维甲酸、地塞米松和生长激素释放激素联合诱导生长激素细胞分化

目的探讨维甲酸(RA)、地塞米松(Dex)和生长激素释放激素(GHRH)在诱导生长激素(GH)细胞分化中的作用。方法原代培养大鼠胚胎垂体细胞,将RA(10-6mol/L)、Dex(50 nmol/L)和GHRH(10-7mol/L)单独或联合应用6 d,诱导大鼠胚胎垂体细胞分化,利用免疫组化检测GH阳性细胞百分比,放射免疫分析检测培养液的上清液中GH含量。结果与对照组相比,单独应用RA显著增加GH阳性细胞的百分比和GH的含量(P<0.05),而单独应用Dex和GHRH均无此作用(P>0.05);RA诱导4 d后,再加入Dex诱导2 d,其诱导效应较单独应用RA明显增强(P<0.05),而加入GHRH则不起作用(P>0.05);RA和Dex联合诱导4 d后,再加入GHRH诱导2 d,其诱导效应较RA和Dex联合诱导明显增强(P<0.01)。结论RA能够促进GH细胞的早期分化,Dex和GHRH则不能;RA能够与Dex、GHRH发挥协同效应,诱导GH细胞分化。     

地塞米松体外诱导大鼠胚胎垂体生长激素细胞的实验研究

目的探讨糖皮质激素和生长激素释放激素(GHRH)对大鼠胚胎垂体生长激素(GH)细胞分化的作用。方法利用免疫组化、放射免疫分析、RT-PCR和免疫电镜等方法,研究糖皮质激素体外诱导GH细胞分化的最佳浓度、所需要的最短时间及GHRH在大鼠胚胎垂体细胞分化中的作用。结果在体外培养的大鼠胚胎垂体细胞中,地塞米松能提高GH mRNA的表达水平,增加GH细胞内分泌颗粒的积聚。当GHRH浓度达到1 ×10-7 mol/L 时,可以增强地塞米松对GH细胞的诱导分化作用。结论地塞米松能够促进GH细胞的体外分化,并具有一定的剂量依赖性。GHRH与地塞米松具有协同效应,共同促进GH细胞的分化与分泌。     

蛋白激酶Cσ介导GHRP-6诱导的大鼠生长激素腺瘤GH3细胞CREB磷酸化

目的探讨生长激素释放肽-6(GHRP-6)对大鼠生长激素腺瘤细胞GH3细胞cAMP反应元件结合蛋白(CREB)磷酸化水平的影响及其细胞内信号传导机制。方法用GHRP-6处理大鼠生长激素腺瘤GH3细胞并检测其对GH3细胞CREB磷酸化水平的影响;初步筛选参与GHRP-6激活的细胞内信号通路的蛋白激酶C(PKC)亚型;然后应用RNA干扰技术确认发挥中介作用的PKC亚型;Western免疫印迹法检测蛋白质磷酸化水平。结果 GHRP-6诱导的CREB磷酸化呈时间依赖性;PKCσ特殊抑制剂楸毒素可以明显减弱GHRP-6的这种效应;PKCσ表达沉默后GHRP-6诱导的CREB磷酸化水平明显降低;并且GHRP-6可以诱导PKCσ磷酸化。结论 PKC,特别是PKCσ,介导了GHRP-6诱导的CREB磷酸化。此结果为了解垂体生长激素腺瘤细胞内信号通路间"交叉通讯"提供了新的依据。     

