二仙汤及其拆方对GT_(1-7)细胞株GnRH分泌的影响

目的 :GT1 7细胞株是转入了猴病毒 40的 T抗原 (SV40 T)癌基因的促性腺激素释放激素 (Gn RH)神经内分泌细胞株。本实验观察补肾方二仙汤及其“温肾”、“滋阴”两个拆方对 GT1 7细胞株释放 Gn RH的影响。方法 :(1) 3个月龄的 SD雄性大鼠予成人每公斤体重的 10倍剂量灌服中药 4日 ,于末次给药后 1或 2小时腹主动脉取血 ,制备成药物血清。 (2 )氯胺 T法标记 Gn RH,建立稳定可靠的放免标准曲线。 (3) GT1 7细胞用含10 %、30 %或 5 0 %药物血清的培养液孵育 2 4或 48小时 ,收集上清液做放射免疫测定。结果 :(1)末次给药后 1、2小时取血的二仙汤药物血清均能刺激 Gn RH释放 ,以 1小时给药血清效果最好。 (2 ) 10 %浓度的药物血清为最有效剂量。 (3)二仙汤全方及其两个拆方均能促进 Gn RH释放 ,以全方效果最显著。结论 :中医“肾主生殖”的功能似涉及下丘脑 Gn RH神经元及其调控性腺轴的功能 ,二仙汤及其全方能够直接调节 Gn RH的分泌。

二仙汤及其药物血清对GT1-7细胞增殖及胰岛素样生长因子-1 mRNA的影响

目的 GT1-7细胞株是促性腺激素释放激素(GnRH)神经内分泌细胞株,本实验观察补肾方二仙汤及其温肾、滋阴两个拆方药物血清对 GT1-7细胞株增殖及胰岛素样生长因子-1(IGF-I)mRNA 的影响。方法运用 Northern blot 方法观察 IGF-I mRNA 水平的变化;用 MTT 法观察 GT1-7细胞增殖情况。结果 (1)二仙汤及其温肾、滋阴两个拆方药物血清都能增加 IGF-I mRNA 的表达,其中以滋阴组作用最显著;(2)全方组和滋阴组药物血清能促进 GT1-7细胞增殖,而温肾组无明显变化。结论补肾方二仙汤可能通过作用于 IGF-I 的mRNA 水平而影响 GT1-7细胞的增殖和 GnRH 释放,温肾组和滋阴组作用于该细胞的机制可能有所不同。

GT1-7细胞及其在生殖相关研究中的应用

GT1-7细胞是GT1细胞株的亚株,通过转基因技术从小鼠下丘脑分离获得的Gn RH神经元细胞系,具有高度分化的神经内分泌细胞典型特征。下丘脑Gn RH合成和释放对生殖功能具有重要作用,而Gn RH神经元在脑内数量少且呈弥散分布,体内研究较困难。目前,GT1-7细胞是研究Gn RH神经元的理想离体细胞模型,在生殖相关研究中广泛使用。文章详细阐述GT1-7细胞获得过程、功能特性及其在生殖系统研究中应用,以及影响GT1-7细胞活性的信号通路,以期对动物生殖调控研究提供参考。

雌激素对GT1-7细胞GnRH分泌及相关基因表达的影响

【目的】研究低浓度雌激素对下丘脑GnRH分泌以及ERα、GnRH等6个生殖相关基因表达的影响.【方法】采用体外培养GT1-7细胞,对照组添加溶剂(无水乙醇),试验组添加雌激素(100pmol/L),培养1、3、6、12、18、24、30、36h收集细胞和培养上清液,利用酶联免疫法(ELISA)测定收集上清液中GnRH浓度,并利用相对荧光定量法测定目的基因的表达.【结果】与对照组相比,试验组GnRH分泌呈增加趋势,培养12h左右GnRH分泌达到峰值(P<0.05);ERα、GnRH、Kiss-1、GPR54mRNA相对表达量先增加后降低,4种基因mRNA依次在18h(P<0.01)和24h(P<0.05)、6h(P<0.01)、3h(P<0.01)、6h和12h(P<0.05)表达显著增加.nNOS和c-fos mRNA相对表达量较对照组呈降低趋势,这2种基因mRNA依次在30h和36h(P<0.05)、3h(P<0.01)表达显著降低.【结论】100pmol/L雌激素能够促进GT1-7细胞分泌GnRH,这种作用可能是通过调控ERα、GnRH、Kiss-1、GPR54、nNOS以及c-fos mRNA的表达实现的.

