Optimized THP-1 differentiation is required for the detection of responses to weak stimuli

Objectives: The differentiation of THP-1 monocytes into macrophages is mainly conducted at a phorbol 12-myristate 13-acetate (PMA) concentration of 10–400 ng/ml. However, this concentration might be high enough to upregulate the expressions of some genes in differentiated macrophages, which could overwhelm gene expression increases induced by other stimuli. The present study was performed to optimize the PMA concentration required to differentiate monocytes whilst minimizing gene upregulation. Methods: THP-1 cells were treated with 2.5–100 ng/ml PMA and analyzed for the extent of cell adherence, the surface marker of macrophages, and stable differentiation without undesirable gene upregulation. The stably differentiated THP-1 cells at the minimum PMA concentration were treated with 10 ng/ml LPS or 125 nM amyloid beta (Aβ_1-42). Results: The treatment of THP-1 with 5 ng/ml PMA was found to be sufficient to induce stable differentiation without undesirable gene upregulation. These macrophages differentiated at 5 ng/ml responded well to secondary weak stimuli like 10 ng/ml LPS or 125 nM of amyloid beta (Aβ_1-42). Conclusions: This finding suggests that THP-1 cells are well differentiated by 5 ng/ml PMA, and that the resulting differentiated macrophages respond well to secondary weak stimuli without being overwhelmed by undesirable gene upregulation induced by PMA. Received 10 March 2006; returned for revision 9 August 2006; accepted by G. Wallace 11 August 2006     

妊娠滋养细胞肿瘤组织中cyclin D1、Rb蛋白产物的表达

目的：探讨增殖相关基因cyclin D1、Rb在妊娠滋养细胞肿瘤中的表达变化。方法：采用免疫组化S－P法,检测20例葡萄胎、15例侵蚀性葡萄胎、15例绒毛膜癌组织中两种基因蛋白产物的表达。结果：在恶性滋养细胞肿瘤组织中,cyclinD1的阳性表达率随临床期别的增高呈递增趋势,而Rb呈递减趋势,二者在Ⅲ期的阳性表达率与Ⅰ期及Ⅱ期相比差异均具有显著性（P＜0．05）;化疗可降低cyclinD1在恶性滋养细胞肿瘤中的阳性表达,而对Rb无影响。结论：cyclin D1的过表达,Rb的缺失在妊娠滋养细胞肿瘤的发展中可能有重要的生物学意义。     

前列腺癌p21CIP1/WAF1、Rb及PCNA的表达及意义

目的研究p21^CIP1/WAF1、Rb及PCNA在人前列腺癌标本中的表达及三者相关性,阐述它们与前列腺癌病理分级及临床分期的关系。方法收集36例确诊前列腺癌石蜡包埋存档标本作为研究对象,采用免疫组化SABC法对其p21^CIP1/WAF1、Rb及PCNA进行检测,并应用图象分析仪判定,统计学处理,对前列腺癌的病理分级及临床分期进行了对比分析及相关性研究。结果p21^CIP1/WAF1、Rb及PCNA的免疫组化阳性染色为棕黄色和／或棕褐色,定位于细胞浆或细胞核。所得数据均经统计学处理。在不同病理分级、临床分期之间差异均有显著性,同时还发现PCNA与p21^CIP1/WAF1及Rb之间存在显著负相关性。但未发现p21^CIP1/WAF1、与Rb有显著相关性。结论p21^CIP1/WAF1、Rb表达与前列腺癌组织的病理分级、临床分期呈负相关性,在发病机制中可能涉及到p21^CIP1/WAF1、Rb和PCNA凋节通路的异常。同时,作为一种检测方法,可用于判定前列腺癌恶性程度及进程的有价值的指标,对诊治康复也可提供有意义的帮助。     

Epiregulin induces human SK-N-BE cell differentiation through ERK1/2 signaling pathway

Abstract

Epidermal growth factor (EGF) and other EGF-related growth factors, such as transforming growth factor-α, are able to stimulate neuroblastoma (NB) cell proliferation. Epiregulin (Epi) is a growth factor belonging to the EGF family known to be more potent than EGF in mediating mitogenic signals. In this study, we tested the ability of Epi to stimulate a human NB cell line (SK-N-BE) proliferation. Surprisingly, Epi (50–1000?ng/ml) induced a reduction in SK-N-BE proliferation along with a morphological differentiation, associated with an increase in MMP-9 expression. Moreover, Epi-induced differentiation was inhibited by ERK1/2 phosphorilation inhibition. In conclusion, Epi could represent a novel and useful tool to oppose NB cell proliferation.     

