细胞因子对GH3细胞中人生长激素基因表达的影响

为了研究细胞因子IL 11、睫状神经营养因子 (CNTF)和转化生长因子 (TGF β)对大鼠垂体GH3 细胞中人生长激素 (hGH)的基因启动子活性的影响及其与垂体特异性转录因子Pit 1蛋白的关系 ,首先建立含hGH基因启动子 (- 4 84～ 30bp)和荧光素酶融合基因的稳定转化GH3 细胞系 ,然后用细胞因子刺激 ,检测细胞培养液和细胞裂解液中GH的含量 ,反映它们对GH分泌和合成的影响 ;检测GH3 细胞内荧光素酶的变化 ,说明细胞因子对hGH基因启动子活性的作用。将Pit 1蛋白表达质粒 (pcDNA pit 1 cDNA)单独转染或与Pit 1反义寡核苷酸 (Pit 1OND)共转染于稳定转化的GH3 细胞中 ,观察加入细胞因子后荧光素酶的变化 ,探讨细胞因子的作用与Pit 1蛋白的关系。结果表明 ,IL 11(2 0nmol/L)、CNTF(10nmol/L)能刺激大鼠垂体GH3 细胞中GH的分泌和合成 ,增强GH3 细胞中荧光素酶的表达 ,分别增加到对照组的 12 6 %、136 %。TGF β(5nmol/L)能减少GH的分泌和合成 ,抑制荧光素酶的表达到对照组的 77%。Pit 1蛋白过表达和表达被抑制对细胞因子的调节作用没有影响。这说明IL 11、CNTF和TGF β可通过调节大鼠垂体GH3 细胞中hGH基因启动子活性影响GH的合成 ,Pit 1蛋白可能不参与这些调节作用。     

干扰素γ增强GH3细胞中人生长激素基因表达机制

为了研究干扰素γ(IFN γ)对大鼠垂体GH3细胞中人生长激素 (hGH)基因启动子活性的影响及其可能的作用机制 ,采用荧光素酶报告基因方法 ,将含hGH基因启动子 (- 4 84～ 2bp)和荧光素酶报告基因的表达质粒pGL3 4 84 Luc单独转染或与垂体特异性核转录因子Pit 1蛋白表达质粒 (pcDNA Pit 1 cDNA)或Pit 1反义寡核苷酸 (Pit 1OND)共转染于大鼠垂体GH3细胞中 ,观察加入IFN γ及细胞内信号转导途径的抑制剂后GH3细胞中荧光素酶表达的变化 ,反映其对hGH启动子活性的影响 ;将含不同长度hGH基因启动子序列的荧光素酶表达质粒pGL3 3 80 Luc(- 3 80～ 2bp)、pGL3 2 5 0 Luc(- 2 5 0～ 2bp)、pGL3 1 3 2 Luc(- 1 3 2～ 2bp)和 pGL3 6 6 Luc(- 6 6～2bp)分别转染GH3细胞 ,观察它们对IFN γ的反应 ,以寻找IFN γ影响hGH基因启动子活性的关键序列。结果表明 ,IFN γ (1 0 5u/L ,1 0 6u/L)均能促进大鼠垂体GH3细胞中荧光素酶的表达 ,最高达对照组的 1 3 1 % (P <0 .0 0 1 ) ;在胞内信号转导抑制剂中 ,只有丝裂原活化蛋白激酶 (MAPK)信号转导途径抑制剂PD980 5 9(4 0 μmol/L) ,能完全阻断IFN γ的促进作用 ;Pit 1蛋白过表达和表达被抑制对IFN γ的促进作用没有影响 ;含不同长度hGH基因启动子序列质粒中 ,只有 pGL3 3 8     

人生长激素基因逆转录病毒载体的构建及其对3T3和ES细胞的转化与表达

为了研究生长激素基因在异源细胞中的表达,构建了携带人生长激素基因（ｈＧＨ）的逆转录病毒载体ｐＩＮＳ－ＧＨ,并将其导入小鼠成纤维细胞３Ｔ３和小鼠胚胎干细胞（ＥＳ细胞）ＣＣＥ中。ｐＩＮＳ－ＧＨ转化３Ｔ３细胞,获得了高效表达的克隆,即用放射免疫法检测到克隆培养基中有大量的人生长激素蛋白富积,如ＧＨＳＮＣ２０,富积率高达３７８４ｎｇ／ｍｌ,而且外源基因的整合很稳定。不同的３Ｔ３转化克隆的表达率有显著的差异,Ｓｏｕｔｈｅｒｎ杂交的结果表明,这种差异与外源基因整合的拷贝数无关。ｐＩＮＳ－ＧＨ转化ＣＣＥ细胞没有获得明显表达的克隆,而且外源基因的整合也不稳定。转化的ＥＳ细胞克隆总ＤＮＡ的Ｓｏｕｔｈｅｒｎ杂交结果表明,ｈＧＨ基因的确整合到细胞基因组中。目前尚未发现明显的外源ＤＮＡ重排的迹象。     