不同浓度维甲酸诱导体外培养人羊膜上皮细胞分化为神经样细胞的研究

目的探讨维甲酸诱导人羊膜上皮细胞向神经样细胞分化。方法以DMEM/F12培养基培养原代人羊膜上皮细胞,并将细胞分为5组:对照组;1×10-8mol·L-1维甲酸组;1×10-7mol·L-1维甲酸组;1×10-6mol·L-1维甲酸组;1×10-5mol·L-1维甲酸组。各维甲酸组经维甲酸处理7 d后,用免疫组化检测神经干细胞巢蛋白(nestin)、微管相关蛋白2(MAP-2)、神经胶质纤维酸性蛋白(GFAP)和细胞全能性标记物Oct-4表达,并计数阳性细胞比例。结果与对照组比较,各维甲酸组显著提高人羊膜上皮细胞分化为神经样细胞的比例,其中1×10-6mol·L-1维甲酸组阳性细胞比例最高;结论维甲酸体外能诱导人羊膜上皮细胞向神经样细胞分化,最适诱导浓度为1×10-6mol·L-1。     

α-干扰素和溴隐亭对垂体生长激素腺瘤细胞的作用

目的研究α-干扰素(IFN-α)和溴隐亭(BC)对人垂体腺瘤细胞激素分泌的影响。方法将垂体生长激素(GH)腺瘤(11例)进行体外细胞培养,每例培养细胞随机分成IFN-α组、BC组、IFN-α BC组及对照组,每组3管,观测药物干预后细胞激素分泌量。结果干预2d和4d时,IFN-α对6/10例和7/10例垂体腺瘤细胞GH分泌有明显抑制,与对照组比较GH分泌量减少22%～54%(P<0.05或P<0.01)和30%～61%(P<0.01),其中,4/6例和5/6例侵袭性垂体腺瘤细胞GH分泌受到明显抑制,与对照组比较GH分泌量减少22%～38%(P<0.05或P<0.01)和30%～55%(P<0.01);BC对5/10例垂体腺瘤细胞GH分泌无明显抑制作用,而IFN-α对其中4例有明显抑制GH分泌效应,与对照组比较GH分泌量减少22%～54%(P<0.05或P<0.01)和30%～61%(P<0.01)。结论IFN-α对大多数垂体GH腺瘤,尤其是侵袭性垂体腺瘤细胞GH分泌有抑制作用,对大多数BC耐药的垂体腺瘤细胞GH分泌亦有抑制效应。     

维甲酸诱导胚胎大鼠脑海马神经干细胞向神经元的分化

背景：目前认为直接进行神经干细胞移植后细胞虽能存活,但是只分化成神经胶质细胞,并不能分化成有功能的神经元。 目的：探讨维甲酸诱导对胚胎大鼠脑海马神经干细胞向神经元分化的作用。 设计、时间及地点：细胞学体外实验,于2008-09/2009-02在辽宁医学院科技实验楼完成。 材料：胚龄13.5 d的SD大鼠由辽宁医学院实验动物中心提供。 方法：分离胎鼠脑海马组织,胰蛋白酶消化法体外培养获得神经干细胞。将原代和传代细胞以1×107 L-1接种到培养孔中,分别进行常规贴壁分化培养和维甲酸诱导分化培养。 主要观察指标：神经干细胞的鉴定,光镜及免疫组化检测神经干细胞诱导分化结果。 结果：免疫组织化学检测结果显示,原代和传代后得到的神经干细胞团均呈巢蛋白阳性。常规贴壁分化培养7 d后,神经元多呈椭圆型和近似三角形,胞体大,细胞边缘清楚,胞体上有多个突起;而维甲酸诱导分化培养后,神经元数量增加,形态清楚,但细胞胞体上突起较少。与常规贴壁分化培养比较,维甲酸诱导分化培养后神经干细胞向神经元分化率明显升高(P < 0.01),神经干细胞向神经胶质细胞分化率明显降低(P < 0.01)。 结论：从大鼠胚胎脑海马组织中分离得到可自我复制和多向分化的神经干细胞,维甲酸体外诱导后可以增加其向神经元方向分化的比例。     

