Forskolin对成骨样细胞蛋白激酶C及三磷酸肌醇的影响

Ｆｏｒｓｋｏｌｉｎ（ＦＳＫ）是一种植物二萜类化合物,为腺苷酸环化酶的特异激活剂。实验发现：ＦＳＫ和作为参照的诱导分化剂维甲酸（ＲＡ）单独或联合应用均可升高胞浆蛋白激酶Ｃ（ＰＫＣ）活性,并降低膜ＰＫＣ活性。ＦＳＫ可使表皮生长因子（ＥＧＦ）诱导的细胞内三磷酸（ＩＰ３－１,４,５）水平降低至对照组的４４．４％至６７％;ＦＳＫ与ＲＡ合用可显著降低成骨样细胞特征蛋白碱性磷酸酶（ＡＫＰ）的活性,以上结果表明,     

模拟失重对人成骨样细胞凋亡的影响

为了探讨失重对人成骨样细胞凋亡情况的影响及对相关分子的作用,采用双向多样本回转器模拟失重效应,将培养的人成骨样细胞MG-63随机分为静止对照组、水平旋转对照组和失重实验组(用回转器模拟失重条件),在实验的12 h取细胞用流式细胞仪检测早期凋亡情况,同时检测bcf-2、NF-κB(p65)mRNA和P53的表达.结果显示,在模拟失重12 h时,MG-63细胞表现出一定的早期凋亡趋势,且bcl-2、NF-κB(p65)的表达明显降低,P53表达增加,提示失重可能通过影响这几种凋亡相关因子的表达,启动成骨细胞凋亡,从而破坏骨形成和骨吸收之间的平衡.成骨细胞凋亡的启动可能是航天员骨丢失的原因之一.     

甲状腺素及维甲酸对成骨样细胞甲状旁腺素受体的调节作用

研究了甲状腺素及维甲酸夺大鼠成骨样细胞ＲＯＳ１７／２．８细胞株甲状旁腺素受体的调节作用实验结果表明：细胞经Ｔ３／Ｔ４处理后,可显增高ＰＴＨ受体结合率及碱性磷酸酶活性,以及ＰＴＨ受体ｍＲＮＡ的表达,细胞经ＲＡ处理后,则相反地降低了ＰＴＨ受体结合率及碱性磷到酶活性。     

牵张作用对成骨样细胞MG63的线粒体跨膜电位的影响

目的:探讨在低渗透压形成的静牵张应力环境下线粒体跨膜电位与细胞增殖分化及凋亡的关系。方法:用成骨样细胞MG63细胞株进行体外培养、扩增,在对数生长期采用不同的低渗透压对细胞进行刺激,检测不同作用务件下线粒体跨膜电位(ΔΨm)、细胞增殖比例(S期百分比)以及凋亡指数。结果:240mOsm组ΔΨm呈上升趋势,4h达到峰值,6h逐渐下降,但仍高于对照组;163 mOsm组ΔΨm在6 h时明显降低。277和240 mOsm组S期百分比在6 h和8 h达到峰值(26.54±0.71,28.10±0.39:26.96±0.33,28.55±0.26)。三个实验组的凋亡峰均提前,且大于对照组,尤以163 mOsm组为最(54.87±0.78)。结论:成骨样细胞MG63ΔΨm的变化与时间和力学刺激强度有一定的依赖性,预示线粒体跨膜电位的变化与细胞增殖活性之间存在一定的关系。     

毛喉萜对大鼠成骨样细胞内Ca^2＋释出的影响

毛喉萜是一种二萜类化合物,为细胞中腺苷酸环化酶的激活剂,用毛喉萜处理大发习成骨样细胞,发现它可即时激发细胞内Ｃａ＾２＋水平的增高,当细胞经毛喉萜长期处理（１－２天）后,其对ＰＴＨ激发细胞内Ｃａ＾２＋释出的交应也呈增高反应,但毛喉萜的即刻反应与其长期效应的作用机理可能并不相同,鉴于毛喉萜具有抑制增殖的作用,其对细胞内Ｃａ＾２＋ 水平的调节作用或与其对细胞的抑增殖促分化有关。     