人参皂甙Rg1和Rb1药理作用的比较

人参作为一味珍贵的中药,在中医临床已有二千多年的应用历史。历经几个世纪的化学和药理研究,大量科学数据已无可辩驳地证明,人参及其有效成分具有广泛的生物活性和医疗用途。除中国、韩国、日本外,欧洲、北美、前苏联等国家的医药工作者均颇为重视,对人参的独特作用表现出了越来越大的兴趣和热情。当今世界,每隔几年就要举行一次国际人参研究学术讨论会,数十个国家近千名科学家与会发表论文,使人参的药理学研究进入了细胞、亚细胞、分子水平,因而人参的许多新的药理作用及其机制也逐渐被阐明。人参中的有效成分包括皂甙类、非皂甙…     

Neuroprotective effect of ginsenoside Rb1 on glutamate-induced neurotoxicity: With emphasis on autophagy

Ginsenoside Rb1 has been demonstrated with neuroprotective effects, but the mechanisms remain unclear. This study aimed to probe the effects and mechanisms of ginsenoside Rb1 on activation of autophagy in glutamate-injured neurons. Ginsenoside Rb1 of exponential concentrations (1.2, 12, 120 μM) or autophagy inhibitor 3-methyladenine (5 mM) was added to culture medium for cortical neurons after being treated with glutamate. Cell viability was measured by MTT assay. Autophagosomes formation was observed with transmission electron microscope. Autophagy marked protein LC3 was detected with immunofluorescence and visualized under laser confocal microscopy. Changes of autophagy related protein Beclin-1 were measured with Western blot. We found that ginsenoside Rb1 protected cortical neurons from glutamate-induced cell injury. Autophagy was activated after glutamate treatment, with both autophagosomes and punctate LC3 increased significantly compared with control. Beclin-1 was elevated in glutamate-treated cells. Formation of autophagosome and punctate LC3 was attenuated by ginsenoside Rb1. The level of Beclin-1 in ginsenoside Rb1 treated cells was simultaneously decreased compared with glutamate-treated cells. These results suggested that inhibition of autophagy could be responsible for neuroprotective effects of ginsenoside Rb1 in glutamate-induced injury. Down-regulation of Beclin-1 may play an important role in this process.     

Distinct regulation of myoblast differentiation by intracellular and extracellular fibroblast growth factor-1

We studied the role of fibroblast growth factor (FGF)-1 in the physiology of myoblast differentiation. We found that, while endogenous FGF-1 in L6-10 rat myoblasts did not suppress the progress of differentiation, the addition of FGF-1 to the culture medium suppressed it. Moreover, L6-10 cells stably transfected with full length FGF-1 undergo enhanced differentiation. The latter was well correlated with myogenin expression and myotube formation. Constitutive expression of a mutant FGF-1 (FGF-1U) that lacked a nuclear localization signal, promoted the differentiation of the myoblasts even more strongly. Furthermore, the expression of FGF-1U in an inducible expression system enhanced myogenin expression promptly. In L6-10 transfectants expressing a dominant-negative mutant of FGF receptor, stable transfection of FGF-1 promoted differentiation as it did in parent cells. Studies with FGF receptors and MAP kinase suggest that both are involved in the effect of FGF-1 when it is supplemented to culture medium but not during the effect of endogenous FGF-1 synthesized in cells. We conclude that intracellular (endogenous) and extracellular (exogenous) FGF-1 have differential effects on the regulation of myogenic differentiation of L6-10 cells.     