曲格列酮减少CyclinD1的表达抑制体外培养GH3细胞增殖的实验研究

目的 探讨过氧化物酶体增殖激活受体-γ(PPAR-γ)高亲和力配体-噻唑烷二酮类药物曲格列酮对大鼠垂体腺瘤GH3细胞系增殖的影响.并初步探讨其作用机制. 方法 不同浓度的曲格列酮作用于GH3细胞,用MTT法检测各组GH3细胞生长情况,用流式细胞技术检测各组GH3细胞周期的变化,用半定量RT-PCR方法 检测各组GH3细胞CyclinD1基因mRNA的表达.结果 曲格列酮干预GH3细胞72 h后.以浓度效应关系抑制GH3细胞增殖,并使GH3细胞明显被阻滞于G1/S检测点,CyclinD1 mRNA表达明显减少,与对照组比较差异有统计学意义(P<0.05). 结论 曲格列酮能明显抑制大鼠垂体腺瘤细胞的增殖,其分子机制可能是其与PPAR-γ结合后导致CyclinD1 mRNA表达减少,从而抑制了细胞增殖,促进肿瘤细胞死亡.     

α-干扰素和奥曲肽对垂体生长激素腺瘤的作用

目的　研究α 干扰素 (IFN α)和奥曲肽 (SMS)对人垂体腺瘤细胞激素分泌的影响。方法　将垂体生长激素 (GH)腺瘤 (11例 )标本进行体外细胞培养 ,每例培养细胞随机分成IFN α组、SMS组、IFN α +SMS组及对照组 ,每组 3管 ,观测药物干预后细胞激素分泌量的变化。结果　干预 2天和 4天时 ,IFN α对 6 0 % (6 /10例 )和 70 % (7/10例 )垂体腺瘤细胞GH分泌有明显抑制 ,与对照组比较GH分泌量减少 2 2 %～ 5 4 %和 30 %～ 6 1% (P <0 .0 5和P <0 .0 1) ,其中 ,6 7% (4/6例 )和 83% (5 /6例 )侵袭性垂体腺瘤细胞GH分泌受到明显抑制 ,与对照组比较GH分泌量减少 2 2 %～ 38%和 30 %～ 5 5 % (P <0 .0 5和P <0 .0 1) ;SMS对 4 0 % (4/10例 )垂体腺瘤细胞GH分泌无明显抑制作用 ,而IFN α对其中 3例有明显抑制GH分泌效应 ,与对照组比较GH分泌量减少 38%～ 5 4 %和 4 3%～ 6 1% (均P <0 .0 1)。结论　IFN α对大多数垂体GH腺瘤 ,尤其是侵袭性垂体腺瘤激素分泌有抑制作用 ,对大多数SMS耐药的垂体腺瘤细胞GH分泌亦有抑制效应     

腺病毒介导D2S基因联合溴隐亭治疗对GH3细胞生长的抑制作用

目的 探讨腺病毒介导多巴胺2型受体短链亚型基因(D2S)联合溴隐亭对大鼠垂体生长激素腺瘤GH3细胞株体外生长活性的影响.方法 应用携带人D2S基因及增强型绿色荧光蛋白(EGFP)报告基因的复制缺陷型腺病毒载体pAd - D2S - EGFP感染大鼠GH3细胞,RT - PCR、Western - blot法检测转染后细胞D2S mRNA及蛋白的表达,并采用CCK -8法检测转染D2S基因后联合溴隐亭药物干预治疗,对GH3细胞生长抑制及诱导凋亡作用,运用光镜及荧光显微镜检测联合作用后细胞形态变化.结果 GH3细胞转染D2S基因后检测到D2S基因的转录及蛋白表达较对照组增高.腺病毒转染D2S基因联合溴隐亭干预后第2天,体外培养的GH3细胞存活率明显下降(32.76±3.88)％,与单纯加药组的(101.78±2.05)％及空载体组的(91.79±3.80)％相比,差异有统计学意义(P＜0.01).光镜下及荧光显微镜结合Hoechst核染色观察到细胞形态出现皱缩脱落、死亡、核碎片等改变.结论 腺病毒介导D2S基因联合溴隐亭能有效抑制GH3细胞的增殖.     