瘦素对GH3细胞分泌和凋亡的影响

本文旨在探讨瘦素(leptin)对垂体瘤GH3细胞的生长激素(growth hormone,GH)分泌的作用及可能机制。我们观察了leptin对GH3细胞生长激素的分泌、细胞的增殖和凋亡的影响,结果显示:leptin(1、10和100 nmol/L)对GH3细胞的基础GH分泌有抑制作用(P<0.05),并存在剂量依赖效应。用10 nmol/L的leptin作用30 min、1和3 h对GH分泌无明显影响,而作用1、2和3 d则可抑制GH分泌(P<0.05)。应用噻唑蓝(MTT)比色分析法和流式细胞仪研究leptin对GH3细胞增殖和凋亡的影响,我们发现leptin对GH3细胞的增殖有抑制作用,并存在剂量依赖效应;同时leptin可减低GH3细胞的S期细胞比例,而G1期的细胞比例明显增加,进入2相和4相的凋亡细胞比例增加。上述结果表明,leptin可抑制GH3 细胞的基础GH分泌,其作用可能是通过抑制GH3细胞的DNA合成,促进GH3细胞的凋亡,从而影响GH的分泌。     

人胎垂体生长激素样免疫反应细胞的发育

为探讨人胎垂体生长激素细胞发育规律,本文用免疫细胞化学结合图像分析,研究１９例不同胎龄人胎垂体生长激素细胞的形态、大小、光密度变化。结果如下：①胎１６周已存在生长激素免疫反应细胞,随胎龄增加,细胞多排列成团或索,并由周边部向远侧部的中央延伸。②随胎龄增加,生长激素样免疫反应细胞数量逐渐增多,细胞逐渐增大,胞浆逐渐增多,核浆比值逐渐变小。至胎龄３２周,生长激素样免疫反应细胞以大型细胞为主,多为圆形或卵圆形。③生长激素样免疫反应细胞的光密度随胎龄增加而逐渐增高,２６周达高峰,以后呈下降趋势,但仍高于２４周的水平,结果提示：垂体生长激素细胞的发育是从幼稚到成熟的过程,生长激素细胞的功能在胎２４－２６周最旺盛。     

非小细胞性肺癌患者Runx3基因启动子区甲基化状态研究

目的:分析非小细胞性肺癌(NSCLC)中Runx3基因启动子区甲基化状态.方法:运用甲基化特异性PCR技术检测62例NSCLC组织和癌旁正常肺组织中Runx3基因启动子甲基化,并分析该基因启动子甲基化Runx3基因mRNA和蛋白表达的影响及其与临床特征之间的关系.结果:NSCLC组织中Runx3基因异常甲基化率(48.4%)显著高于癌旁正常肺组织中Runx3基因的异常甲基化率(17.7%,P＝0.000);发生完全或者不完全甲基化的NSCLC组织或者正常肺组织中Runx3基因mRNA和蛋白表达显著降低.Runx3基因启动子区高甲基化和肿瘤分化程度及临床分期有相关性(P＝0.041和0.009),而与NSCLC患者性别、年龄、有无吸烟史及肿瘤类型等临床特征无关(P=0.400,0.301,0.290和0.965).结论:Runx3基因启动子区异常甲基化是导致Runx3基因在NSCLC中表达下调的重要因素,有望成为NSCLC早期辅助诊断的分子标志物之一.     

EphA3基因启动子区域定位表达及活性分析

目的构建含有不同长度EphA3基因启动子片段的报告基因载体,研究其在293T细胞和MEF细胞中的转录活性。方法以Balb／C小鼠基因组DNA为模板,扩增不同长度的EphA3基因启动子片段,并克隆进入荧光素酶报告基因质粒pGL3-Basic真核表达载体内。酶切鉴定及基因测序无误后,将重组质粒和pRL—CMV内对照质粒共转染293T和MEF细胞,分析不同长度的OhA3基因启动子片段的转录活性。结果酶切和测序鉴定表明表达载体构建成功,EphA3基因的核心启动子区域位于-279bp～＋110bp之间,在293T细胞和MEF细胞中其转录活性相似。结论成功构建了荧光素报告基因重组质粒,并确定了BphA3基因的核心启动子区域。     

