本文選題：Kisspeptin　切入點(diǎn)：GPR54　出處：《北京協(xié)和醫(yī)學(xué)院》2015年博士論文　論文類型：學(xué)位論文

【摘要】：近十年的研究闡明：下丘腦Kisspeptin神經(jīng)元分泌Kisspeptin多肽,作用于促性腺激素釋放激素(Gonadotropin-releasing hormone, GnRH)神經(jīng)元上的Kisspeptin受體-GPR54,刺激GnRH分泌,進(jìn)而調(diào)控垂體-性腺軸功能。下丘腦Kisspeptin-GPR54系統(tǒng)成為生殖內(nèi)分泌疾病病理生理機(jī)制研究以及臨床治療藥物開發(fā)應(yīng)用的熱門靶點(diǎn)。本文第一部分,研究脂多糖(Lipopolysaccharide, LPS) 對(duì) Kisspeptin-GPR54系統(tǒng)表達(dá)的影響,進(jìn)而闡述LPS抑制性腺軸功能的病理生理機(jī)制；第二部分,比較GnRH脈沖輸注與人絨毛膜促性腺激素/人絕經(jīng)期促性腺激素(Human Chorionic Gonadotropin/Human Menopausal Gonadotropin, HCG/HMG)聯(lián)合肌注治療男性特發(fā)性低促性腺激素性性腺功能減退癥(Idiopathic hypogonadotropic hypogonadism,IHH)生精的療效,并分析影響生精療效的因素。第一部分：脂多糖通過降低下丘腦Kisspeptin-GPR54系統(tǒng)表達(dá)抑制性腺軸功能研究背景：LPS可抑制性腺軸功能；但其病理生理學(xué)機(jī)制尚不清楚。LPS導(dǎo)致的應(yīng)激狀態(tài)(伴糖皮質(zhì)激素升高),也參與LPS抑制性腺軸功能。下丘腦Kisspeptin-GPR54系統(tǒng)統(tǒng)領(lǐng)調(diào)控下丘腦-垂體-性腺軸功能,可能介導(dǎo)LPS及其所致應(yīng)激狀態(tài)對(duì)性腺軸功能的抑制。研究目的：觀察LPS和地塞米松(Dexamethasone, Dex)對(duì)Kisspeptin-GPR54系統(tǒng)表達(dá)的影響。研究方法：1.體外培養(yǎng)MCF7細(xì)胞系,用LPS(10μg/ml和20 gg/m1)和Dex(10-6 mol/L和10-7 mol/L)干預(yù)72 h,評(píng)價(jià)干預(yù)6 h、24 h、48h和72 h后Kisspeptin和GPR54 mRNA (Real-Time PC R)和蛋白表達(dá)量(Western印跡)的變化；2.成年雄性小鼠去勢(shì)4周后,檢測(cè)去勢(shì)前后LH水平和下丘腦視前區(qū)Kisspeptin和GPR54免疫組化表達(dá)；用LPS (100μg/Kg)和Dex (1mg/Kg)干預(yù)去勢(shì)成年雄性小鼠4周,檢測(cè)干預(yù)前后LH水平和下丘腦視前區(qū)Kisspeptin和GPR54免疫組織化學(xué)染色變化。研究結(jié)果：1.在MCF7細(xì)胞系中,和對(duì)照組相比,LPS (10μg/ml和20μg/ml)和Dex(10-6mol/L和10-7mol/L)均顯著降低Kisspeptin mRNA(P0.05)和蛋白表達(dá)；LPS(10μg/ml和20μg/ml)和Dex(10-6mol/L口10-7 mol/L)均顯著增加GPR54 mRNA(P均0.05)和蛋白表達(dá)；2.與正常成年雄性小鼠比較,去勢(shì)4周后成年雄性小鼠LH水平顯著升高(P0.01),下丘腦Kisspeptin表達(dá)顯著增加(P0.05),下丘腦GPR54表達(dá)無(wú)變化(P0.05)。在去勢(shì)成年雄性小鼠中,與對(duì)照組比較,LPS和Dex均顯著降低去勢(shì)成年雄性小鼠LH水平(均P0.01)；LPS降低(P0.05)而Dex不改變(P0.05)去勢(shì)成年雄性小鼠下丘腦視前區(qū)Kisspeptin和GPR54免疫組織化學(xué)染色表達(dá)。研究結(jié)論：LPS可以通過降低下丘腦Kisspeptin-GPR54系統(tǒng)表達(dá)抑制性腺軸功能。地塞米松不通過改變下丘腦Kisspeptin-GPR54系統(tǒng)表達(dá)發(fā)揮其抑制性腺軸功能。進(jìn)一步支持Kisspeptin神經(jīng)元是成年雄性小鼠睪酮負(fù)反饋調(diào)節(jié)作用部位。和Dex(10-6mol/L和10-7mol/L)均顯著降低Kisspeptin mRNA(P0.05)和蛋白表達(dá)；LPS(10μg/ml和20μg/ml)和Dex(10-6mol/L口10-7 mol/L)均顯著增加GPR54 mRNA(P均0.05)和蛋白表達(dá)；2.