低氧对小鼠破骨细胞分化影响的时间依赖关系

背景:平原人群到高原后不久会引起骨量丢失,在高原发生骨折会引起延迟愈合甚至不愈合.与低海拔地区相比,高海拔地区骨吸收增加,骨量减少.目的:通过观察不同时间点低氧对小鼠破骨细胞分化的影响,探讨低氧对破骨细胞分化影响可能的分子机制.方法:取小鼠骨髓细胞,用分化培养基分别在20%氧体积分数(常氧组)、和3%氧体积分数(低氧组)中培养.骨髓细胞纯化后加入到第3～5代成骨细胞进行共培养,随机分组,每组样本数为4,培养第1,3,5,7天,用TRIzol提取细胞总RNA,用半定量反转录PCR检测骨保护素、RANK及RANKLmRNA的表达;用western blotting检测RANK蛋白的表达情况.结果与结论:与常氧组相比,骨保护素mRNA的表达随着缺氧时间的延长而降低,RANKmRNA和RANK蛋白的表达随着缺氧时间的延长而升高,RANKLmRNA的表达随着缺氧时间的延长而升高.结果表明,低氧可促进破骨样细胞的分化,尤其在缺氧第5天,破骨细胞生成最多.     

双膦酸盐对破骨细胞分化中组织蛋白酶K及骨吸收功能的影响

背景：有研究表明双膦酸盐可抑制破骨细胞的骨吸收功能,但对其骨吸收功能关键细胞因子组织蛋白酶K是否产生作用,至今少有报道。目的：观察双膦酸盐对破骨细胞分化中组织蛋白酶K及骨吸收功能影响。方法：用小鼠单核巨噬细胞RAW264.7诱导培养破骨细胞。实验分2组：对照组加入质量浓度100μg/L核因子κB受体活化因子配体进行诱导至收获细胞,双膦酸盐组在对照组的基础上加入10-7 mol/L阿仑膦酸盐处理至收获细胞。培养第7天检测各组破骨细胞生成和骨吸收功能,培养72 h免疫荧光检测两组组织蛋白酶K表达差异,Western blot检测组织蛋白酶K蛋白表达情况。结果与结论：两组均有抗酒石酸酸性磷酸酶阳性多核破骨细胞生成,并在牙本质磨片上形成吸收陷窝;但对照组抗酒石酸酸性磷酸酶阳性多核细胞数目、吸收陷窝数目及陷窝面积均大于双膦酸盐组（P〈0.01）。免疫荧光检测组织蛋白酶K表达对照组强于双膦酸盐组（P〈0.01）;Western blot检测组织蛋白酶K表达双膦酸盐组低于对照组（P〈0.01）。结果证实,双膦酸盐通过抑制组织蛋白酶K因子的表达,阻碍破骨细胞的骨吸收功能。     

柚皮甙干预鼠颅顶骨破骨细胞的形成及功能

背景：柚皮甙能诱导骨形态发生蛋白2的基因表达,促进成骨细胞系的增殖和分化。体外细胞实验提示柚皮甙有抑制破骨细胞的形成及抗骨质疏松的作用。目的：建立含有柚皮甙的鼠颅顶骨培养模型,观察不同剂量下柚皮甙对破骨细胞增殖分化的影响。方法：实验选取出生4d的SD乳鼠颅顶骨,在分别含有O,1,10,100mg／L的柚皮甙的培养基中培养,于培养的第1,3,7,10天测定柚皮甙对鼠颅顶骨中破骨细胞标志酶抗酒石酸酸性磷酸酶阳性细胞数及培养基中钙离子浓度的影响。结果与结论：第1天各组间抗酒石酸酸性磷酸酶阳性细胞数及培养基中钙离子浓度无明显变化,第3,7天各组间抗酒石酸酸性磷酸酶阳性细胞数随柚皮甙质量浓度增加而减少,而培养基中钙离子浓度变化明显增加,到第10天这种变化趋势最为明显。说明柚皮甙能影响鼠颅顶骨中抗酒石酸酸性磷酸酶阳性细胞数量及其功能,并且这种作用成明显的时间剂量相关性。提示柚皮甙不仅能促进成骨细胞的增殖,同时也能减少破骨细胞的分化或加速其凋亡。     