甲状旁腺素对成骨样细胞增殖的调节作用

甲状旁腺素（ＰＴＨ）是调节钙磷代谢的经典激素,有报道ＰＴＨ对其靶细胞－成骨细胞有促增殖分化作用。经多层次、多水平的实验研究证实,ＰＴＨ对成骨样细胞ＲＯＳ１７／２．８确有促增殖作用。（１）细胞计数、ＭＴＴ［３－（４,５－ｄｉｍｅｔｈｙｌｔｈｉａ－ｚｏｌ－ｚ－ｙｉ）２,５－ｄｉｐｈｅｎｙｌｔｅｔｒａｚｏｌｉｕｍｂｒｏｍｉｄｅ］测定及ＳＲＢ（ｓｏｄｉｕｍｒｈｏｄａｍｉｎｅＢ,ＳＲＢ）染色均显示经ＰＴＨ（１０－９ｍｏｌ／Ｌ）处理的细胞,其数目明显增加;（２）３Ｈ－ＴｄＲ参入增加;（３）与增殖相关的原癌基因（ｃ－ｆｏｓ、ｃ－ｊｕｎ、ｃ－ｋｉ－ｒａｓ和ｃ－ｍｙｃ）的表达增强;（４）成骨细胞特征性蛋白－碱性磷酸酶活性降低．这些结果不仅表明该激素具有非经典样作用,同时意味着激素也参与其靶细胞增殖分化的调节作用     

蛋白激酶C的分子异质性

蛋白激酶C(PKC)是由多种亚类组成的蛋白质大家族;这个家族成员具有各自独立的酶学特性、不同的组织表达及胞内定位;在加工与调节对外来信号起反应的生理与病理应答过程中,不同亚类的激酶有不同的功能。     

非编码RNA调控血管平滑肌细胞成骨样表型转化的研究进展

分化成熟的血管平滑肌主要功能是收缩血管、调节血管周径及血压等.在高磷、高糖、维生素D₃、炎症等因素的作用下,平滑肌细胞可转分化为成骨样细胞参与血管钙化的形成,诱发心脑血管不良事件.非编码RNA是经基因转录但不翻译为蛋白质的一类RNA总称,其通过调控多种细胞活动来参与机体的生理和病理过程.已有研究表明,非编码RNA可通过调控血管平滑肌细胞成骨样表型转化影响血管钙化的发生、发展.本文从微小RNA、长链非编码RNA、环状RNA几方面综述非编码RNA在血管平滑肌成骨样表型转化中的调节作用,有助于进一步了解血管钙化的分子机制以及发现防治血管钙化的新靶点.     

过表达Runx2促进C2C12细胞成骨分化

利用Tet-on(Tetracycline-on)基因表达系统,通过强力霉素(doxycycline,DOX)诱导Runx2基因在C2C12细胞中的表达,探究Runx2促成骨分化功能,为其分子机制的研究提供一个理想的实验平台.先后将调控质粒pTet-on和反应质粒pTRE-Flag-Runx2转染入C2C12细胞,并用G418和潮霉素分别进行2轮筛选,运用实时荧光定量PCR选择对强力霉素诱导敏感的细胞克隆.用不同浓度DOX诱导C2C12/Tet/pTRE-Flag-Runx2细胞,蛋白免疫印迹检测Runx2的表达,确定DOX的最佳诱导浓度与时间,并检测C2C12细胞的成骨分化能力.结果表明,诱导细胞最佳DOX浓度为10μg/ml;最佳诱导时间为12h;诱导后Runx2基因高表达,C2C12细胞向成骨方向分化(P0.05).成功建立Tet调控Runx2基因表达C2C12细胞系,为进一步研究Runx2基因功能分子机制提供理想的细胞模型.     