Rb1延缓人脐静脉内皮细胞早熟性衰老与caveolin-1表达的关系

目的:血管内皮细胞衰老是动脉粥样硬化发生的病理生理机制之一。本研究旨在探讨人参皂苷Rb1延缓人脐静脉内皮细胞(HUVECs)早熟性衰老与窖蛋白1(caveolin-1)表达的关系,为延缓HUVECs衰老提供新的靶点。方法:建立60μmol/L过氧化氢(H_2O_2)诱导的HUVECs早熟性衰老模型,根据细胞形态学的变化、衰老相关β-半乳糖苷酶(SA-β-Gal)染色阳性率和细胞周期评估内皮细胞衰老,采用Western blot和激光共聚焦显微成像的方法检测caveolin-1的变化,观察人参皂苷Rb1对HUVECs衰老的作用及其相关的分子机制。结果:60μmol/L H_2O_2可成功地诱导内皮细胞衰老,早熟性衰老的HUVECs体积变大,SA-β-Gal活性明显增加,细胞发生G_1期阻滞,细胞增殖受抑制,caveolin-1表达增多。与H_2O_2处理组相比,人参皂苷Rb1预处理延缓HUVECs早熟性衰老,SA-β-Gal染色阳性细胞百分比降低,G_0/G_1期细胞比例下降,caveolin-1表达减少。结论:人参皂苷Rb1可通过抑制caveolin-1的表达延缓H_2O_2诱导的HUVECs早熟性衰老。     

cyclinD1,Rb基因蛋白在乳腺癌中的表达

目的：探讨ｃｙｃｌｉｎＤ１基因及Ｒｂ基因的表达与乳腺癌发生发展的关系。方法：应用ＡＢＣ免疫组化法检测２４例良性乳腺组织及５８例乳腺癌中ｃｙｃｌｉｎＤ１及Ｒｂ蛋白表达。结果：乳腺癌中ｃｙｃｌｉｎＤ１过表达阳性率５８．６２％（３４／５８）显著高于良性乳腺组织中的１６．６７％（４／２４），Ｐ＜０．０５。ｃｙｃｌｉｎＤ１过表达出现于导管原位癌并持续于浸润、转移等进展过程中，与年龄、肿瘤大小、组织学类型及淋巴结状态无相关性，但与组织学分级负相关。乳腺癌中Ｒｂ蛋白表达阳性率为３６．２％（２１／５８），显著低于良性乳腺组织的７５％（１８／２４），Ｐ＜０．０５；未见Ｒｂ失表达与临床病理参数间存在相关性，Ｒｂ表达与ｃｙｃｌｉｎＤ１过表达呈正相关。结论：ｃｙｃｌｉｎＤ１过表达及Ｒｂ失表达是乳腺癌发生中的重要事件且前者是一早期分子事件；ｃｙｃｌｉｎＤ１过表达发挥作用可能部分依赖于Ｒｂ蛋白的存在；提示细胞周期调控异常参与乳腺癌的发生。     