一氧化氮外源性供体L-arginine对破骨细胞骨吸收功能的影响

目的:一氧化氮在维持机体多个系统的生理功能中起重要作用,许多慢性疾病可造成一氧化氮产生减少,此时一氧化氮供体是一种必要的补充。观察一氧化氮外源性供体L-arginine对体外培养破骨细胞增殖及骨吸收功能的影响。方法:实验于2005-06/2006-05在四川大学华西医院生物治疗国家重点实验室干细胞与组织工程研究室完成。选择出生1d的清洁级SD大鼠乳鼠,采用骨髓诱导法体外培养破骨细胞,培养液内分别加入0.3,0.6,1.0g/L不同浓度的L-arginine,并以等体积三蒸水作为对照。培养7d后,以抗酒石酸酸性磷酸酶染色观察破骨细胞数目、形态,MIAS-2000图像分析仪检测骨片上骨吸收陷窝的数目和面积,并用扫描电镜观察不同浓度L-arginine对骨吸收陷窝的影响。结果:①破骨细胞的一般形态:破骨细胞较其他细胞大,形态不规则,呈油煎蛋形、长条形、腊肠形或漏斗形等,细胞内可见几个至几十个核不等。抗酒石酸酸性磷酸酶染色酶活性部分酒红色,颗粒状。②抗酒石酸酸性磷酸酶阳性细胞数:各组破骨细胞数目随着L-arginine浓度增加而减少(P<0.05)。③骨吸收陷窝的面积和数目:骨片培养7d,吸收陷窝计数的结果显示,0.3g/L以上浓度L-arginine对破骨细胞吸收功能均有明显抑制作用,并呈剂量相关性。0.3g/LL-arginine组陷窝面积为对照组的91%(P<0.05),0.6g/LL-arginine组为对照组的80%(P<0.05),1.0g/LL-arginine组为对照组的69%(P<0.01)。结论:采用骨髓诱导法培养的破骨细胞数量多、纯度高,且具有明显的骨吸收功能。L-arginine抑制破骨细胞增殖,抑制破骨细胞骨吸收功能,并呈剂量相关性。     

不同质量浓度阿仑膦酸钠干预牙槽骨吸收模型兔牙周组织破骨细胞分化因子和骨保护素的表达

背景：有研究表明阿仑膦酸钠能够防治骨质疏松症,但其应用到口腔牙周组织干预牙槽骨吸收较为罕见。目的：建立兔牙槽骨吸收模型,观察不同质量浓度阿仑膦酸钠干预下炎症牙周组织破骨细胞分化因子和骨保护素的表达。方法：将大耳白兔建立牙槽骨吸收模型,建模成功后随机分成5组,胶原＋0.5g/L阿仑膦酸钠组、胶原＋1g/L阿仑膦酸钠组、胶原＋2g/L阿仑膦酸钠组、胶原组、对照组。各组分别于用药后2和4周用免疫组织化学方法检测破骨细胞分化因子和骨保护素表达情况,并进行药效评价。结果与结论：破骨细胞分化因子和骨保护素阳性细胞在5组成骨细胞、破骨细胞、成纤维细胞中均有表达,胞浆呈棕色或棕褐色颗粒状着色。对照组和胶原组牙槽嵴吸收区破骨细胞分化因子和骨保护素比率明显大于其他3组（P〈0.05）,胶原＋1g/L阿仑膦酸钠组在用药后2和4周均高于胶原＋0.5g/L阿仑膦酸钠组（P〈0.05）。结果证实,阿仑膦酸钠能够降低牙槽骨破骨细胞分化因子和骨保护素的比率,应用含1g/L阿仑膦酸钠胶原海绵载体更适合抑制牙槽骨吸收。     

不同质量浓度阿仑膦酸钠干预牙槽骨吸收模型兔牙周组织破骨细胞分化因子和骨保护素的表达

背景:有研究表明阿仑膦酸钠能够防治骨质疏松症,但其应用到口腔牙周组织干预牙槽骨吸收较为罕见。目的:建立兔牙槽骨吸收模型,观察不同质量浓度阿仑膦酸钠干预下炎症牙周组织破骨细胞分化因子和骨保护素的表达。方法:将大耳白兔建立牙槽骨吸收模型,建模成功后随机分成5组,胶原+0.5g/L阿仑膦酸钠组、胶原+1g/L阿仑膦酸钠组、胶原+2g/L阿仑膦酸钠组、胶原组、对照组。各组分别于用药后2和4周用免疫组织化学方法检测破骨细胞分化因子和骨保护素表达情况,并进行药效评价。结果与结论:破骨细胞分化因子和骨保护素阳性细胞在5组成骨细胞、破骨细胞、成纤维细胞中均有表达,胞浆呈棕色或棕褐色颗粒状着色。对照组和胶原组牙槽嵴吸收区破骨细胞分化因子和骨保护素比率明显大于其他3组(P<0.05),胶原+1g/L阿仑膦酸钠组在用药后2和4周均高于胶原+0.5g/L阿仑膦酸钠组(P<0.05)。结果证实,阿仑膦酸钠能够降低牙槽骨破骨细胞分化因子和骨保护素的比率,应用含1g/L阿仑膦酸钠胶原海绵载体更适合抑制牙槽骨吸收。     

