menopausal gonadotropin (Human Chorionic Gonadotropin/Human Menopausal Gonadotropin, HCG/HMG) combined with intramuscular injection for treating male idiopathic low gonadotropin Primality hypogonadism (Idiopathic hypogonadotropic, hypogonadism, IHH) efficacy of spermatogenesis, and analyze the influencing factors of spermatogenic efficacy. The first part: lipopolysaccharide by reducing the hypothalamic Kisspeptin-GPR54 system inhibits expression of gonadal function research background: LPS can inhibit the function of gonadal axis; but its pathophysiology is unclear due to.LPS stress (with glucocorticoid increase), is also involved in LPS inhibition of gonadal function. The hypothalamic Kisspeptin-GPR54 system and regulation of hypothalamic pituitary gonadal axis function, may mediate LPS induced stress on gonadal function. Objective: To observe the inhibition of LPS and dexamethasone (Dexamethasone, Dex) on expression of Kisspeptin-GPR54 system research. Methods: 1. in vitro MCF7 cells cultured with LPS (10 g/ml and 20 gg/m1) and Dex (10-6 mol/L and 10-7 mol/L) 72 h intervention, evaluation Price intervention 6 h, 24 h, 72 48h and H Kisspeptin and GPR54 mRNA (Real-Time PC R) and protein expression (Western blot) changes; 2. adult male mice 4 weeks after castration, castration and detection of serum LH levels before and after the preoptic area Kisspeptin and immunohistochemical expression of GPR54 with LPS (100; U g/Kg) and Dex (1mg/Kg) intervention in castrated adult male mice 4 weeks before and after the intervention, detection of the level of LH and the preoptic area Kisspeptin and GPR54 immunohistochemical staining. Results: 1. of the MCF7 cells, compared with control group, LPS (10 g/ml and 20 g/ and Dex (ML) 10-6mol/L and 10-7mol/L) were significantly decreased in Kisspeptin mRNA (P0.05) and protein expression; LPS (10 g/ml and 20 g/ml) and Dex (10-6mol/L 10-7 mol/L) significantly increased GPR54 mRNA (P 0.05) and protein expression; and 2. normal adult male mice 4 weeks after castration, adult LH male mouse 鏄捐憲鍗囬珮(P0.01),涓嬩笜鑴慘isspeptin琛ㄨ揪鏄捐憲澧炲姞(P0.05),涓嬩笜鑴慓PR54琛ㄨ揪鏃犲彉鍖，

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