HIF-1α介导人参皂甙Rb1对缺氧心肌细胞葡萄糖代谢的调节

目的:探讨人参皂甙Rb1(Gs-Rb1)是否通过缺氧诱导因子1α(HIF-1α)和(或)AMP激活的蛋白激酶α(AMPKα)调节缺氧心肌细胞的葡萄糖代谢而改善其生存能力。方法:将乳鼠心肌细胞随机分为对照组、缺氧组(1%O_2、94%N_2和5%CO_2)和缺氧干预组(即Gs-Rb1组、Ara-A组、Gs-Rb1+Ara-A组、YC-1组、Gs-Rb1+YC-1组、Ara-A+YC-1组和Gs-Rb1+YC-1+Ara-A组),Gs-Rb1、Ara-A和YC-1浓度分别为200μmol/L、500μmol/L和5μmol/L,均干预8 h。MTT法检测细胞存活率;Western blot法半定量检测AMPKα、p-AMPKα、HIF-1α和葡萄糖转运体4(GLUT-4)的蛋白水平;ELISA检测己糖激酶(HK)、磷酸果糖激酶(PFK)和乳酸脱氢酶(LDH)的活性。结果:Gs-Rb1显著改善缺氧心肌细胞的生存能力,该作用可被Ara-A和YC-1抑制,且YC-1和Ara-A具有协同效应。Gs-Rb1增加缺氧心肌细胞AMPK活性的效应可被Ara-A或YC-1抑制。Ara-A和YC-1能不同程度抑制Gs-Rb1上调缺氧心肌细胞HIF-1α表达的效应。Gs-Rb1显著上调缺氧心肌细胞膜GLUT-4的表达,但该作用可被Ara-A或YC-1抑制,Ara-A和YC-1联用时尤为显著。Gs-Rb1显著增加缺氧心肌细胞HK、PFK和LDH等的活性,但该作用可被Ara-A和YC-1抑制,且YC-1和Ara-A具有协同抑制效应。结论:人参皂甙Rb1改善缺氧心肌细胞生存能力与其促进葡萄糖摄取和增强葡萄糖糖酵解有关,这些效应可被HIF-1α和(或)AMPK调节,且HIF-1α和AMPK对此的调节具有协同作用。     

人参皂甙Rb1对大鼠急性心肌梗死后左室重构的影响

目的 观察人参皂甙Rb1对大鼠急性心肌梗死（AMI）后左室重构（VR）的影响，并对其作用机制作一初步探讨。方法 结扎Wistar大鼠左冠状动脉前降支，将大鼠随机分为Rb1治疗组、单纯手术组及假手术组。手术4周后．采用高频彩色多普勒超声观察各组大鼠左室舒张末期内径（LVDD）、舒张末期容积（LEDV）、左室重量指数（LVMI）、射血分数（EF）、短轴缩短率（FS）的变化；病理切片HE染色观察大鼠心肌细胞和间质的变化；放免法检测左心室肌肾素活性（RA）和血管紧张素Ⅱ（AngⅡ）含量。结果 与假手术组比较，单纯手术组大鼠LVDD、LEDV、LVMI显著增加。EF、FS等指标显著降低，光镜下心肌细胞肥大，间质增生明显，心脏RA和AngⅡ水平均明显升高（P〈0．05）；经人参皂甙Rb1干预后，LVDD、LEDV、LVMI降低，EF、FS升高，心肌RA、AngⅡ浓度降低，与单纯手术组相比，差异显著（P〈0．05）。结论 人参皂甙Rb，对AMI大鼠左室重构具有治疗作用，其作用机制可能与抑制肾素-血管紧张素系统（RAS）活性有关。     

p16、Rb和cyclin D1蛋白在横纹肌肉瘤中的表达及其意义

目的：探讨细胞周期相关蛋白表达异常与横纹肌肉瘤（RMS）的关系。方法：应用S－P免疫组化方法观察p16,Rb和cyclinD1蛋白在RMS中的表达状态。结果：p16,Rb和cyclinD1蛋白表达阳性率分别为55．3％,61．7％和51．1％,在胚胎性横纹肌肉瘤（ERMS）,p16和Rb蛋白表达阳性率为75．0％（21／28）和67．9％（19／28）,明显高于在腺泡状横纹肌肉瘤（ARMS）中的表达（P＜0．05）,p16蛋白阳性率在I,Ⅱ级横纹肌肉瘤分别为75．0％和73．3％,高于Ⅲ级横纹肌肉瘤（45．0％）;p16蛋白阴性及cyclinD1蛋白过表达组,局部浸润,复发或转移发生率高于p16蛋白阳性及cyclinD1蛋白阴性组;11／47例（23．4％）出现两种以上蛋白表达异常。结论：P16,Rb和cyclinD1蛋白异常可能与部分横纹肌肉瘤发生发展有关,且横纹肌肉瘤的发生可能涉及两种以上基因异常。     