與正常成年雄性小鼠比較,去勢(shì)4周后成年雄性小鼠LH水平顯著升高(P0.01),下丘腦Kisspeptin表達(dá)顯著增加(P0.05),下丘腦GPR54表達(dá)無(wú)變化(P0.05)。在去勢(shì)成年雄性小鼠中,與對(duì)照組比較,LPS和Dex均顯著降低去勢(shì)成年雄性小鼠LH水平(均P0.01)；LPS降低(P0.05)而Dex不改變(P0.05)去勢(shì)成年雄性小鼠下丘腦視前區(qū)Kisspeptin和GPR54免疫組織化學(xué)染色表達(dá)。研究結(jié)論：LPS可以通過降低下丘腦Kisspeptin-GPR54系統(tǒng)表達(dá)抑制性腺軸功能。地塞米松不通過改變下丘腦Kisspeptin-GPR54系統(tǒng)表達(dá)發(fā)揮其抑制性腺軸功能。進(jìn)一步支持Kisspeptin神經(jīng)元是成年雄性小鼠睪酮負(fù)反饋調(diào)節(jié)作用部位。第二部分：GnRH脈沖輸注與HCG/HMG聯(lián)合肌注治療男性IHH患者生精療效比較和影響因素分析研究目的：比較GnRH脈沖式皮下輸注和HCG/HM G聯(lián)合肌注治療男性IHH的生精療效,并分析影響生精療效的因素。研究方法：回顧性分析2010年5月至2014年10月在北京協(xié)和醫(yī)院內(nèi)分泌科門診就診的男性IHH患者92例�；颊咦栽高x擇一種治療方案,并據(jù)此將92例IHH患者分成兩組：GnRH脈沖式治療組(GnRH組,n=40);HCG/HMG聯(lián)合治療組(HCG/HMG組,n=52)。觀察GnRH組在治療后第1天、第3天、第5天、第7天和每月的LH及FSH水平；每月隨診觀察兩組患者血總睪酮(Serum total testosterone, TT)水平、睪丸體積(Testicular volume, TV)和精子生成例數(shù)的變化情況。并比較GnRH組和HCG/HMG組在治療過程中TT水平、TV、精子生成率和精子初現(xiàn)時(shí)間的差異；并分析影響生精療效的因素。研究結(jié)果：1.所有患者均治療3個(gè)月以上。GnRH組和HCG/HMG組隨訪中位時(shí)間分別為8.2(3.0-18.4)個(gè)月和9.2(3.0-18.6)個(gè)月,差別無(wú)統(tǒng)計(jì)學(xué)意義(P=0.413)。2.GnRH組治療3天時(shí),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)水平較治療前顯著升高。GnRH組治療2個(gè)月時(shí)與治療前比較,TT(1.0±1.0 vs.8.2±7.3 nmol/L, P0.01)水平顯著升高,TV(2.3±1.5 vs.6.0±2.5ml,P=0.001)顯著增大。GnRH組患者末次隨診TT水平(7.4±5.2)nmol/L和TV(8.1±4.0)m1分別較治療前TT水平(1.0±1.0) nmol/L和TV (2.3±1.5) ml顯著升高和增大,均P0.01。HCG/HMG組末次隨診TT水平(14.4±48.0)nmol/L和TV(7.6±4.2)m1分別較治療前TT水平(0.8±0.6)nmol/L口TV (2.3±2.1) ml顯著升高和增大,均P0.01。GnRH組和HCG/HMG組分別有20人(20/40,50.0%)和15人(15/52,28.8%)生成精子,組間差異有統(tǒng)計(jì)學(xué)意義(P=0.038)。精子初現(xiàn)時(shí)間方面,GnRH組(6.5±3.1)月短于HCG/HMG組(10.8±3.7)月(P=0.001)。精子初現(xiàn)時(shí),GnRH組和HCG/HMG組的TT水平分別為(9.0±5.1)nmol/L和(14.8±8.8)nmol/L(P=0.034)。3.在GnRH組和HCG/HMG組,各自生成精子和未生成精子IHH患者基礎(chǔ)TV比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(均P0.05)；在GnRH組和HCG/HMG組,各自IHH患者Logistic回歸分析基礎(chǔ)TV對(duì)生精療效影響,差異無(wú)統(tǒng)計(jì)學(xué)意義(均P0.05)。研究結(jié)論：GnRH脈沖式皮下輸注治療男性IHH 比 HCG/HMG聯(lián)合肌注更早產(chǎn)生精子�；A(chǔ)睪丸體積不影響GnRH脈沖輸注和HCG/HMG聯(lián)合肌注生精療效。
[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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