Wnt-1基因在神经干细胞向神经元分化过程中的表达

目的研究Wnt-1基因在全反式维甲酸(ATRA)诱导人胚胎神经干细胞分化为神经元过程中的表达及其作用。方法用1μmol/L的维甲酸诱导人胚胎神经干细胞,诱导7d后,做NSE免疫荧光染色,计数分化为神经元的比例。于ATRA诱导前、诱导3d和诱导7d,提取细胞的总RNA。用半定量RT-PCR法检测Wnt-1基因的表达情况。结果与对照组相比,全反式维甲酸能显著提高人胚胎神经干细胞分化为神经元的比例(P<0.01)。Wnt-1基因在维甲酸诱导后明显上调(P<0.001)。结论Wnt-1基因在全反式维甲酸诱导人胚胎神经干细胞分化为神经元的过程中起正调控作用,Wnt-1基因与神经元的分化关系密切。     

Regulational effect of ghrelin on growth hormone secretion from perifused rat anterior pituitary cells

Ghrelin, a novel growth hormone (GH)-releasing peptide, was recently isolated from the rat stomach as an endogenous ligand to growth hormone secretagogue receptor (GHS-R). Ghrelin specifically stimulates the release of GH from the rat anterior pituitary gland, but the regulational effect of ghrelin on GH secretion has not yet been clarified. We used a perifusion system to examine the single effect and combined effects of ghrelin with growth hormone-releasing hormone (GHRH) and somatostatin on GH secretion from rat anterior pituitary cells. The increase in GH concentration due to ghrelin stimulation showed a transitory peak that was almost the same as that previously reported for GHS, but apparently distinct from that of GHRH. Ghrelin (10(-10) M to 10(-8) M) stimulated GH secretion from the rat anterior pituitary cells in a dose-dependent manner. Serial ghrelin stimulation of the dispersed cells at 1-h intervals decreased the GH response, but the response recovered with stimulation at 3-h intervals, indicating that ghrelin strongly desensitized cells. Costimulation with ghrelin and GHRH elicited neither a synergistic nor an additive GH response from the rat pituitary cells. Furthermore, pretreatment to anterior pituitary cells with somatostatin strongly abolished ghrelin- and/or GHRH-stimulated GH secretion. In this study, we demonstrated that ghrelin caused weaker GH secretion than that caused by GHRH, and we also showed that costimulation with GHRH had no additive or synergistic effect on GH secretion, suggesting that ghrelin indirectly affects coordinated GH release from pituitary gland, as found in vivo.     

Retinal growth hormone in perinatal and adult rats

Growth hormone (GH) mRNA and protein have recently been localized in the neural retina, of embryonic chicks, in which exogenous GH promotes cell survival. GH is also expressed in the rat CNS, in which it has neuroprotective roles, although its presence in the rat neural retina is unknown and is the focus of the present study. GH immunoreactivity, to a 22-kDa protein, was present in extracts of fetal (embryonic day [ED]17) eyes and in extracts from the neural retinas of newborn pups, comparable to GH immunoreactivity in pituitary extracts. The GH immunoreactivity in the neural retina was widespread but was most intense in large rounded cells in the retinal ganglion cell (RGC) layer and in the optic fiber layer derived from the axons of the RGCs. A 693-bp cDNA was also generated by the RT-PCR of RNA extracted from the eyes of ED17 rats and from the neural retinas and eyes of newborn rats, when amplified in the presence of oligonucleotide primers for the rat GH cDNA. Expression of the GH gene in the neural retina was also shown by specific in situ hybridization of an antisense GH riboprobe to cells in the neural retina, particularly those in the RGC layers of fetal and adult rat eyes. These results demonstrate GH expression in the neural retinas of fetal, newborn, and adult rats, in which retinal GH might have neuroprotective roles.     

