蛋氨酸脑啡肽—阿片受体激活免疫细胞抗肿瘤活性的研究进展

蛋氨酸脑啡肽(MENK)是人体中的一种神经递质,其阿片受体广泛存在于免疫细胞表面。MENK与阿片受体相结合,通过调节cAMP-PKA信号通路、Ca2+-钙调蛋白、蛋白激酶C等多种物质的表达,参与到免疫细胞的成熟分化。进一步研究提示,适当剂量的MENK通过激活T细胞、NK细胞、LAK细胞等发挥抗肿瘤的作用。此外,MENK-阿片生长因子受体(OGFr)通路的过表达,直接抑制肿瘤发展与血管生长。     

甲硫氨酸脑啡肽和抗δ阿片受体抗体对小鼠骨髓瘤细胞信号传递系统的作用

为了探讨阿片肽与细胞表面受体结合后所产生的生物效应及其机制 ,用不同浓度甲硫氨酸脑啡肽 ( MENK)及抗 δ阿片受体单克隆抗体处理小鼠的骨髓瘤细胞 ( NS- 1 ) ,然后测定蛋白激酶A( PKA) ,蛋白激酶 C( PKC)活性及三磷酸肌醇 ( IP3 )含量 .研究结果表明 ,NENK可升高细胞胞浆及胞膜 PKA的活性 ,且这一作用可被抗δ阿片受体抗体所拮抗 .MENK对 PKC影响呈双向反应 ,0 .1 μmol/L MENK可以升高胞浆 PKC活性 ,但却明显降低胞膜 PKC活性 ;在 MENK浓度为 1 0μmol/L时则情况刚好相反 .1 μmol/L的 MENK可明显降低胞浆及胞膜 PKC活性 ,抗体可拮抗这种下调作用 .MENK可降低细胞内 IP3 的含量 ,且这一作用可被抗δ阿片受体抗体所拮抗 .由此可以推论 :MENK在与 δ阿片受体结合后 ,可以经过多种信号传导系统来调节细胞功能 ,从而产生不同的生物效应 .     

蛋氨酸脑啡肽抑制人胃癌细胞BGC823增殖作用及免疫调节机制

采用不同浓度梯度的蛋氨酸脑啡肽(methionine enkephalin,MENK)体外作用于人胃癌细胞BGC823后,探讨对其增殖影响及其作用机制,为胃癌的免疫治疗提供理论依据。体外培养人胃癌细胞株BGC823,PCR检测阿片受体OGFr的表达;用不同浓度(0、1、2、3、4 mg/mL)的MENK体外作用于BGC823细胞24、48、72、96 h后,MTS检测MENK对其增殖影响;流式细胞术和Annexin V-FITC/PI双染法检测4 mg/mL MENK体外处理48、72 h后BGC823细胞凋亡变化。结果显示,人胃癌BGC823细胞有阿片受体OGFr的表达;MENK可抑制BGC823细胞增殖,且随着剂量的增加和时间的延长,其抑制作用逐渐增强(P0.05);4 mg/mL MENK48 h处理组与空白组相比细胞凋亡率增加,72 h处理组与48 h处理组结果一致(P0.05)。结果表明,MENK可抑制BGC823细胞增殖,具有显著的剂量依赖性和时间依赖性,且可通过诱导细胞凋亡抑制BGC823细胞的增殖。     

三碘甲腺原氨酸对大鼠成骨细胞增殖的影响

目的:探讨三碘甲腺原氨酸(T3)对大鼠成骨细胞增殖的影响。方法:采用骨组织块法原代分离培养新生SD大鼠颅骨成骨细胞,然后配置含不同浓度(10-7mol/L、10-8mol/L、10-9mol/L)T3的培养基,与大鼠成骨细胞共培养4d,采用噻唑蓝(MTT)法检测细胞增殖能力,采用细胞化学染色法测定成骨细胞ALP活性。结果:T3可促进大鼠成骨细胞增殖,并呈剂量依赖性;随T3浓度的增高,成骨细胞表达ALP活性增强(P<0.05)。结论:10-7mol/L～10-9mol/L浓度的T3可通过刺激成骨细胞的增殖,对预防和治疗骨质疏松发挥一定作用。     

