甘丙肽对GT1-7细胞GnRH释放的调节作用

目的：GT1-7细胞是替代研究GnRH神经元的理想细胞模型。本实验研究甘丙肽1型，2型受体mRNA在GT1-7细胞中的表达及对GnRH的调节作用。方法：（1）采用逆转录-聚合酶链反应（RT-PCR）法观察甘丙肽受体mRNA在GT1-7中的表达；（2）将不同浓度的甘丙肽以不同时间与GT1-7细胞卵育，用RIA法测定细胞上清液中GnRH含量。结果：（1）GT1-7细胞同时表达甘丙肽1型和2型受体mRNA;(2)甘丙肽能刺激GnRH释放，且呈明显的量效关系。结论：甘丙肽可通过其受体直接作用下丘脑GnRH神经元，而对生殖功能起调节作用。     

GnRH拮抗剂在体外受精-胚胎移植中的应用

目的探讨促性腺激素释放激素（GnRH）拮抗剂方案在体外受精-胚胎移植（IVF—ET）促超排卵中的应用效果。方法回顾性比较分析本中心2006年8月～2007年8月接受ⅣF—ET助孕治疗的患者中采用GnRH拮抗剂方案的54例患者和采用GnRH激动剂长方案的135例患者，观察其临床效果。结果两组Gn用量、HCG日内膜厚度、受精率、卵裂率之间比较无显著性差异；两组患者Gn使用天数、HCG日E2值、获卵数、冷冻率、种植率、妊娠率、OHSS发生率之间比较有显著性差异。结论GnRH拮抗剂联合促性腺激素促超排卵方案缩短用药时间，减少费用，并可显著降低OHSS的发生率，但冷冻率、妊娠率较GnRH激动剂长方案偏低。     

GnRH拮抗剂与GnRH激动剂短方案IVF-ET结局的比较

目的比较GnRH antagonist与GnR Hagonist短方案的IVF-ET结局。方法2006年8月至2007年8月GnR Hantagonist治疗组54人和GnR Hagonist短方案对照组132人,记录促性腺激素的用量及其用药天数、hCG日子宫内膜厚度和激素水平、获卵数、受精率、卵裂率、优胚率、妊娠率和OHSS发生率等指标。结果两组促性腺激素的用量及其用药天数、获卵数、受精率、卵裂率、着床率和妊娠率相比较均无显著差异（P〉0.05）。GnR Hantagonist组在hCG日激素水平低,与对照组比较其差异有统计学意义。结论行GnR Hantagonist方案IVF-ET助孕治疗与传统的GnR Hagonist短方案比较,其hCG日雌激素水平下降可能是OHSS发生率显著下降的主要因素;但卵泡的发育、卵母细胞的受精率、卵裂率及妊娠率和着床率均不受影响。GnR Hantagonist的使用为IVF-ET助孕药物提供了一种新的选择。     

GnRH类似物对大鼠回肠组织胰高血糖素释放的影响

目的 :研究促性腺激素释放激素 (GnRH)类似物 (阿拉瑞林 )对大鼠回肠L细胞释放胰高血糖素的影响。方法 :应用放射免疫分析法对体内和体外大鼠回肠进行观察。结果 :大鼠回肠灌注GnRH类似物后 ,血中及肠液中胰高血糖素的含量较对照组明显升高 ;体外孵育大鼠回肠组织后 ,在一定浓度范围内 ,孵育液中胰高血糖素含量随GnRH类似物浓度升高而升高 ;当浓度高于一定范围时 ,则随浓度升高而降低。GnRH类似物浓度为 1 .0× 1 0 - 4mol/L时孵育液中胰高血糖素含量是升高的。结论 :GnRH可能对大鼠回肠L细胞分泌胰高血糖素呈现双向调节作用。但是GnRH类似物为 1 .0× 1 0 - 4mol/L ,不论是体内还是体外 ,都可能对肠道分泌胰高血糖素表现促进作用     