Effects of retinoic acid on NB 69 human neuroblastoma cells and fetal rat mid brain neurons

Summary Retinoids are chemical compounds which play important roles in ontogenetic development and cranio-caudal differentiation in animals, but their effect on phenotypic expression of neurotransmitters are unknown. We studied the pharmacological and morphological effects of retinoic acid (RA) on two types of immature vertebrate neurons, the human derived neuroblastoma cells, NB69, and fetal rat mid brain neurons in culture. The pharmacological effects of RA on the cultures and their relation to catecholamine and acetylcholine neurotransmission were evaluated according the levels of catecholamines, tyrosine hydroxylase (TH) activity, TH immunostaining, and choline acetyltransferase (CAT) activity, respectively. RA reduces catecholamine levels and TH activity in NB69 cells and the number of dopamine neurons in cultures derived from rat fetal mid brain. The detrimental effect of RA on mid brain neurons is dose- dependent; limited to TH+ cells at low concentrations (100 to 500 nM) and toxic for all types of cells at high concentrations (1 to 2 M). RA increases CAT activity in NB 69 cells and produces phenotypic differentiation of these to a more mature neuronal phenotype with more prolonged neurite extensions. Therefore, RA may play a trophic positive role in the differentiation of immature cells to cholinergic neurons; this contrasts with the detrimental effects of RA on catecholamine neurons.     

A novel hypothalamic-dispersed pituitary co-perifusion model for the study of growth hormone secretion.

This study presents a novel, in vitro, hypothalamic-dispersed pituitary co-perifusion system (HPPS) developed to examine the influence of the hypothalamus on pituitary growth hormone (GH) secretion in a controlled environment. In this perifusion system, dispersed rat pituitary cells were loaded onto Biogel P-2 (P-2) beads in a 0.5-ml plexiglas chamber and were submerged in a 37 degrees C water bath. After stabilization, two hypothalami were placed into each chamber on a thin layer of P-2 beads and the chamber was re-equilibrated. To test the system, pituitary cells were stimulated either directly with growth hormone-releasing factor (GRF) or indirectly via the hypothalamus, with clonidine, an alpha 2-adrenergic (alpha 2) receptor agonist. Perifusion of HPPS or pituitary cells with GRF (40 ng/ml) induced a substantial endogenous GH surge. Clonidine (2 x 10(-8) M) treatment stimulated a GH surge in HPPS chambers, but not in chambers containing only pituitary cells. Thus, somatotrophs respond to hypothalamic factors released in response to clonidine and not directly to alpha 2 stimulation. To determine if the components involved in GH feedback are present in the perifusion system, HPPS chambers were sequentially perifused with hGH, clonidine, and GRF. hGH pretreatment suppressed the clonidine but not the GRF-induced GH surge(s) observed in chambers perifused with clonidine and GRF only. In chambers only containing pituitary cells, GH was only increased in response to GRF when sequentially perifused with all three substances. This study demonstrates the dynamic interaction between the hypothalamus and pituitary in the regulation of GH secretion in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

转录因子YY1对垂体瘤侵袭性的影响

目的观察转录因子YY1(Yin-Yang 1)在垂体腺瘤中的表达情况,并进一步分析沉默YY1对大鼠垂体瘤GH3细胞侵袭性的影响。方法运用免疫组化技术观察YY1在不同类型垂体腺瘤组织中的表达情况。Western blot实验检测siRNA沉默大鼠垂体瘤GH3细胞中的基因YY1后,YY1蛋白及基质金属蛋白酶MMP-2、MMP-9的表达水平。采用Transwell chamber细胞侵袭实验观察沉默YY1对GH3细胞侵袭的影响。结果 YY1在侵袭性垂体腺瘤中表达升高;沉默YY1后,GH3细胞侵袭能力下降35.7%(P0.05),MMP-2、MMP-9蛋白表达量下降(P0.05)。结论转录因子YY1在侵袭性垂体腺瘤中的表达升高,并通过上调MMP-2、MMP-9的表达来促进GH3细胞侵袭性。     