三碘甲腺原氨酸对大鼠成骨细胞增殖的影响

目的:探讨三碘甲腺原氨酸(T3)对大鼠成骨细胞增殖的影响.方法:采用骨组织块法原代分离培养新生SD大鼠颅骨成骨细胞,然后配置含不同浓度(10-7mol/L、10-8mol/L、10-9mol/L )T3的培养基,与大鼠成骨细胞共培养4d,采用噻唑蓝(MTT)法检测细胞增殖能力,采用细胞化学染色法测定成骨细胞ALP活性.结果:T3可促进大鼠成骨细胞增殖,并呈剂量依赖性;随T3浓度的增高,成骨细胞表达ALP活性增强(P＜0.05).结论:10-7mol/L～10-9mol/L浓度的T3可通过刺激成骨细胞的增殖,对预防和治疗骨质疏松发挥一定作用.     

E3泛素连接酶Smurf家族在骨形态发生蛋白和转化生长因子信号转导中的作用

泛素-蛋白酶体降解系统广泛存在于各种真核细胞中,参与调控细胞多种生理进程.作为该系统中行使调控降解功能的核心成员,E3泛素连接酶的重要作用已经越来越引起人们的重视.BMP和TGF-β是骨组织中调控成骨细胞和软骨细胞增殖、分化和凋亡的关键分子,通过不同的信号通路体系调控骨生理代谢,参与骨组织的多种生理进程.最近的研究表明,泛素-蛋白酶体降解系统在骨细胞和骨组织中具有十分重要的作用,E3泛素连接酶Smurf作为这一系统的核心,参与调控骨组织中BMP和TGF-β两个家族的分子信号转导过程.在前期成果的基础上,结合最新的研究进展,系统阐述了骨组织中E3泛素连接酶的发现,及其调控BMP和TGF-β信号通路的机制以及其对成骨细胞和软骨细胞增殖和分化的影响.     

P物质在BMSCs来源的成骨细胞与内皮细胞体外联合培养中的作用

目的：观察P物质（substanceP,SP）在BMSCs来源的成骨细胞与内皮细胞体外联合培养中的作用,研究P物质作用于种子细胞的最适浓度指导组织工程骨修复骨缺损。方法：采用新生新西兰大白兔胎兔（雌雄不限）密度梯度离心法分离骨髓间充质干细胞行体外培养和连续传代,获得较纯的BMSCs。取生长状态良好的第3代BMSCs行成骨诱导培养及成血管内皮细胞诱导培养并鉴定。将诱导7d的两种细胞按2：1比例混合培养,待细胞传至2代加入不同浓度的sP作为实验组,以正常未加sP的细胞培养基为对照组。培养后1、3、5、7d采用CCK-8法测定细胞增殖并绘制生长曲线,观察细胞生长数量,测定碱性磷酸酶活性及观察细胞周期分布。结果：浓度范围从1×10^-12-1×10^-6mol／L的sP对联合共培养的成骨细胞增殖和活性都有促进作用,在浓度为1×10^-8mol／L对联合共培养的成骨细胞增殖和活性的作用功效最强。结论：在体外直接联合共培养的体系中,SP对新种子细胞促进效果明显,其在1×10^-8mol／L对联合共培养的成骨细胞增殖和活性作用最强。     