利用GT1-7细胞体外研究雌激素对GnRH分泌及其相关基因表达的影响

雌激素对GnRH神经元活性及其合成分泌具有重要的调控作用。为了体外模拟雌激素对GnRH合成分泌的抑制作用,使用1μmol/L的雌激素处理GT1-7细胞0、6、12、18、24和30 h,分别检测GnRH的分泌及相关基因GnRH、ERα、KISS-1和GPR54的表达。结果显示:雌激素处理GT1-7细胞GnRH分泌量相对于对照组先升高(6和12 h,P>0.05)后极显著降低(18和30 h,P<0.01)。雌激素处理组GnRH mRNA表达在整个培养期间相对于对照组极显著降低(P<0.01),ERα(12和18 h,P<0.01)、KISS-1(12 h,P<0.01)和GPR54表达也极显著降低(6、12和24 h,P<0.01)。结果表明:1μmol/L雌激素能够抑制GT1-7细胞GnRH的分泌,可能是通过降低ERα、GnRH、KISS-1及GPR54等基因的表达来实现的。

Kisspeptin和N-氨甲酰谷氨酸对GT1-7细胞GnRH分泌的影响

GT1-7细胞是体外研究GnRH分泌的理想模型。为了揭示kisspeptin和N-氨甲酰谷氨酸(NCG)对GnRH分泌活性的影响,体外培养GT1-7细胞,研究kisspeptin-10和NCG单独或组合培养对细胞增殖及GnRH分泌的影响。结果表明:kisspeptin-10和NCG单独处理均可促进GT1-7细胞增殖,组合培养对细胞增殖具有协同促进作用;kisspeptin-10单独处理可促进GnRH分泌,NCG单独处理可抑制GnRH分泌,组合处理后kisspeptin-10可补偿NCG对GnRH分泌的抑制,在一定浓度kisspeptin-10存在的条件下,NCG能够呈剂量依赖性促进GnRH的分泌。揭示了kisspeptin和NCG对GnRH分泌的促进作用。

Expression and significance of B7-H1 in peripheral blood dendritic cells from patients with bladder cancer

Objective： The aim of this study was to study the expression and the clinical significance of B7-H1 on dendritic cells （DCs） in peripheral blood from patients with bladder cancer. Methods： Peripheral blood mononuclear cell were disparted from 30 bladder cancer patients and 7 healthy controls by density gradient centfifugation and then co-cultured. The expres- sion of B7-H1 on DCs were analyzed by flow cytometry. Results： Expression of BT-H1 on DCs in bladder cancer was higher than healthy controls （P 〈 0.01）. And the expression were strongly associated with the pathological grade and clinical stage of bladder cancer （P 〈 0,05）. Conclusion： The up-regulation of B7-H1 on DCs was strongly associated with neoplastic progres-sion of bladder cancer. B7-H1/programmed death （PD）-1 signal pathway may also play an important role in immune escape of bladder cancer during initial phase of T cell immune response.