GnRH在宫颈鳞状细胞癌中的表达及意义

目的:从蛋白质及核酸水平研究GnRH(促性腺激素释放激素)在宫颈鳞状细胞癌中的表达,以探讨其存在的临床意义。方法:采用免疫组化和原位杂交两种实验方法,分别从蛋白质和核酸水平检测45例宫颈鳞状细胞癌及20例慢性宫颈炎中GnRH的表达。结果:在20例慢性宫颈炎宫颈上皮组织中GnRH及GnRH mRNA表达均为阴性,而在45例宫颈鳞状细胞癌组织中,GnRH阳性表达率为80%,GnRH mRNA阳性表达率为64.44%,即无论是在蛋白质水平还是在核酸水平比较两者均显示差异有显著性;定性及定量比较GnRH及GnRH mRNA在不同临床期别的宫颈癌组织中的表达情况,结果为GnRH定性表达率Ⅰ期50%(6/12),Ⅱa期85.7%(18/21),Ⅱb-期100%(12/12);GnRH mRNA定性表达率,Ⅰb期33.3%(4/12),Ⅱa期71.4%(15/21),Ⅱb-期83.3%(10/12);GnRH及GnRH mRNA在定性上表达有显著差别(P<0.05)。结论:宫颈鳞状细胞癌组织存在较高的GnRH,其有可能参与宫颈鳞状细胞癌发生、发展,为进一步研究通过抑制GnRH的产生,减少宫颈癌的发生及治疗宫颈癌提供了一定的理论基础。     

脑室注射L-谷氨酸对成年大鼠下丘脑GnRH含量的影响

目的 :观察脑室注射L -谷氨酸 (L -Glu)对成年雄性Wistar大鼠下丘脑促性腺激素释放激素(GnRH)含量的影响。方法 :摘取下丘脑组织 ,匀浆化 ,用RIA法检测匀浆上清液中GnRH的含量。结果 :脑室分别注射 0 0 1176、0 1176、1 176 0 μg/ 2 0 μl-1 -1L -Glu后 40分钟 ,下丘脑GnRH含量依次为 1 5 9± 0 41、0 88± 0 34、0 70± 0 42ng/ 10mg湿重 ,均显著低于盐水对照组 (P <0 .0 1) ;脑室注射 0 1176 μg/ 2 0 μl-1·-1L -Glu后 2 0、40、12 0分钟 ,下丘脑GnRH含量依次为 0 99± 0 37、0 88± 0 34、1 2 6± 0 39ng/ 10mg湿重 ,亦均显著低于盐水对照组 (P <0 .0 1)。脑室注射L -Glu对下丘脑GnRH含量的降低作用呈现剂量与时间依从关系。而脑室注射3H -Glu 2 μCi/ 2 0 μl-1·-1后 40分钟发现 ,大脑、小脑、垂体、下丘脑内侧基底部 (MBH)和视前区 (POA) 5个不同部位脑组织中以MBH对3H -Glu的摄取量最大 (10 6 9 82± 490 33cpm/ 10mg湿重 )。结论 :L -Glu可能参与了大鼠下丘脑GnRH神经元功能活动的调节。     

人胎胰腺GnRH免疫反应细胞

目的：探讨促性腺激素释的激素(GnRH)免疫反应细胞在人胎胰腺的存在部位和数量变化。方法：用免疫组织化学SABC法,对37例第10-32w人胎胰腺内的GnRH-IR细胞进行观察,并用体视方法分析其数量变化。结果：人胎胰腺GnRH-IR细胞出现于第13w,其数密度随胎龄增加而增大;分布于胰岛及外分泌部的腺泡上皮、导管上皮细胞间。位于胰岛的GnRH-IR细胞呈圆形、卵圆形或多边形。位于腺泡上皮细胞间的GnRH-IR细胞多为锥体形,外分泌部的GnRH-IR细胞均为开放型细胞。结论：胰腺GnRH-IR细胞于胚胎第13w出现,广泛存在于内、外分泌部,其数量随胎龄增加而增加。     