雌激素对成骨细胞增殖及IGF-2表达的调控作用

目的 探讨雌激素对大鼠成骨细胞功能的影响及胰岛素样生长因子2（insulin-like growth factor 2,IGF-2）表达的调控机理。方法应用1×10^-10mol／L、1×10^-8mol／L、1×10^-6mol／L浓度的雌二醇（estradiol,172）分别作用原代培养的新生大鼠颅盖骨成骨细胞24h;采用MTT法和对硝基酚磷酸盐法检测成骨细胞增殖能力和细胞中碱性磷酸酶（ALP）活性;应用实时定量PCR和Western印迹杂交分析成骨细胞中IGF-2的mRNA和蛋白质的表达规律。结果1×10^-6mol／L浓度的磁使大鼠成骨细胞增殖能力和ALP活性分别增加了67％和55％,使IGF-2的mRNA与蛋白质的表达水平分别增加了90％和140％,差异有统计学意义（P〈0．05）。结论雌激素对成骨细胞中IGF-2基因的表达具有正性调控作用。成骨细胞中IGF-2的高表达可能与雌激素调节成骨细胞增殖和分泌功能相关。     

肿瘤坏死因子-α和白介素-2对成骨细胞增殖的影响

成骨细胞在骨组织改建中至关重要。本试验体外培养人骨肉瘤细胞系MG63,MTT方法检测白介素-2(Interleukin-2,IL-2)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)对成骨细胞增殖的影响。结果表明,当IL-2的浓度大于100U/mL,其能够促进成骨细胞增殖(P0.05),一定浓度(50～1000U/mL)的TNF-α对成骨细胞的增殖也有促进作用。     

FGFs/FGFRs系统对骨调节的研究进展

成纤维细胞生长因子家族(fibroblast growth factors,FGFs)及其受体FGFRs系统影响骨骼发育和形成过程,FGF与细胞表面FGFR结合,激活信号通路调控多种细胞生长、分化和凋亡。骨是FGF的重要靶器官,研究表明FGFs/FGFRs系统对骨组织成骨细胞、破骨细胞、软骨细胞的增殖和分化起重要调控作用,本文就FGFs/FGFRs系统对骨组织调节研究进展进行综述。     

Effects of insulin and insulin-like growth factor 1 on osteoblast proliferation and differentiation: differential signalling via Akt and ERK

Insulin and insulin-like growth factor 1 (IGF-1) are evolutionarily conserved hormonal signalling molecules, which influence a wide array of physiological functions including metabolism, growth and development. Using genetic mouse studies, both insulin and IGF-1 have been shown to be anabolic agents in osteoblasts and bone development primarily through the activation of Akt and ERK signalling pathways. In this study, we examined the temporal signalling actions of insulin and IGF-1 on primary calvarial osteoblast growth and differentiation. First, we observed that the IGF-1 receptor expression decreases whereas insulin receptor expression increases during osteoblast differentiation. Subsequently, we show that although both insulin and IGF-1 promote osteoblast differentiation and mineralization in vitro, IGF-1, but not insulin, can induce osteoblast proliferation. The IGF-1-induced osteoblast proliferation was mediated via both MAPK and Akt pathways because the IGF-1-mediated cell proliferation was blocked by U0126, an MEK/MAPK inhibitor, or LY294002, a PI3-kinase inhibitor. Osteocalcin, an osteoblast-specific protein whose expression corresponds with osteoblast differentiation, was increased in a dose- and time-dependent manner after insulin treatment, whereas it was decreased with IGF-1 treatment. Moreover, insulin treatment dramatically induced osteocalcin promoter activity, whereas IGF-1 treatment significantly inhibited it, indicating direct effect of insulin on osteocalcin synthesis.     