瘦素对GT1-7细胞GnRH释放的影响

目的 :GT1- 7细胞是替代研究GnRH神经元的理想细胞模型 ,本文观察瘦素 (Leptin)对GT1- 7细胞GnRH释放的影响。方法 :采用氯胺T法放射性核素标记物12 5I标记GnRH ,建立了稳定灵敏的GnRH放射免疫分析方法。将不同浓度的瘦素 ,以不同的时间与GT1- 7细胞孵育 ,用RIA法测定GT1- 7细胞上清液中GnRH含量。结果 :结果表明瘦素能直接作用于GT1- 7细胞株 ,促进GnRH释放 ,以浓度为 2 0ng/ml,孵育 15分钟作用最明显。结论 :瘦素可能通过直接作用于下丘脑GnRH神经元 ,而对生殖功能起调节作用。

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细胞孵育 ,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 。

PCB118对GT1-7细胞GnRH分泌及Kisspeptin/GPR54信号通路蛋白表达的影响

为研究多氯联苯118(polychlorinated biphenyl 118,PCB118)对GT1-7细胞的毒性作用,探讨其对促性腺激素释放激素(gonadotropin-releasing hormone,GnRH)分泌的影响及相关机制,在PCB118作用GT1-7细胞后,分析了细胞存活率、GnRH分泌水平以及Kisspeptin/GPR54信号通路中关键蛋白的表达水平。结果表明,PCB118(0.05~50000nmol·L^-1)分别作用GT1-7细胞6、12、24和48h后,细胞存活率随PCB118浓度的增加和作用时间的延长而显著降低。但只有在高浓度(50000nmol·L^-1)条件下,PCB118才能显著促进GT1-7细胞释放乳酸脱氢酶(lactate dehydrogenase,LDH)。PCB118作用GT1-7细胞6h后,5、500和50000nmol·L^-1组能显著抑制GnRH的分泌,并降低Kisspeptin/GPR54信号通路中GnRH、G蛋白偶联受体54(G-protein-coupled receptor 54,GPR54)、磷脂酶C(phospholipase C,PLC)和蛋白激酶C(protein kinase C,PKC)等关键蛋白的表达水平;作用24h,500和50000nmol·L^-1PCB118能显著抑制GnRH的分泌,并降低GnRH、GPR54、PLC和PKC等关键蛋白的表达水平。研究表明:PCB118对GT1-7细胞具有细胞增殖毒性作用,呈现一定的剂量和时间效应;PCB118能在一定程度上抑制GT1-7细胞GnRH的合成与分泌,并下调Kisspeptin/GPR54信号通路中GnRH、GPR54、PLC和PKC等关键蛋白的表达水平,推测Kisspeptin/GPR54信号通路可能参与介导了PCB118对GT1-7细胞GnRH分泌的抑制作用。

miR-505-3p对GT1-7细胞株基因表达谱的影响

目的了解微小RNA 505-3p(miR-505-3p)对下丘脑GnRH神经元GT1-7稳转细胞株表达谱的影响。方法用慢病毒转染GT1-7细胞获得稳定表达miR-505-3p的细胞株,感染实验分为3组:空白对照组(未经处理的GT1-7细胞),阴性对照组(感染pLenti6.3-nc空病毒),实验组(感染pLenti6.3-miR-505-3p病毒)。采用实时荧光定量PCR(Quantitative Real-time PCR,qRT-PCR)技术检测稳定细胞株中miR-505-3p的表达丰度,并进一步利用芯片检测对照组和实验组细胞的表达谱结合生物信息学方法探寻差异表达基因。采用GO分析和Pathway分析差异表达基因,解析miR-505-3p的功能。结果在稳定表达miR-505-3p的GT1-7细胞中,165个基因的表达量变化在2倍以上,这些基因主要参与细胞粘附、GTP分解代谢、神经系统发育等生物过程。结论 miR-505-3p可能通过影响下丘脑GnRH神经元中相应靶基因的表达调控细胞粘附、GTP分解代谢、神经系统发育等生物过程,和间隙连接、轴突导向和胃酸分泌等信号通路。