左旋18-甲基炔诺酮对大鼠下丘脑GnRH神经元的影响

目的：观察孕激素类避孕药—左旋18-甲基炔诺酮(LNG)对大鼠下丘脑GnRH神经元的影响。方法：正常雌性大鼠分为长期给药组(灌服LNG90d)和停药组(停药后正常喂养20d)。免疫组织化学方法显示下丘脑GnRH阳性神经元。结果：长期给药组与对照组相比斜角带(db)、视前区(MPA)内GnRH阳性神经元构成比发生改变,棘型神经元减少,阳性神经元光密度减低,阳性纤维膨体密度降低,正中隆起(ME)处阳性纤维密度增加。停药后基本恢复正常。结论：从形态学上证实LNG作用于下丘脑水平;停药后GnRH阳性神经元形态学变化可恢复正常,表明LNG具有高效安全性。     

GnRH激动剂主动免疫母羊对生殖激素分泌的作用

目的:探讨GnRH激动剂(GnRHa)主动免疫对绵羊生殖激素合成与分泌的作用,并深入研究GnRH-A免疫调节动物生殖功能的机理。方法:42只5～6月龄母绵羊(Ovis aries)随机分为6组(n=7),EG-Ⅰ、EG-Ⅱ和EG-Ⅲ分别于0和14天皮下注射阿拉瑞林抗原200、300和400μg;EG-Ⅳ和EG-Ⅴ分别皮下注射阿拉瑞林抗原200、300、0、7、14和21天各一次,共4次;对照组在0和14天皮下注射抗原溶媒(除不用阿拉瑞林外,其余成分和制备方法与阿拉瑞林抗原相同)2.0 ml。无菌采集不同时段的血液,分离血清。以ELISA测定血清GnRH抗体浓度,用激素检测试剂盒(ELISA)分别测定血清GnRH、FSH、LH和E2浓度。结果:①阿拉瑞林首次免疫7天后,各实验组的抗体浓度逐渐升高,EG-Ⅰ、EG-Ⅱ和EG-Ⅲ分别在28、28和35天达到峰值(P<0.05),EG-IV和EG-V则在在45天达到峰值(P<0.01),至60天时仍明显高于对照组(P<0.05)。14～60天间EG-IV和EG-V抗体浓度均高于EG-Ⅰ、EG-Ⅱ和EG-Ⅲ(P<0.05)。②EG-Ⅰ、EG-Ⅱ的GnRH在21和28天抵谷值(P<0.05),EG-Ⅲ、EG-Ⅳ和EG-Ⅴ则在45天抵谷值(P<0.01),且以EG-Ⅴ为最低。谷值之后逐渐上升趋势,70天时达到免疫注射前水平。③实验组血清FSH浓度始终高于对照组(P<0.05)。EG-Ⅰ、EG-Ⅱ和EG-Ⅲ于28、28和35天达到峰值(P<0.05),而EG-IV和EG-V在60天达到高峰值(P<0.01)。④实验组绵羊血清LH呈下降趋势,EG-Ⅰ、EG-Ⅱ和EG-Ⅲ分别在21、21和28天达到谷值(P<0.01),EG-Ⅳ和EG-Ⅴ在35天达到谷值(P<0.01)。35天时EG-Ⅳ和EG-Ⅴ低于EG-Ⅰ、EG-Ⅱ和EG-Ⅲ。⑤各组的血清E2含量无显著差异。结论:GnRH激动剂(阿拉瑞林)抗原主动免疫可促进GnRH抗体的生成,抑制母羊GnRH和LH的合成与分泌,增强FSH的合成与分泌,且随着注射剂量和注射次数的增加,这种作用更加明显,而对血清E2无明显影响。     