Beta 3-adrenergic receptors regulate retinal endothelial cell migration and proliferation

Sympathetic nerves may play a role in vascular disorders of the eye. In the present study, we hypothesized that activation of beta3-adrenergic receptors on retinal endothelial cells would promote migration and proliferation of these cells, two markers of an angiogenic phenotype. We show, for the first time, expression of beta3-adrenergic receptors on cultured retinal endothelial cells. Activation of these receptors with BRL37344, a specific beta3-adrenergic receptor agonist, promoted migration that was blocked by inhibitors of phosphatidylinositol 3-kinase (PI3K), the mitogen activated protein kinase component MEK, and matrix metalloproteinases (MMPs) 2 and 9. BRL37344 stimulated proliferation, which could be blocked by inhibitors of Src, PI3K, and MEK. These cells also express the beta1-adrenergic receptor with no beta2-adrenergic receptor expression observed. Stimulation of the beta1-adrenergic receptor with xamoterol, a specific partial agonist, did not promote proliferation or migration. These results support the hypothesis that beta3-adrenergic receptors play a role in proliferation and migration of cultured human retinal endothelial cells.     

Studies on the receptors mediating responses of osteoblasts to thrombin

The serine protease thrombin stimulates proliferation in osteoblasts, but decreases alkaline phosphatase (ALP) activity, a marker of osteoblast differentiation. Three thrombin receptors have been identified, protease activated receptor (PAR)-1, PAR-3 and PAR-4; we have previously demonstrated that mouse osteoblasts express PAR-1 and PAR-4. The effect of thrombin on osteoblast proliferation and differentiation was studied to determine which of the thrombin receptors is responsible for the primary effects of thrombin. Primary mouse calvarial osteoblasts from PAR-1-null and wild-type mice, and synthetic peptides that specifically activate PAR-1 (TFFLR-NH2) and PAR-4 (AYPGKF-NH2) were used. Both the PAR-1-activating peptide and thrombin stimulated incorporation of 5-bromo-2'-deoxyuridine (two to four-fold, P < 0.001) and reduced alkaline phosphatase activity (approximately three-fold, P < 0.05) in cells from wild-type mice. The PAR-4-activating peptide, however, had no effect on either alkaline phosphatase activity or proliferation in these cells. Neither thrombin nor PAR-4-activating peptide was able to affect osteoblast proliferation or alkaline phosphatase activity in cells isolated from PAR-1-null mice. The results demonstrate that thrombin stimulates proliferation and inhibits differentiation of osteoblasts through activation of PAR-1. No other thrombin receptor appears to be involved in these effects.     

Effects of insulin and insulin‐like growth factor 1 on osteoblast proliferation and differentiation: differential signalling via Akt and ERK

Insulin and insulin‐like growth factor 1 (IGF‐1) are evolutionarily conserved hormonal signalling molecules, which influence a wide array of physiological functions including metabolism, growth and development. Using genetic mouse studies, both insulin and IGF‐1 have been shown to be anabolic agents in osteoblasts and bone development primarily through the activation of Akt and ERK signalling pathways. In this study, we examined the temporal signalling actions of insulin and IGF‐1 on primary calvarial osteoblast growth and differentiation. First, we observed that the IGF‐1 receptor expression decreases whereas insulin receptor expression increases during osteoblast differentiation. Subsequently, we show that although both insulin and IGF‐1 promote osteoblast differentiation and mineralization in vitro, IGF‐1, but not insulin, can induce osteoblast proliferation. The IGF‐1‐induced osteoblast proliferation was mediated via both MAPK and Akt pathways because the IGF‐1‐mediated cell proliferation was blocked by U0126, an MEK/MAPK inhibitor, or LY294002, a PI3‐kinase inhibitor. Osteocalcin, an osteoblast‐specific protein whose expression corresponds with osteoblast differentiation, was increased in a dose‐ and time‐dependent manner after insulin treatment, whereas it was decreased with IGF‐1 treatment. Moreover, insulin treatment dramatically induced osteocalcin promoter activity, whereas IGF‐1 treatment significantly inhibited it, indicating direct effect of insulin on osteocalcin synthesis. Copyright © 2012 John Wiley & Sons, Ltd.     