肿瘤坏死因子α对大鼠成骨样细胞增殖与甲状旁腺素受体表达的影响

大鼠成骨样细胞ＲＯＳ１７／２．８经肿瘤坏死因子α处理后,发现其ｃ－ｍｙｃ,ｃ－ｆｏｓ,ｃ－ｊｕｎ等原癌基因ｍＲＮＡ的表达降低,细胞增殖明显受到抑制;同时,其ＰＴＨ受体的表达以及ＰＴＨ经受体刺激后引起的细胞内ｃＡＭＰ的增加也受到抑制。提示ＴＮＦα可抑制大鼠在骨要细胞ＲＥＯＳ１７／２．８的增殖和分化。     

毛喉萜对大鼠成骨样细胞内Ca~（2＋）释出的影响

毛喉萜是一种二萜类化合物,为细胞中腺苷酸环化酶的激活剂。用毛喉萜处理大鼠成骨样细胞,发现它可即时激发细胞内Ｃａ~（２＋）水平的增高。当细胞经毛喉萜长期处理（１－２天）后,其对ＰＴＨ激发细胞内Ｃａ~（２＋）释出的效应也呈增高反应。但毛喉萜的即刻反应与其长期效应的作用机理可能并不相同。鉴于毛喉萜具有抑制增殖的作用,其对细胞内Ｃａ~（２＋）水平的调节作用或与其对细胞的抑增殖促分化有关。     

Cyclic AMP-Independent Inhibition of Voltage-Sensitive Calcium Channels by Forskolin in PC12 Cells

Abstract: Forskolin has been used to stimulate adenylyl cyclase. However, we found that forskolin inhibited voltage-sensitive Ca²⁺ channels (VSCCs) in a cyclic AMP (cAMP)-independent manner in PC12 cells. Ca²⁺ influx induced by membrane depolarization with 70 m M K⁺ was inhibited when cells were preincubated with 10 µ M forskolin. Almost maximum inhibitory effect on Ca²⁺ influx without any significant increase in cellular cAMP level was observed in PC12 cells exposed to forskolin for 1 min. In addition, the forskolin effect on Ca²⁺ influx was not affected by the presence of 2',5'-dideoxyadenosine, an inhibitor of adenylyl cyclase that reduces dramatically forskolin-induced cAMP production. 1,9-Dideoxyforskolin, an inactive analogue of forskolin, also inhibited ∼80% of Ca²⁺ influx induced by 70 m M K⁺ without any increase in cAMP. The data suggest that forskolin and its analogue inhibit VSCCs in PC12 cells and that the inhibition is independent of cAMP generation.     

RFP134对UMR106细胞膜脂流动性的影响(英文)

癌细胞具有与正常细胞不同的膜脂流动性,导致细胞对生长因子和癌基因产物反应敏感;引起细胞增殖失控。本实验室从植物中发现一种二萜类活性物质──RFP134,在细胞周期和信号传递等多方面表现出有抑制癌细胞增殖,促进细胞分化的作用。本文以大鼠成骨肉瘤细胞（UMR106）和正常大鼠成骨细胞为模型,研究其对癌细胞膜脂流动性的影响。细胞系UMR106由美国麻省总医院内分泌室赠送。成骨细胞由本实验室分离培养。以不同浓度（20、40、60、80、100μM／L）的RFP134,在同一时间处理细胞,或以最适浓度（50μM／L）在不同时间作用于细胞。DPH为荧光标记物,测得的荧光偏振值和微粘度值为膜膜流动性指标。结果显示,无论在恒定的时间、以不同浓度的RFP134作用于UMR106细胞（Fig.1B）,或以恒定的浓度、在不同时间处理UMR106细胞（Fig．1D）,结果均表现为显著降低膜脂流动性。前者,RFP134作用于细胞时,细胞荧光偏振值与微粘度值逐步升高,其变化呈量效关系;而后者,呈时效关系。但在最适浓度与最佳作用时间,荧光偏振值和微粘度值达饱和状态。在同样条件下,RFP134对正常成骨细胞的膜脂流动性影响极小。即：荧光偏振值和微粘度值均在正常范围内保持恒定（Fig．1A;Fig．1C）。RFP134降低癌细胞的膜脂流动性     

Effect of Forskolin and Prostaglandin E1 on Stimulus Secretion Coupling in Cultured Bovine Adrenal Chromaffin Cells