GnRHa长方案促超排卵对种植窗ICAM-1表达的影响及其机制的探讨

目的研究Decapty1/HMG/HCG长方案促超排卵对子宫内膜ICAM-1表达的影响,分析ICAM-1变化与血清E2、P水平之间的相关性,探讨该方案高获卵率而低妊娠率的原因,为临床改善促排卵方案,提高IVF-ET的妊娠率提供理论依据。方法将小白鼠分为NC组和COH组,用免疫组化SP法观察两组小鼠pd1~pd6子宫内膜ICAM-1蛋白的表达。用罗氏电化学发光法检测两组小鼠pd1~pd6血清E2、P的水平。结果NC组中ICAM-1于pd4表达最明显（P（0.01）,COH组中ICAM-1于pd3表达最明显（P（0.01）。ICAM-1蛋白的表达高峰和血清高峰点P/E2呈正相关关系（r=0.756,P（0.01）。结论Decapty1/HMG/HCG可能通过改变血清E2、P水平,使子宫内膜种植窗开放异常,导致子宫内膜发育与胚胎发育不同步,降低子宫内膜接受性,这可能是影响IVF-ET妊娠率的重要原因。     

Kisspeptin-10 elicits triphasic cytosolic calcium responses in immortalized GT1-7 GnRH neurones

Kisspeptins, which are alternatively called as metastin since they were originally identified as products of metastasis suppressor gene KiSS-1, are the natural ligands for the G protein-coupled receptor 54 (GPR54). Kisspeptins are the most potent activators of hypothalamic-pituitary-gonadal (HPG) axis reported to date. The pulsatile pattern of GnRH release, which results in the intermittent release of gonadotropic hormones from the pituitary, has a critical importance for reproductive function but the factors responsible from this release pattern are not known. Therefore, the pattern of kisspeptin-induced intracellular signaling and the role of PKC in the intracellular signaling cascade were investigated by fluorescence calcium imaging using the immortalized GnRH-secreting GT1-7 hypothalamic neurons. Kisspeptin-10 caused a triphasic change characterized by an initial small increase followed by a significant decrease and increase in intracellular free calcium concentrations ([Ca(2+)](i)). The changes in [Ca(2+)](i) were significantly attenuated by pre-treatment with protein kinase C inhibitor. The compatibility of appeared mirrored-patterns of kisspeptin-10-induced changes in [Ca(2+)](i) concentrations in these neurons and GnRH secretion confirm the importance of intracellular calcium flux downstream from GPR54 through PKC signaling pathway.     

Heme oxygenase expression and activity in immortalized hypothalamic neurons GT1-7

Heme oxygenase (HO), the main enzyme deputed to heme metabolism, has been identified as two main isoforms called HO-1 and HO-2. HO-1 is inducible and plays a main role in the cellular oxidant/antioxidant balance whereas HO-2 is constitutive and involved in the physiological metabolism of heme. However, it is noteworthy to mention that HO contribute to the regulation of the hypothalamic release of neuropeptides such as corticotrophin-releasing hormone and arginine-vasopressin and could modulate the pulsatile release of gonadotropin releasing hormone (GnRH). GT1-7 cells are immortalized hypothalamic neurons and a valuable tool to evaluate hypothalamic neuroendocrine control of reproduction. The aim of this work was to investigate and characterize the presence of HO isoforms in the GT1-7 hypothalamic neurons. Hemin, a well-known inducer of HO-1, significantly increased HO activity, whereas dexamethasone did not modify HO-2 activity. Moreover, hemin and DEX, in combination, did not have any additive effect on HO activity in GT1-7 neurons. Furthermore, basal HO-1 immunoreactivity identified in GT1-7 cells, was significantly up-regulated by hemin. Conversely, no HO-2 immunoreactivity was detected. Taken together, these results suggest the presence of functional HO-1 in GT1-7 immortalized hypothalamic neurons and open new avenues about the use of this cell line for the study of HO modulation of GnRH secretion and reproduction.     