Th细胞及其分化调节

幼稚CD4^＋T细胞可分化为Th1和Th2细胞，Th1主要产生IL-2、IFN-γ、TNF，增强吞噬细胞介导的抗感染机制，促进细胞免疫，也在器官特异性自身免疫疾病中起作用；Th2细胞主要产生IL-4、IL-5、IL-10、IL-13，促进B细胞增殖、分化和产生抗体，增强B细胞介导的体液免疫应答，在变态反应和机体抗寄生虫免疫中发挥作用。Th细胞分化主要由局部环境中的细胞因子及细胞内关键转录因子调控。转录因子STAT1、STAT4、IRF1和T—bet促使Th1细胞分化；转录因子STAT6、IRF4和GATA-3促使Th2细胞分化。     

Molecular-Signaling Pathways of Ginsenosides Rb in Myocardial Ischemia-Reperfusion Injury: A Mini Review

Reperfusion injury following myocardial ischemia remained a challenge for optimal treatment of myocardial infarction. Ginsenosides Rb (G-Rb), the primary components of ginsenoside, have been reported to exert cardioprotective effects via numerous mechanisms. G-Rb1 mediate cardioprotective effects via various signaling pathways, including mitochondrial apoptotic pathway, PI3K/Akt/mTOR, HIF-1α and GRF91, RhoA, p38α MAPK, and eNOS. G-Rb2 activates the SIRT-1 pathway, while G-Rb3 promotes both JNK-mediated NF-κB and PERK/Nrf2/HMOX1. Generally, ginsenosides Rb1, 2, and 3 modulates oxidative stress, inflammation, and apoptosis, contributing to the improvement of structural, functional and biochemical parameters. In conclusion, G-Rb, particularly G-Rb1, have vast potential as a supplement in attenuating reperfusion injury. Translation into a clinical trial is warranted to confirm the beneficial effects of G-Rb.     

人参皂苷Rb1对缺氧诱导N9细胞应激活化反应的影响

目的 探讨人参皂苷Rb1对缺氧诱导的小胶质细胞活化的的影响. 方法 通过人参皂苷Rb1对缺氧状态下N9细胞的干预,检测细胞形态、增殖活力改变.采用ELISA法、荧光探针DAF-FM DA、Griess Reagent法检测细胞TNF-α、O-2产量以及NO含量改变的影响.借助化学发光法、免疫荧光法分别检测各组细胞线粒体膜电位、细胞色素C含量. 结果 无论是预防性给药还是治疗性给药,人参皂苷Rb1能明显降低缺氧诱导活化的N9细胞NO、O-2以及TNF-α产量,抑制线粒体膜电位的降低,缓解细胞内细胞色素C含量的改变程度. 结论 人参皂苷Rb1均能在一定程度上下调由于缺氧活化导致神经毒性因子的高表达,稳定细胞线粒体的结构和功能,抑制缺氧诱导的N9细胞活化.     

辅助性T细胞分化发育的表观遗传学调控

CD4+初始T细胞分化发育成在机体免疫防御中发挥重要作用的辅助性T细胞(Th)亚群,此过程受控于由细胞因子和转录因子所组成的复杂涮控网络.表观遗传学通过程序性地使得祖细胞内特定基因的表达和关闭,从而调控核内转录因子作用的发挥,同时使细胞保留以应对环境信号改变而发生相应改变的潜能.     

IL‐13Rα1 is a surface marker for M2 macrophages influencing their differentiation and function

In this study, we examined the role IL‐13 receptor alpha 1 (IL‐13Rα1) plays in macrophage differentiation and function. The findings indicate that IL‐13Rα1 is expressed on the M2 but not on the M1 subset of macrophages and specifically heterodimerizes with the IL‐4Rα chain to form a type II receptor, which controls the differentiation and function of these cells. Indeed, BM cells from IL‐13Rα1^+/+ and IL‐13Rα1^?/? mice yield equivalent numbers of macrophages when cultured under M2 polarizing conditions. However, IL‐13Rα1^?/? BM cells yield a much higher number of macrophages than IL‐13Rα1^+/+ BM cells when the differentiation is carried out under M1‐polarizing conditions. Further analyses indicated that macrophages that express IL‐13Rα1 also display surface markers associated with an M2 phenotype. In addition, the IL‐13Rα1⁺ macrophages were highly efficient in phagocytizing zymosan bioparticles both in vitro and in vivo, and supported differentiation of naïve T cells to a Th2 phenotype. Finally, when stimulated by IL‐13, a cytokine that uses the heteroreceptor, the cells were able to phosphorylate STAT6 efficiently. These previously unrecognized findings indicate that IL‐13Rα1 serves as a marker for M2 macrophages and the resulting heteroreceptor influences both their differentiation and function.     