不同氧环境中破骨细胞特异性基因的表达

背景：课题组以往研究证实,氧浓度过低乃至处于低氧时（体积分数2%O2）,破骨前体细胞的增殖及破骨细胞的分化和功能都受到抑制,但体外培养的破骨细胞在不同氧环境下的基因表达未见有相关报道。目的：实验拟观察不同氧环境下破骨细胞各特异性基因的表达规律,探寻氧环境改变影响破骨细胞分化的表达机制。方法：将破骨前体细胞株经质量浓度均为10μg/L的核因子κB受体活化因子配体和巨噬细胞集落刺激因子联合诱导成为成熟的破骨细胞,然后分别置于常氧、组织氧、低氧（体积分数20%,7%,2%）条件下培养,用抗酒石酸酸性磷酸酶染色检测破骨细胞形成的变化,并分别在培养第1-7天收集细胞,通过定量PCR方法检测核因子κB受体活化因子,肿瘤坏死因子受体相关因子6,抗酒石酸酸性磷酸酶,组织蛋白酶K基因mRNA的表达。结果与结论：低氧条件下抗酒石酸酸性磷酸酶阳性的破骨细胞数显著低于组织氧和常氧培养时形成的抗酒石酸酸性磷酸酶阳性细胞数（P〈0.05）。不同氧环境下,破骨细胞中核因子κB受体活化因子mRNA的表达无明显改变,组织氧和常氧培养下肿瘤坏死因子受体相关因子6 mRNA的表达在培养第5天最高（P〈0.05）。随着氧浓度的降低,抗酒石酸酸性磷酸酶和组织蛋白酶K mRNA表达时间延后,组织氧培养条件下此2者的表达可以保持在最高水平。结果证实,与常氧和低氧条件相比,破骨细胞在组织氧培养条件下培养时更易促进其分化,从而维持其活性和功能。     

双膦酸盐对破骨细胞分化及抗酒石酸酸性磷酸酶的影响

背景：抗酒石酸酸性磷酸酶是破骨细胞分化及骨吸收功能的特异性标志酶,是破骨细胞分化成熟的标志。目的：观察双膦酸盐对破骨细胞分化及骨吸收功能相关因子抗酒石酸酸性磷酸酶的影响。方法：小鼠单核巨噬细胞RAW264.7诱导培养破骨细胞。实验分2组：对照组开始时加入质量浓度100μg/L核因子kB受体活化因子配体进行诱导至收获细胞,双膦酸盐组在对照组的基础上加入10-7 mol/L阿仑膦酸盐处理至收获细胞。培养第7天检测各组破骨细胞生成和骨吸收功能,免疫荧光检测两组抗酒石酸酸性磷酸酶表达的差异,Western blot检测抗酒石酸酸性磷酸酶蛋白表达情况。结果与结论：各组细胞均有抗酒石酸酸性磷酸酶阳性多核破骨细胞生成,并在牙本质磨片上形成吸收陷窝;但对照组抗酒石酸酸性磷酸酶阳性多核细胞数目、吸收陷窝数目及陷窝面积均大于双膦酸盐组（P〈0.01）。免疫荧光检测显示,对照组抗酒石酸酸性磷酸酶表达均强于双膦酸盐组（P〈0.01）。Western blot检测显示,双膦酸盐组抗酒石酸酸性磷酸酶蛋白的表达低于对照组（P〈0.01）。说明双膦酸盐通过抑制抗酒石酸酸性磷酸酶蛋白的表达,阻碍破骨细胞分化生成及骨吸收功能。     

V-ATPase a3转运系统影响破骨细胞、骨吸收及骨折愈合的分子机制

背景：V-ATPase a3转运系统在破骨细胞对于骨吸收机制中起重要作用。目的：归纳总结V-ATPase a3转运系统在骨折修复中的表达,以及V-ATPase a3转运系统抑制剂对于骨折愈合的影响,进而能更好的指导临床。方法：在国内外期刊数据库中检索近10年内国内外文献,按检索关键词检索相关文献,筛选出符合纳入标准的文献,对其文献进行质量评估后采纳。结果与结论：V-ATPase a3转运系统广泛存在于真核细胞的细胞质膜和细胞器膜上,V-ATPase a3有2个结构域V0和V1,V0结构域是质子转运的通道,V1结构域主要是水解ATP供能。V-ATPase a3转运系统集中存在于破骨细胞皱褶缘上,逆浓度梯度转运H＋,为破骨细胞提供酸性环境,溶解无机物,为水解酶创造微环境降解有机物,参与骨吸收。因而V-ATPase a3转运系统在骨折的修复与重塑中选作研究靶点。     