Functional expression of 5-HT2A receptor in osteoblastic MC3T3-E1 cells

In the previous study, we reported the gene expression for proteins related to the function of 5-hydroxytryptamine (5-HT, serotonin) and elucidated the expression patterns of 5-HT₂ receptor subtypes in mouse osteoblasts. In the present study, we evaluated the possible involvement of 5-HT receptor subtypes and its inactivation system in MC3T3-E1 cells, an osteoblast cell line. DOI, a 5-HT_2A and 5-HT_2C receptor selective agonist, as well as 5-HT concentration-dependently increased proliferative activities of MC3T3-E1 cells in their premature period. This effect of 5-HT on cell proliferation were inhibited by ketanserin, a 5-HT_2A receptor specific antagonist. Moreover, both DOI-induced cell proliferation and phosphorylation of ERK1 and 2 proteins were inhibited by PD98059 and U0126, selective inhibitors of MEK in a concentration-dependent manner. Furthermore, treatment with fluoxetine, a 5-HT specific re-uptake inhibitor which inactivate the function of extracellular 5-HT, significantly increased the proliferative activities of MC3T3-E1 cells in a concentration-dependent manner. Our data indicate that 5-HT fill the role for proliferation of osteoblast cells in their premature period. Notably, 5-HT_2A receptor may be functionally expressed to regulate mechanisms underlying osteoblast cell proliferation, at least in part, through activation of ERK/MAPK pathways in MC3T3-E1 cells.     

Functional redundancy of type II BMP receptor and type IIB activin receptor in BMP2-induced osteoblast differentiation

Signaling pathways for bone morphogenetic proteins (BMPs) are important in osteoblast differentiation. Although the precise function of type I BMP receptors in mediating BMP signaling for osteoblast differentiation and bone formation has been characterized previously, the role of type II BMP receptors in osteoblasts is to be well clarified. In this study, we investigated the role of type II BMP receptor (BMPR-II) and type IIB activin receptor (ActR-IIB) in BMP2-induced osteoblast differentiation. While osteoblastic 2T3 cells expressed BMPR-II and ActR-IIB, loss-of-function studies, using dominant negative receptors and siRNAs, showed that BMPR-II and ActR-IIB compensated each other functionally in mediating BMP2 signaling and BMP2-induced osteoblast differentiation. This was evidenced by two findings. First, unless there was loss of function of both type II receptors, isolated disruption of either BMPR-II or ActR-IIB did not remove BMP2 activity. Second, in cells with loss of function of both receptors, restoration of function of either BMPR-II or ActR-IIB by transfection of the wild-type forms, restored BMP2 activity. These findings suggest a functional redundancy between BMPR-II and ActR-IIB in osteoblast differentiation. Results from experiments to test the effects of transforming growth factor β (TGF-β), activin, and fibroblast growth factor (FGF) on osteoblast proliferation and differentiation suggest that inhibition of receptor signaling by double-blockage of BMPR-II and ActR-IIB is BMP-signaling specific. The observed functional redundancy of type II BMP receptors in osteoblasts is novel information about the BMP signaling pathway essential for initiating osteoblast differentiation.     

Zinc Upregulates the Expression of Osteoprotegerin in Mouse Osteoblasts MC3T3-E1 Through PKC/MAPK Pathways

Zinc is an essential element for bone formation; however, its role in osteoblast has not been well understood. In the present study, we hypothesized that zinc could increase osteogenetic function by stimulating osteoblast proliferation and osteoprotegerin (OPG) activity. To test this hypothesis, osteoblastic MC3T3-E1 cells were cultured and treated with various concentrations of zinc (0, 10, 30, 50, 70, 110, 130, and 150 μM) for 24 and 48 h. 3-[4,5-dimethylthiazol-2-y]-2,5-diphenyltetrazolium bromide assay showed that cell proliferation was significantly stimulated with 50 μM zinc treatment. Furthermore, under the same treatment condition, OPG expression was significantly increased as evidenced by the results of RT-PCR and ELISA. However, the zinc-induced OPG expression was significantly attenuated when MC3T3-E1 cells were co-treated with either protein kinase C (PKC) inhibitor, GF109203X, or the Inhibitor of mitogen-activated extracellular signal-regulated kinase 1 (MEK1), PD98059. Moreover, OPG expression was further increased when MC3T3-E1 cells were treated with PMA (the activator of protein of kinase C) in the presence of zinc. These results suggested that zinc would increase osteogenic function by stimulating PKC and MAPK signaling pathways.     