人生长激素基因在小鼠细胞中的调节表达

我们已经提过,在人生长激素基因周围是否存在着一些序列,它们使生长激素基因可以被糖皮质激素所诱导。用同转化法将含有人生长激素基因的重纽克隆引入鼠成纤维细胞染色体,在有糖皮质激素存在下,同转化体可被诱导出8到5倍的人生长激素mRNA,对人生长激素蛋白的分泌也有相似的诱导。诱导所需的DNA序列位于DNA5’端,有500个核苷酸。把该DNA6’端片段融合到一个非激素敏感基因的结构基因中,例如胸苷激酶,可使该基因对糖皮质激素的诱导产生反应。     

丝裂原活化蛋白激酶在人冠状动脉平滑肌细胞向三维纤维蛋白胶迁移中的作用

目的:观察三维纤维蛋白(Fb)胶对体外培养的人冠状动脉平滑肌细胞(HCASMC)的趋化作用及其信号转导机制.方法:采用胶原酶消化法培养HCASMC,倒置相差显微镜观察其向三维Fb胶中迁移的能力及ERK、p38、JNK信号通路对其迁移能力的影响.Western blot检测Fb对HCASMC p-ERK、p-p38和p-JNK表达的调控.结果:向Fb胶中迁移的HCASMC呈长梭型,细胞数量增加时形成环形管腔样结构.纤维蛋白原(Fg)浓度为0.8 g/L～6.4 g/L时,HCASMC向胶中迁移的数量呈浓度依赖性增加,并随培育时间的延长逐渐增多.用Western Blot分析显示Fb以时间依赖性方式诱导ERK、p38及JNK活化,三者的选择性抑制剂PD98059 50 μmol/L、SB20358010 μmol/L及SP600125 20 μmol/L可分别抑制其活化,但对HCASMC向胶中迁移的抑制能力不尽相同.PD9805950 μmol/L对HCASMC迁移无明显影响,而SB203580 10 μmol/L和SP600125 20 μmol/L均可抑制HCASMC向Fb胶迁移,且后者抑制作用更强.结论:Fb胶通过激活细胞JNK和p38(而不是ERK信号通路)促进HCASMC向Fb胶中迁移,这种机制可能在动脉粥样硬化血栓形成及再狭窄过程中发挥重要作用.     

Thyroid-stimulating hormone and growth hormone release alterations induced by mosquito larvae proteins on pituitary cells

Mosquito larvae crude extract has shown to modulate cell proliferation of different mouse epithelial as well as human mononuclear cell populations in vivo and in vitro. A soluble fraction of the extract, with a molecular weight ranging from 12 to 80 kD, also showed an inhibitory effect on the proliferation of mouse hepatocytes. This effect disappeared after heating the extract at 90 degrees C for 60 min, suggesting that some proteinaceous molecule is involved. We report the effect of dialysed extract (MW >12 kD) on the concentration of both thyroid-stimulating hormone (TSH) and growth hormone (GH) in an incubation medium of pituitary cells from normal and oestrogenised rats. Time- and dose-dependent response of both hormones resulted in increasing TSH levels. Concentrations of GH were lower in the treated than in control pituitary cells. The time elapsed until the finding of differences suggests the presence in the mosquito extract of some protein binding the hormone. The differences were not due to lethal toxic effects since the Trypan blue viability test showed no differences between control and treated cells. Furthermore, the effect disappeared when the extract had previously been heated at 90 degrees C for 60 min. Finally, our results suggest the presence of some proteins in the mosquito Culex pipiens L. larvae, which would act as a pituitary hormone regulator.     

Administration of human pancreatic growth hormone-releasing factor (GRF) analogs enhances responsiveness of cultured rat pituitary cells to GRF

The aim of this study was to investigate whether anterior pituitary responsiveness to human pancreatic growth hormone-releasing factor containing 29 amino acids (GRF-29) can be modulated by GRF-29 itself. Male rats were injected (sc) daily for 3 days with 50 ug of GRF-29, or were treated twice daily for 14 days with 5 ug of [D-Ala-2]-GRF-29 (a potent GRF agonist). Control animals were injected with saline. After the last injection, pituitaries were removed, dispersed, cultured for 96 h and then challenged with either GRF-29 or [D-Trp-6]-LHRH (a LHRH agonist). Cultured cells from analog-treated rats were more responsive to GRF-29 stimulation than were cells obtained from controls. In contrast, neither treatment altered the response to [D-Trp-6]-LHRH. These studies indicate that periodic administration of GRF analogs can increase hypophyseal GRF responsiveness. Such control may be an important component in the physiological regulation of GH secretion and has important implications for potential therapeutic uses of GRF analogs.     