Effect of angiotensin 1-7 on endothelial cell injury caused by oxidative stress

Objective:To study the effects of angiotensin 1-7 (Ang1-7) on endothelial cell injury caused by oxidative stress.Methods: Human umbilical vein endothelial cells (HUVECs) were cultured and divided into blank control group, hydrogen peroxide and different Ang1-7 dose groups (1, 2 and 4 μmol/L Ang1-7 groups). The cell proliferation activity, the contents of antioxidant enzymes in cell culture medium, and the contents of endoplasmic reticulum stress molecules in cells were determined.Results: After 6, 12, 18 and 24 h of treatment, CCK-8 proliferation activity values of hydrogen peroxide group were significantly lower than those of blank control group, CCK-8 proliferation activity values of 1, 2 and 4 μmol/L Ang1-7 groups were significantly higher than those of hydrogen peroxide group, and the larger the Ang1-7 dose, the higher the CCK-8 proliferation activity values;after 24 h of treatment, SOD, GSH-Px, HO-1 and CAT contents in cell culture medium of hydrogen peroxide group were significantly lower than those of control group, and GRP78, XBP1 and CHOP contents in cells were significantly higher than those of control group;SOD, GSH-Px, HO-1 and CAT contents in cell culture medium of 1, 2 and 4 μmol/L Ang1-7 groups were significantly higher than those of hydrogen peroxide group, GRP78, XBP1 and CHOP contents in cells were significantly lower than those of hydrogen peroxide group, and the larger the Ang1-7 dose, the more significant the changes of above molecules in cell culture medium and cells.Conclusion: Angiotensin 1-7 has protective effect on the endothelial cell injury caused by oxidative stress.

Angiotensin-(1-7)Changes Apoptosis-Related Genes Expression in Human Breast Cancer Cell Line T47D

Angiotensin-(1-7) [Ang-(1-7)] is a heptapeptide of the renin-angiotensin system with vasodilator and anti-proliferative properties. In the present study, we aim to investigate whether Ang-(1-7) induces apoptosis in breast cancer cells and whether the altered expression of apoptosis-related genes is involved in this process. Human breast cell line T47D was treated with angiotensin-(1-7) and angiotensin II (Ang II). Cell proliferation and apoptosis were quantified using hemocytometer and flow cytometry, respectively. The expression of 84 apoptosis-related genes was evaluated through qPCR array. Ang-(1-7), as opposed to Ang II, decreased proliferation and increased apoptosis in T47D cells. Moreover, many pro-apoptotic genes were up-regulated, such as BAK1, BAX, BCL2L1, BID and BIK. In addition, some anti-apoptotic genes as AKT1 and XIAP were down-regulated by heptapeptide. Although a deeper study should be performed, our results support the hypothesis that Ang-(1-7) could change the expression of several genes related to apoptosis, interfering directly in the molecular pathways associated with the survival of breast cancer cells.

Angiotensin-(1-7)and Human Chorionic Gonadotropin(hCG)Modulate the Nuclear Transcription Factors or Nuclear Receptors Genes in the Tumorigenic Undifferentiated Breast Cancer Cell Line SKBR3

Breast cancer is the most common cancer among women. Angiotensin-(1 - 7) [Ang-(1 - 7)] has been correlated with cancer antiproliferative and apoptotic effects, similar properties of the human Chorionic Gonadotrofin (hCG). The aims of this work are to evaluate the role of Ang-(1 - 7) and of hCG in modulating the expression of Nuclear Receptors and Coregulators related genes in the tumorigenic breast cell line SK-BR3. Three experimental groups were created: control, hCG and hCG + Ang-(1 - 7). Cells were treated for 11 days and then had their RNA extracted. Samples were loaded into PCR Array plates containing 84 genes relate to Nuclear Receptors and Coregulators pathways. Gene expression data were used to construct canonical pathways (MetacoreTM). hCG and hCG + Ang-(1 - 7) treatments markedly modulate the expression of Nuclear Receptors and Coregulators related genes. hCG differentially expressed 17% of the genes, being 29% upregulated and 71% downregulated. Meanwhile, hCG + Ang-(1 - 7) changed the expression of 30% of the genes on the plate, among these genes 56% were upregulated and 44% downregulated. Among these differentially expressed genes, we highlight Esr1, Nr2f2, and Nr2f1, Esr1, Hdac5, and Nr4A1 (>4 fold). Finally MetaCore analysis based on Gene Ontology (GO) generated six networks for hCG and ten networks for the combined treatment. All generated networks are related to regulation of apoptosis or to Programmed Cell Death processes. In summary, our results herein demonstrate that the modulation of sexual hormones and of other nuclear factor genes expression might underlie the tumorigenic protection effect and the induction of cell differentiation caused by the hormones hCG and Ang-(1 - 7), especially in Cancer Stem Cells.