Treatment of adrenal chromaffin cells with forskolin (0.1-10 microM) stimulated cyclic AMP levels, reduced the maximal stimulation of release of noradrenaline by nicotine, and increased release in response to elevated external potassium and the calcium ionophore A23187. The presence of the phosphodiesterase inhibitor Ro 20-17-24 with forskolin potentiated both the stimulation of cyclic AMP and the inhibition of nicotine-induced noradrenaline release. Dibutyryl cyclic AMP, and the elevation of cyclic AMP with prostaglandin E1, also attenuated nicotine-stimulated release. However, when the stimulation of intracellular cyclic AMP production by prostaglandin E1 was potentiated by low levels of forskolin, there was not a concomitant potentiation of effect on noradrenaline release. Dideoxyforskolin, an analogue of forskolin which does not stimulate adenylate cyclase, inhibited both potassium- and nicotine-stimulated release, probably by a mechanism unrelated to the action of forskolin in these experiments. Using Fura-2 to estimate free intracellular calcium levels, both forskolin and dideoxyforskolin (at 10 microM) reduced the calcium transient in response to nicotine. These results support a model in which elevation of cyclic AMP inhibits the activation of nicotinic receptors, but augments stimulus secretion coupling downstream of calcium entry. The data, however, do not indicate a simple relationship between total intracellular cyclic AMP levels and the attenuation of nicotinic stimulation of release.     

Adenosine Receptors Activate Adenylate Cyclase and Enhance Secretion from Bovine Adrenal Chromaffin Cells in the Presence of Forskolin

Cells of the adrenal medulla release not only catecholamines but also high concentrations of neuropeptides and nucleotides. Chromaffin cells, like many neuronal cells, have a diversity of receptors: adrenergic receptors, peptide receptors, histamine receptors, and dopamine receptors. We recently reported that these cells have nucleotide receptors that can mediate inhibition of the secretory response. The present studies show that adenosine, in the presence of enabling concentrations of forskolin, can potently enhance response to nicotinic stimulation. Neither adenosine nor forskolin alone produces a significant effect. A marked rise in intracellular cyclic AMP (cAMP) concentration is associated with the enhancement of secretion caused by forskolin plus adenosine. A phosphodiesterase inhibitor, Ro 20-1724, used together with forskolin produces significant increases in both cellular cAMP content and catecholamine secretion. However, the adenosine agonist 5'-N-ethylcarboxyadenosine elevates cellular cAMP content in the presence of forskolin without having any positive effect on secretion. This finding suggests that the rise in cAMP level may not be the sole cause of the increase in secretion by adenosine.     

甲状旁腺激素PTH1—34对成骨细胞的机械应答的影响

目的：探讨PTH对成骨细胞在机械载荷状态下的功能影响。方法：人成骨样细胞株MG63复苏培养,采用163mOsm低渗液作为刺激源,实验分为三组,A组为单纯用DMEM培养基培养;B组为低渗液对成骨细胞分别作用0．5h、2h、4h和6h;C组为低渗处理同期加入hPTH1-34（20ng／L）。检测细胞内钙离子浓度[Ca2＋]、细胞凋亡指数以及增殖活力的变化。所得结果采用单因素方差分析法。结果：随着培养时间延长,[Ca2＋]i升高,以B组升高最为明显,（与A组比较p〈0．05）,A组和C组比较p〉0．05;细胞凋亡指数在B组4h和6h为最高（29．38±0．336;54．87±0.781）,明显高于A组和C组,且与两组相比均p〈0．05,C组4h凋亡率低于A组（p〈0．05）,其它时间组统计学比较p〉0．05。细胞增殖活力随着培养时间增加逐渐增加,与A组和C组同期相比,B组较低,且p〈0.05。结论：过强的低渗牵张作用对细胞有损害作用,[Ca2＋]i降低,凋亡率增加且降低增殖活性;PTH通过维持[Ca2＋]i浓度,对细胞的功能具有保护作用。     

中药固真方对UMR106细胞周期蛋白表达及细胞周期的影响

应用流式细胞技术和 Western blot技术 ,观察了中药固真方对大鼠成骨肉瘤细胞 UMR1 0 6细胞的细胞周期及细胞周期蛋白 cyclin E和 cyclin A表达的影响 .结果显示 :固真方处理 UMR1 0 6细胞后 2 4 h,S期细胞百分比达高峰 ,占 49.6% ,而对照组 30 h时 ,S期细胞才增高 ,达 43.2 % .cy-clin E的表达在固真方处理 8h时增高 ,而对照组在 1 6h时 ,cyclin E表达才达高峰 .cyclin A的表达在给药组处理 2 4 h时最高 ,而对照组在 30 h时 ,cyclin A表达才增高 .揭示固真方可缩短细胞周期的时程 ,加速细胞周期的运行 ,从而促进细胞增殖 .     