全反式维甲酸对人乳腺癌MCF-7细胞凋亡的影响

目的:探讨全反式维甲酸(ATRA)对人乳腺癌MCF-7细胞凋亡的影响及作用途径.方法:在人乳腺癌细胞株MCF-7培养基中加入一定浓度ATRA和PKC-δ的专一抑制剂rottlerin(RO)并分组,通过SubG1assay by FACS、琼脂糖凝胶电泳检测基因组DNA ladder来观察ATRA对MCF-7的影响.结果:在浓度为5μM的ATRA作用下,MCF-7的凋亡率显著高于其它各组(P＜0.01),并可观察到明显梯状DNA.结论:ATRA能够诱导乳腺癌MCF-7细胞凋亡,但受PKC-δ的专一抑制剂RO的抑制.     

血管紧张素-(1-7)与血管紧张素Ⅱ对THP-1 巨噬细胞NPC1表达及胆固醇流出的影响

目的: 研究血管紧张素-(1-7) 与血管紧张素Ⅱ(AngⅡ)对C型1类尼曼-匹克蛋白(NPC1)表达及胆固醇流出率的影响,探讨Ang-(1-7)对AngⅡ在胆固醇逆转运方面的拮抗作用。方法: 体外培养的人THP-1单核细胞经佛波酯(PMA)诱导48 h,使之分化为巨噬细胞,随机分为对照组、AngⅡ组、Ang-(1-7)组、AngⅡ+Ang-(1-7)组和AngⅡ+ Ang-(1-7) + Ang-(1-7)受体阻断剂A-779组。运用逆转录-多聚酶链反应(RT-PCR)和Western blotting蛋白印记技术分别检测NPC1 mRNA与蛋白的表达水平,应用液体闪烁计数仪检测胆固醇流出的变化。结果: 与对照组相比,AngⅡ能引起THP-1源性巨噬细胞NPC1 mRNA与蛋白质表达的下调及胆固醇流出的减少( P< 0.05);Ang-(1-7)使NPC1蛋白和mRNA表达升高,胆固醇流出增多(P<0.05) ;混合刺激组中, 不同浓度的Ang-(1-7) ( 100-10 000 nmol/L)呈剂量依赖性地减轻AngⅡ对THP-1源性巨噬细胞 NPC1蛋白和mRNA表达的抑制作用,促进细胞胆固醇流出,与AngⅡ组相比差异显著(P<0.05) ;加入A-779 组与AngⅡ组比较无显著差异(P>0.05)。结论: Ang-(1-7) 通过其特异性受体Mas拮抗AngⅡ抑制的THP-1巨噬细胞NPC1的表达,并呈浓度依赖性,进而促进巨噬细胞内胆固醇流出,抑制动脉粥样硬化的发生发展。     

Immunohistochemical expression of gonadotropin releasing hormone receptor in human breast carcinoma

Gonadotropin releasing hormone (GnRH) analogs can cause regression of hormone-dependent breast carcinomas via the specific GnRH receptor (GnRH-R). In an attempt to obtain a better understanding of GnRH actions in human breast carcinoma, the expression of GnRH-R was examined immunohistochemically in 58 invasive ductal carcinomas and correlated with various clinicopathological parameters. GnRH-R was immunolocalized in the cytoplasm of carcinoma cells in 37 of 58 invasive ductal carcinoma cases (64%). Immunoreactivity for GnRH-R was also detected focally in the cytoplasm of morphologically normal glandular epithelia adjacent to the carcinoma. A significant correlation was observed between the immunohistochemical expression of GnRH-R and estrogen receptor labeling index (LI; P = 0.030) or progesterone receptor LI (P = 0.0074). There was a significant inverse correlation between GnRH-R immunoreactivity and Ki-67 LI (P = 0.012). No significant correlations were detected between GnRH-R and other clinicopathological parameters, including patient age, menopausal status, stage, tumor size, lymph node status, histological grade and prognosis. This study indicates that GnRH-R is widely distributed in human breast carcinoma cells and regulates GnRH actions locally. Breast carcinomas positive for GnRH-R maintain some hormonal regulatory mechanisms, and GnRH actions may lead to a low proliferative rate in human breast carcinoma.     