Angiotensin-（1-7）： new perspectives in atherosclerosis treatment

Angiotensin （Ang）-（1-7） is recognized as a new bioactive peptide in renin-angiotensin system （RAS）. Ang-（1-7） is a counter-regulatory mediator of Ang-II which appears to be protective against cardiovascular disease. Recent studies have found that Ang-（1-7） played an important role in reducing smooth muscle cell proliferation and migration, improving endothelial function and regulating lipid metabolism, leading to inhibition of atherosclerotic lesions and increase of plaque stability. Although clinical application of Ang-（1-7） is restricted due to its pharmacokinetic properties, identification of stabilized compounds, including more stable analogues and specific delivery compounds, has enabled clinical application of Ang-（1-7）. In this review, we discussed recent findings concerning the biological role of Ang-（1-7） and related mechanism during atherosclerosis development. In addition, we highlighted the perspective to develop therapeutic strategies using Ang-（1-7） to treat atherosclerosis.

Ang-(1-7)exerts anti-inflammatory and antioxidant activities on high glucose-induced injury by prohibiting NF-κB-IL-1βand activating HO-1 pathways in HUVECs

Previous reports have suggested that Ang-(1-7)may have a protective effect in endothelial cells against high glucose(HG)-induced cell injury thanks to a modulatory mechanism in the NF-κB signaling pathway.In this study,we have examined whether NF-κB-IL-1βand Heme oxygenase-1(HO-1)pathways contribute to the protection of Ang-(1-7)against hyperglycemia-induced inflammation and oxidative stress in human umbilical vein endothelial cells(HUVECs).Our results indicate that time-varying exposures of HUVECs,from 1 h to 24 h,to high glucose concentrations result in an increased expression of phosphorylated(p)-p65 and HO-1 in a time-dependent manner.As an inhibitor of NF-κB,pyrrolidinedithiocarbamic acid(PDTC)suppressed IL-1βproduction induced by HG.Of note,HUVECs previously treated with Ang-(1-7)(2μM)for 30 min before being exposed to HG concentrations significantly ameliorated the HG-increased in p-p65 and IL-1βexpression;whereas obviously up-regulated the level of HO-1,along with inhibition of oxidative stress,inflammation,and the HG-induced cytotoxicity.Importantly,when HUVECs were previously treated either with PDTC or IL-1Ra for 30 min before being exposed to HG,it significantly prevented damages caused by high glucose concentrations mentioned above,while the treatment of HO-1 inhibitor Sn-protoporphyrin(SnPP)before exposure to both HG and Ang-(1-7)significantly blocked the protective effect exerted by Ang-(1-7)on endothelial cells against injuries induced by HG mentioned above.To conclude,the data of this study showed that activation and inhibition of the NF-κB-IL-1βpathway and HO-1 pathway may constitute an important defense mechanism against endothelial cell damage caused by HG concentrations.We additionally gave new evidence showing that exogenous Ang-(1-7)exerts a protective effect on HUVECs against the HG-induced cell injury via the inhibition and the activation of the NF-κB-IL-1βpathway and the HO-1 pathway,respectively.

Angiotensin-converting enzyme 2 alleviates liver fibrosis through the renin-angiotensin system

The present letter to the editor is related to the study titled‘Angiotensin-converting enzyme 2 improves liver fibrosis in mice by regulating autophagy of hepatic stellate cells’.Angiotensin-converting enzyme 2 can alleviate liver fibrosis by regulating autophagy of hepatic stellate cells and affecting the renin-angiotensin system.