Forskolin对放射性转化细胞TGF基因及部分癌基因表达的影响

3~H-TdR放射性转化细胞(TC3H/10)经1×10~(-5)mol/L Foskolin处理24 h后,TGFα、c-myc、c-K-ras基因的mRNA表达下降,TGFβ、c-fos基因表达无明显变化。非转化细胞(NC3H/10)经相同条件处理,TGFα、TGFβ、c-myc及c-K-ras基因表达无显著改变。提示:Forskolin介导的转化细胞的生长抑制作用与TGFa、cmyc、c-K-ras基因的转录表达下降有关。     

Effects of Phorbol Esters and Forskolin on Basal and Histamine-induced Accumulation of Inositol Phosphates in Cultured Bovine Adrenal Chromaffin Cells

The effect of phorbol esters and forskolin pretreatment on basal and histamine-induced accumulation of inositol phosphates and catecholamine release was examined in cultures of bovine adrenal chromaffin cells. Histamine caused a dose-dependent, Ca2+-dependent accumulation of total inositol phosphates with an EC50 at approximately 1 microM and an eight- to 10-fold increase at 100 microM within 30 min of incubation. Histamine (10 microM) also caused the release of cellular catecholamines amounting to some 2.8% of cellular stores released over a 20-min period. Both the inositol phosphate and catecholamine responses were completely blocked by the H1-antagonist mepyramine and were insensitive to the H2-antagonist cimetidine. Examination of the time course of accumulation of the individual inositol phosphates stimulated by histamine revealed an early and sustained rise in inositol 1,4-bisphosphate content but not inositol 1,4,5-trisphosphate content at 1 min and the overall largest accumulation of inositol monophosphate after 30 min of stimulation. Pretreatment with the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) resulted in a dose-dependent, time-dependent inhibition of histamine-induced inositol phosphate formation and catecholamine secretion. In this inhibitory action, PMA exhibited high potency (IC50 of approximately 0.5 nM), an effect not shared by the inactive phorbol ester 4-alpha-phorbol 12,13-didecanoate. Pretreatment with forskolin, on the other hand, only marginally inhibited the histamine-induced inositol phospholipid metabolism and catecholamine secretion. These data suggest that protein kinase C activation in chromaffin cells may mediate a negative feedback control on inositol phospholipid metabolism.     

Enhancement of Stimulation-Evoked Catecholamine Release from Cultured Bovine Adrenal Chromaffin Cells by Forskolin

Acetylcholine (ACh) increased cyclic AMP levels in cultured bovine chromaffin cells with a peak effect at 1 min after the addition. Pretreatment with forskolin (0.3 microM) enhanced the ACh-evoked cyclic AMP increase. The catecholamine (CA) release induced by ACh was enhanced by forskolin, but forskolin alone did not enhance the CA release. The effect of forskolin increased dose-dependently up to 1 microM, but decreased at higher concentrations. Dibutyryl cyclic AMP (DBcAMP) also enhanced ACh-evoked CA release, but the effect was less potent than that of forskolin. Forskolin enhanced both [3H]norepinephrine ([3H]NE) and endogenous CA release evoked by 30 mM K+ from cells that were preloaded with [3H]NE. The effects of forskolin were substantial when CA release was evoked with low concentrations of ACh or excess K+, but decreased with higher concentrations of the stimulants. Forskolin also enhanced the CA release induced by ionomycin and veratrine, or by caffeine in Ca2+-free medium. The potentiation by forskolin of the ACh-evoked CA release was manifest in low Ca2+ concentrations in the medium, but decreased when Ca2+ concentration was increased. These results suggest that cyclic AMP may play a role in the modulation of CA release from chromaffin cells.