Expression and function of B7-1 (CD80) and B7-2 (CD86) on human epidermal Langerhans cells

In addition to T cell receptor triggering, activation of T cells requires co-stimulatory signals that have been shown to be mainly initiated through CD28. We analyzed the expression and function of the two ligands for CD28, B7-1 (CD80) and B7-2 (CD86), on human Langerhans cells (LC), the antigen-presenting cells from epidermis. Human LC freshly isolated from epidermis (fLC) expressed significant level of B7-2, which was increased upon a short culture in vitro. In contrast, B7-1 was undetectable on fLC but appeared at the cell surface after a 3-day culture in vitro. Pre-incubation of 18-h cultured LC with anti-B7-2 monoclonal antibodies (mAb) was sufficient to abrogate the binding of CTLA4-Ig fusion protein, while a combination of both mAb against B7-1 and B7-2 was necessary to obtain a complete inhibition of CTLA4-Ig binding on 3-day cultured LC, showing the absence of a third CTLA4 ligand. The function of B7-1 and B7-2 on human LC has been analyzed by adding mAb at the beginning of mixed epidermal cell lymphocyte reactions. Anti-B7-2 mAb and CTLA4-Ig, but not anti-B7-1 mAb, strongly inhibited allogeneic, as well as recall antigen-induced T cell proliferation supported by fLC or 3-day cultured LC. Collectively, these results demonstrate that B7-2 is the major ligand for CD28/CTLA4 at the LC surface and that it plays a crucial role in human LC co-stimulatory function with little, if any, dependence on B7-1 expression.     

不同剂量培哚普利对缺血性心功能障碍家兔心功能及ACE2/Ang-(1-9)/Ang-(1-7)轴的影响

 目的: 探讨不同剂量培哚普利对缺血性心功能障碍家兔心功能的影响。方法: 采用结扎冠状动脉前降支的方法制作缺血性心功能障碍家兔模型。利用随机数字表法将30只家兔随机分为培哚普利大、小剂量组和心功能障碍组。大、小剂量组分别给予培哚普利生理盐水溶液(浓度分别为1 g/L、0.33 g/L)2 mL·kg^-1·d^-1灌胃;心功能障碍组给予等量生理盐水灌胃。4周后心脏超声测定心功能;real-time PCR检测血管紧张素转换酶2(angiotensin-converting enzyme 2,ACE2)和血管紧张素2型受体(angiotensin type 2 receptor,AT2R) mRNA表达;ELISA检测家兔血清血管紧张素(angiotensin,Ang)-(1-9)和Ang-(1-7)水平。结果: 与心功能障碍组相比,不同剂量培哚普利均可改善心功能(P<0.01),大剂量培哚普利比小剂量培哚普利改善心功能的效果显著(P<0.05);心功能障碍家兔应用培哚普利后,血清Ang-(1-9)和Ang-(1-7)水平均增高(P<0.01),ACE2和AT2R mRNA表达均增加(P<0.01);与小剂量组相比,大剂量组心肌ACE2和AT2R mRNA表达均增高(P<0.01),血清Ang-(1-9)水平增高(P<0.05),血清Ang-(1-7)水平无明显增加。相关性分析发现,左室射血分数与血清Ang-(1-9)、ACE2及AT2R水平呈正相关关系(P<0.01),与血清Ang-(1-7)水平无相关关系。结论: 大剂量培哚普利较小剂量培哚普利可以更有效改善缺血性心功能障碍家兔心功能,其心功能的改善可能与Ang-(1-9)水平增多,引起AT2R活化相关。