Effect of 17 beta-estradiol on the proliferation of osteoblastic MC3T3-E1 cells via human monocytes

Effects of human monocyte-conditioned medium on the proliferation of osteoblastic MC3T3-E1 cells were investigated in serum-free cultured condition. Monocyte-conditioned medium significantly stimulated osteoblast proliferation at the concentration between 10 and 30%, compared to that in the absence of monocytes. 17 beta-estradiol directly stimulated osteoblast proliferation at the concentrations of 10(-8) and 10(-10)M. On the contrary, the conditioned medium prepared by monocytes cultured in the presence of 17 beta-estradiol at the concentrations of 10(-8) and 10(-10)M significantly inhibited osteoblast proliferation. Present data indicate that in addition to direct effect on osteoblasts, 17 beta-estradiol affected osteoblast proliferation presumably through modulating the release of several local regulators of bone turnover from monocytes. The effect on osteoblastic activity via monocytes might be linked to the coupling of osteoclast and osteoblast actions.     

Effect of elevated extracellular calcium on the proliferation of osteoblastic MC3T3-E1 cells:its direct and indirect effects via monocytes.

There has been evidence that elevated calcium concentration at the resorptive site of the bone directly regulates osteoclast function. In the present study, in order to clarify the role of elevated calcium concentration at the resorptive site in the regulation of osteoblast function, not only direct but also indirect effect via human monocytes of the increase in extracellular calcium (Cae) on the proliferation of osteoblastic MC3T3-E1 cells have been investigated in serum-free condition. The increase in Cae enormously stimulated osteoblast proliferation at the concentration of 3 to 20 mM. When human monocytes were cultured at the elevated Cae concentration, monocyte-conditioned medium-induced stimulation of osteoblast proliferation was significantly amplified. Present data demonstrate that elevated Cae has pronounced stimulatory effect on osteoblast proliferation not only directly but also indirectly via monocytes. Calcium released from bone matrix at the resorptive sites might be linked to the coupling of osteoclast and osteoblast functions.     

Eph B4 receptor signaling mediates endothelial cell migration and proliferation via the phosphatidylinositol 3-kinase pathway

The goals of this study were 2-fold: 1) to determine whether stimulation of Eph B4 receptors promotes microvascular endothelial cell migration and/or proliferation, and 2) to elucidate signaling pathways involved in these responses. The human endothelial cells used possessed abundant Eph B4 receptors with no endogenous ephrin B2 expression. Stimulation of these receptors with ephrin B2/Fc chimera resulted in dose- and time-dependent phosphorylation of Akt. These responses were inhibited by LY294002 and ML-9, blockers of phosphatidylinositol 3-kinase (PI3K) and Akt, respectively. Eph B4 receptor activation increased proliferation by 38%, which was prevented by prior blockade with LY294002, ML-9, and inhibitors of protein kinase G (KT5823) and MEK (PD98059). Nitrite levels increased over 170% after Eph B4 stimulation, indicating increased nitric oxide production. Signaling of endothelial cell proliferation appears to be mediated by a PI3K/Akt/endothelial nitric-oxide synthase/protein kinase G/mitogen-activated protein kinase cascade. Stimulation with ephrin B2 also increased migration by 63% versus controls. This effect was inhibited by blockade with PP2 (Src inhibitor), LY294002 or ML-9 but was unaffected by the PKG and MEK blockers. Eph B4 receptor stimulation increased activation of both matrix metalloproteinase-2 and -9. The results from these studies indicate that Eph B4 stimulates migration and proliferation and may play a role in angiogenesis.