Arg-Phe-amide-related peptides influence gonadotropin-releasing hormone neurons

The hypothalamic Arg-Phe-amide-related peptides, gonadotropin-inhibitory hormone and orthologous mammalian peptides of Arg-Phe-amide, may be important regulators of the hypothalamus-pituitary-gonadal reproductive axis. These peptides may modulate the effects of kisspeptins because they are presently recognized as the most potent activators of the hypothalamus-pituitary-gonadal axis. However, their effects on gonadotropin-releasing hormone neurons have not been investigated. In the current study, the GT1-7 cell line-expressing gonadotropin-releasing hormone was used as a model to explore the effects of Arg-Phe- amide-related peptides on kisspeptin activation. Intracellular calcium concentration was quantified using the calcium-sensitive dye, fura-2 acetoxymethyl ester. Gonadotropin-releasing hormone released into the medium was detected via enzyme-linked immunosorbent assay. Results showed that 100 nmol/L kisspeptin-10 significantly increased gonadotropin-releasing hormone levels （at 120 minutes of exposure） and intracellular calcium concentrations. Co-treatment of kisspeptin with 1 μmol/L gonadotropin-inhibitory hormone or 1 μmol/L Arg-Phe-amide-related peptide-1 significantly attenuated levels of kisspeptin-induced gonadotropin-releasing hormone but did not affect kisspeptin-induced elevations of intracellular calcium concentration. Overall, the results suggest that gonadotropin-inhibitory hormone and Arg-Phe-amide-related peptide-1 may have inhibitory effects on kisspeptin-activated gonadotropin-releasing hormone neurons independent of the calcium signaling pathway.

利用基于CRISPR-Cas9的基因编辑技术对miR-29a在GT1-7细胞中进行基因敲除

目的:本文利用CRISPR-Cas9技术在GT1-7细胞中对miR-29a基因进行基因编辑,用于构建miR-29a基因敲除GT1-7细胞模型。方法:通过构建Cas9稳转的GT1-7细胞株并转染sgRNA质粒用于在靶向miR-29a基因区域引发突变。然后构建EGFP与sgRNA共表达质粒并转染Cas9稳转GT1-7细胞,利用流式细胞仪富集表达绿色荧光蛋白的阳性细胞和分选阳性单克隆细胞。最后利用实时荧光定量PCR(realtimefluorescencequantitativePCR)对富集细胞和单克隆细胞进行miR-29a表达量检测。结果:T7E1检测结果显示CRISPR-Cas9系统有效地在miR-29a基因区域引发了突变。荧光定量PCR结果显示,与对照组相比,富集后阳性细胞miR-29a的表达量整体下降了50%左右(P<0.05)。此外,通过流式筛选获得了一个纯合miR-29a基因敲除细胞克隆,与对照组相比,其miR-29a的表达量下降了75%左右(P<0.05)。结论:本文建立了一种有效编辑GT1-7细胞基因的方法,并采用该方法构建了miR-29a稳定敲除细胞模型。

二代测序应用于检测GT1-7细胞中KISS1和GnRH启动子的DNA甲基化状态的研究

目的:利用二代测序技术检测GT1-7细胞中KISS1和GnRH基因启动子范围内的甲基化状态,并用金标准的亚硫酸氢盐修饰后的克隆测序作为对照,比较二代测序与金标准克隆测序在研究DNA甲基化检测中的差别。方法:提取GT1-7细胞基因组DNA并进行亚硫酸氢盐处理。进行巢式PCR,将PCR产物进行二代测序。同时采用金标准的亚硫酸氢盐修饰后克隆测序的方法作为对照,对相同批次的PCR产物进行克隆测序。结果:PCR产物二代测序结果表明KISS1和GnRH两个基因的27个CpG甲基化位点信息完整,结果准确。挑取10个克隆进行一代测序结果表明序列无丢失,KISS1和GnRH两个基因的27个CpG甲基化位点信息完整。结论:利用高通量的二代测序技术能够有效的对DNA甲基化的PCR产物进行检测,二代测序和克隆测序都是研究DNA甲基化的有效方法,但前者与克隆测序相比每一个读取序列(reads)都相当于一个单克隆,且二代测序每个区段得到成百上千个reads,因此二代测序结果更加精确。