人参皂苷Rg1和Rb1对流感病毒感染细胞的影响及其机制研究

目的 研究人参皂苷Rg1和Rb1对流感病毒感染所致的细胞氧化应激损伤、细胞内转录因子(NF-κB和AP-1)转录活性及前炎症细胞因子IL-8释放的影响.方法 甲型流感病毒H3N2作为刺激因素,以DCFH-DA为探针,流式细胞仪检测细胞内活性氧(iROS)产生水平.利用双荧光素酶顺式报告系统,检测人参皂苷Rg1和Rb1对NF-κB-luc及AP-1-luc相对荧光素酶值的影响.RT-PCR法进一步验证人参皂苷Rg1和Rb1对IL-8 mRNA表达水平的影响.结果 病毒感染后细胞中ROS产生增加45%～55%,而给予有效浓度人参皂苷Rg1和Rb1后,荧光强度降低,与正常细胞内自由基含量基本相同.通过NF-κB及AP-1双荧光素酶报告系统检测发现,与正常对照组比较,病毒感染的细胞中NF-κB及AP-1荧光素酶报告活性明显升高;与病毒损伤组比较,人参皂苷Rg1、Rb1治疗组NF-κB及AP-1荧光素酶报告活性明显受到抑制.RT-PCR结果显示人参皂苷Rg1和Rb1能明显抑制病毒诱导的IL-8 mRNA表达水平的变化,使之趋近于正常对照组水平.结论 人参皂苷Rg1和Rb1可拮抗病毒感染细胞中ROS产生水平、降低氧化应激敏感的信号传导通路NF-κB及AP-1的转录活性,并抑制二者下游靶基因IL-8表达.     

人参皂苷Rb1对体外培养人脂肪干细胞增殖与成骨分化的影响

背景：脂肪干细胞成骨分化受多种因素的影响,中药成骨诱导活性因子在脂肪干细胞研究中有重要意义。 目的：观察人参皂苷Rb1在体外培养条件下对人脂肪干细胞增殖和成骨分化的影响。 方法：体外分离培养人脂肪干细胞,传至第3代后,按2×10³/孔接种至96孔板,分别加入0.5,1.0,2.0,4.0,6.0 μmol/L人参皂苷Rb1培养基200 μL进行培养;设立对照组,仅加入等量普通DMEM培养基。采用XTT比色法测定大鼠脂肪干细胞的生长增殖曲线。通过碱性磷酸酶试剂盒测定细胞碱性磷酸酶活性,放射免疫法检测骨钙素含量,茜素红染色观察钙化结节形成能力。 结果与结论：0.5 μmol/L人参皂苷Rb1可明显促进人脂肪干细胞增殖;随着人参皂苷Rb1浓度的增加,促细胞增殖活性降低,6.0 μmol/L人参皂苷Rb1表现为明显的抑制细胞增殖作用。人参皂苷Rb1呈剂量依赖性促进人脂肪干细胞碱性磷酸酶活性和骨钙素表达。4.0,6.0 μmol/L人参皂苷Rb1诱导钙化结节形成能力优于0.5,1.0和2.0 μmol/L人参皂苷Rb1,对照组人脂肪干细胞未见钙化结节形成。提示人参皂苷Rb1在一定浓度范围内对体外培养条件下的人脂肪干细胞具有促生长增殖作用,但在高浓度时,人参皂苷Rb1对人脂肪干细胞的成骨分化具有促进作用,因此可作为一种良好的成骨诱导活性因子。