Direct pituitary effects of kisspeptin: activation of gonadotrophs and somatotrophs and stimulation of luteinising hormone and growth hormone secretion

Recent, compelling evidence indicates that kisspeptins, the products of KiSS-1 gene, and their receptor GPR54, represent key elements in the neuroendocrine control of reproduction, and that they act primarily by regulating gonadotrophin-releasing hormone (GnRH) secretion at the hypothalamus. Conversely, and despite earlier reports showing GPR54 expression in the pituitary, the potential physiological roles of kisspeptins at this gland have remained elusive. To clarify this issue, cultures of rat pituitary cells were used to evaluate expression of KiSS-1 and GPR54, and to monitor the ability of kisspeptin-10 to stimulate Ca(2+) responses in gonadotrophs and to elicit luteinising hormone (LH) secretion in vitro. The results obtained show that both GPR54 and KiSS-1 are expressed in the pituitary of peripubertal male and female rats. Moreover, kisspeptin-10 induced a rise in free cytosolic Ca(2+) concentration ([Ca(2+)](i)) in approximately 10% of male rat pituitary cells. Intriguingly, kisspeptin-responsive cells included not only gonadotrophs, in which a 62.8 +/- 16.0%[Ca(2+)](i) rise was observed, but also somatotrophs, wherein kisspeptin induced a 60.3 +/- 5.5%[Ca(2+)](i) increase. Accordingly, challenge of dispersed pituitary cells with increasing kisspeptin-10 concentrations induced dose-related LH and growth hormone (GH) secretory responses, which were nevertheless of lower magnitude than those evoked by the primary regulators GnRH and GH-releasing hormone, respectively. In particular, 10(-8) M kisspeptin caused maximal increases in LH release (218.7 +/- 23.6% and 180.4 +/- 7.2% in male and female rat pituitary cells, respectively), and also stimulated maximally GH secretion (181.9 +/- 14.9% and 260.2 +/- 15.9% in male and female rat pituitary cells, respectively). Additionally, moderate summation of kisspeptin- and GnRH-induced LH responses was observed after short-term incubation of male rat pituitary cells. In conclusion, our results provide unequivocal evidence that kisspeptins exert direct pituitary effects in peripubertal male and female rats and suggest a possible autocrine/paracrine mode of action. The precise relevance and underlying mechanisms of this potential new actions of kisspeptins (i.e. the direct modulation of gonadotrophic and somatotrophic axis at the pituitary) deserve further analysis.     

Growth hormone localization in the neural retina and retinal pigmented epithelium of embryonic chicks

It is well-established that growth hormone (GH) is present in the brain, spinal cord, and peripheral nerves of embryonic chicks, prior to the differentiation of pituitary somatotrophs, but its presence and distribution in retinal tissues is controversial. The possible presence of GH and GH mRNA in retinal tissues of early embryos has therefore been further evaluated. A 466-bp fragment of the pituitary GH cDNA, derived from a portion of exon 3 and spanning exons 4 and 5, was amplified by RT-PCR from reverse-transcribed mRNA from the pituitary glands of juvenile chicks and from the whole eye, neural retina, and retinal pigmented epithelium (RPE) of embryonic-day (ED) 9 chick embryos. In ED 9 embryos, GH immunoreactivity was demonstrated in the choroid and neural retina, in which it was particularly abundant in a layer of cells with the location and morphological appearance of retinal ganglion cells. GH immunoreactivity was also present in tissue sections of the RPE that were bleached to remove the melanin pigment. The intense GH staining in the RPE of ED 9 embryos was also revealed using a fluorescein-labeled GH antibody and confocal microscopy. At the ultrastructural level, GH detected by immunogold electron microscopy was present in the cytoplasm of RPE and neural retinal cells of ED 9 embryos. Although not associated with secretory granules, GH in the RPE was particularly associated with the membranes of the melanin granules. These results demonstrate that the neural retina and RPE are extrapituitary sites of GH production in early chick embryos, prior to the differentiation of the pituitary gland.