In vitro pituitary hormone releasing activity of 40 residue human pancreatic tumor growth hormone releasing factor

The hypophysiotropic activities of a synthetic human pancreatic growth hormone releasing factor (hpGRF) with 40 residues was examined in vitro using rat pituitary halves. At concentrations from 10(-10) M to 10(-7) M the peptide stimulated GH release in a dose-dependent manner with the ED50 being 1.2 x 10(-9) M. The concentration of 10(-10) M hpGRF is comparable to the basal hypophyseal portal blood levels of other known hypothalamic hypophysiotropic hormones. However, GH release was enhanced three-fold by concentration as low as 10(-12) M, though no dose-response relationship was observed up to 10(-10) M. Thus, this peptide not only stimulates the release of GH in a dose-dependent manner, but at lower concentrations also maintains elevated GH levels. The release of ACTH, beta-endorphin, LH, and FSH was not affected by hpGRF at any of the concentrations tested. At hpGRF concentrations less than 10(-7) M, the release of TSH and PRL were unaffected. However, at 10(-6) M, TSH release was enhanced about 2.5 fold and prolactin release was elevated slightly.     

Thyroid hormone and apotransferrin regulation of growth hormone secretion by GH1 rat pituitary tumor cells in iron restricted serum-free defined medium

Summary Growth hormone (GH) production by GH₁ rat pituitary tumor cells in iron restricted serum-free defined medium requires apotransferrin (apoTf) and triiodothyronine (T₃). As measured by radioimmunoassay, apoTf plus T₃ induced GH levels 2 to 4-fold above controls. Deletion of either apoTf or T₃ arrested GH secretion. ApoTf/T₃ defined medium regulated GH production as effectively as whole serum. Because glucocorticoids enhance GH secretion in serum containing cultures, the effects of dexamethasone were evaluated in apoTf/T₃ defined medium. The steroid hormone showed no enhancing effects unless the cells were exposed to serum prior to incubation in apoTf/T₃ defined medium. Even under these conditions, the response to dexamethasone remained T₃ dependent. These observations indicate that a yet to be characterized serum factor(s), other than apoTf, regulates the reponse to the steroid hormone. This is the first report of thyroid hormone regulation of GH secretion by rat pituitary tumor cells under completely serum-free chemically defined conditions.     

Expression of human growth hormone in transgenic rice cell suspension culture

Human growth hormone (hGH), a pituitary-derived polypeptide, evidences a wide range of biological functions, including protein synthesis, cell proliferation, and metabolism. A synthetic hGH gene (shGH) has been synthesized on the basis of plant-optimized codon usage via an overlap PCR strategy and located in a plant expression vector under the control of the rice amylase 3D (Ramy3D) promoter, which is induced by sugar starvation. The plant expression vector was introduced into rice calli (Oryza sativa L. cv. Donjin) via particle bombardment transformation methods. The integration of the shGH gene into the chromosome of the transgenic rice callus was verified via genomic DNA PCR amplification and shGH expression in transgenic rice suspension cells was confirmed via Northern blot analysis. The shGH protein was detected in the transgenic rice cell suspension culture medium following induction with sugar starvation, using Western blot analysis. The quantity of shGH that accumulated in the transgenic rice cell suspension medium was 57 mg/l. The shGH accumulated in the transgenic rice cell suspension culture medium evidenced a biological activity similar to that of Escherichia coli-derived recombinant hGH. These results indicate that the shGH was generated and accumulated in the transgenic rice cell suspension culture medium, and manifested biological activity.     

Tilapia adropin: the localization and regulation of growth hormone gene expression in pituitary cells

The peptide hormone adropin, encoded by the energy homeostasis-associated (Enho) gene, plays a role in energy homeostasis and the control of vascular function. The aim of this study was to examine the role of adropin in growth hormone (GH) gene expression at the pituitary level in tilapia. As a first step, the antiserum for the tilapia adropin was produced, and its specificity was confirmed by antiserum preabsorption and immunohistochemical staining in the tilapia pituitary. Adropin could be detected immunocytochemically in the proximal pars distalis (PPD) of the tilapia pituitary. In primary cultures of tilapia pituitary cells, tilapia adropin was effective in increasing GH mRNA levels. However, removal of endogenous adropin by immunoneutralization using adropin antiserum inhibited GH gene expression. In parallel experiments, pituitary cells co-treated with ovine pituitary adenylate cyclase activating polypeptide 38 (oPACAP38) and adropin showed a similar increase level compared to those treated with oPACAP38 alone, whereas insulin-like growth factor 1 (IGF1) not only had an inhibitory effect on basal GH mRNA levels, but also could abolish adropin stimulation of GH gene expression. In pituitary cells pretreated with actinomycin D, the half-life of GH mRNA was enhanced by adropin. Taken together, these findings suggest that adropin may serve as a novel local stimulator for GH gene expression in tilapia pituitary.