全身振动对绝经后妇女骨量和骨代谢的影响

目的:寻找一种新型的、安全有效的防治绝经后骨质疏松的办法。方法:对北京市罗庄里小区28例49—65岁绝经后妇女进行问卷调查、腰椎骨密度(BMD)、双侧股骨上端BMD,护骨素(OPG)、抗酒石酸酸性磷酸酶(TRAP)和骨特异性磷酸酶(BALP)等测定。随机分为实验组和对照组,实验组采用振动干预,对照组无任何干预。振动方案为:频率30—45Hz、20min/次,3次/周,连续3个月。结果:在实验前和实验后,组间对比BMD均无明显变化。组内对比,实验组腰椎和股骨上端BMD较实验前无明显改变(P=0.397);而对照组左侧股骨上端BMD和双侧股骨BMD均值均较实验前降低0.01g/cm2(P<0.05);实验组与对照组血清TRAP、BALP、OPG在振动干预结束后较实验前均无明显变化。两组血清TRAP、OPG在实验前后分别进行组间对比也无明显变化(P>0.05);而实验前,实验组BALP明显高于对照组5.61μg/L(P<0.05),但3个月后,差异扩大到6.42μg/L(P<0.01),但两组的变化值间没有明显差异(P>0.05)。结论:振动方案对绝经后女性血清BALP、TRAP、OPG无明显影响,但可延缓股骨上端骨丢失。     

Exogenous nitric oxide regulates activity and synthesis of vascular endothelial nitric oxide synthase

Background Nitric oxide (NO) – a major signalling molecule of the vascular system – is constitutively produced in endothelial cells (EC) by the endothelial NO synthase (eNOS). Since a reduced NO synthesis is an early sign of endothelial dysfunction and NO delivering drugs are used to substitute the impaired endothelial NO production, we addressed the effect of exogenous NO on eNOS in human umbilical venous endothelial cell cultures. Materials and methods The synthetic NO donor DETA/NO (trade name, but in the following we refer to detNO), that releases NO in a strictly first order reaction with a half life of 20 h, was used in our experiments. Results Short‐term (20–30 min) detNO treatment of EC increases the Ser¹¹⁷⁷ phosphorylation of the constitutively expressed endothelial NOS and the production of endogenous NO generated by eNOS from [³H]arginine. The phosphorylation of eNOS is Akt‐dependent and completely reverted by the phosphatidylinositol‐3 kinase (PI‐3K) inhibitor LY294002. A prolonged continuous exposure of EC to detNO 150 µmol L^?1 over a period of 24–48 h causes a reversible cell cycle arrest at G₁‐phase associated with a larger cell volume and increased cell protein content (hypertrophic phenotype of EC). The eNOS protein and mRNA of the hypertrophic cells and the generation of endogenous NO are reduced but eNOS phosphorylation could still be elevated by stimulation with vascular endothelial growth factor. Conclusions Our data explain clinical studies describing a short‐term but not a long‐term benefit of NO treatment for patients with cardiovascular risk factors. The results could be a rational approach to develop a generation of NO donors accomplishing a retarded release from NO donors that mimic the low continuous pulsatile stress‐induced release of endogenous NO.     

Hypercholesterolemia decreases nitric oxide production by promoting the interaction of caveolin and endothelial nitric oxide synthase

Hypercholesterolemia is a central pathogenic factor of endothelial dysfunction caused in part by an impairment of endothelial nitric oxide (NO) production through mechanisms that remain poorly characterized. The activity of the endothelial isoform of NO synthase (eNOS) was recently shown to be modulated by its reciprocal interactions with the stimulatory Ca2+-calmodulin complex and the inhibitory protein caveolin. We examined whether hypercholesterolemia may reduce NO production through alteration of this regulatory equilibrium. Bovine aortic endothelial cells were cultured in the presence of serum obtained from normocholesterolemic (NC) or hypercholesterolemic (HC) human volunteers. Exposure of endothelial cells to the HC serum upregulated caveolin abundance without any measurable effect on eNOS protein levels. This effect of HC serum was associated with an impairment of basal NO release paralleled by an increase in inhibitory caveolin-eNOS complex formation. Similar treatment with HC serum significantly attenuated the NO production stimulated by the calcium ionophore A23187. Accordingly, higher calmodulin levels were required to disrupt the enhanced caveolin-eNOS heterocomplex from HC serum-treated cells. Finally, cell exposure to the low-density lipoprotein (LDL) fraction alone dose-dependently reproduced the inhibition of basal and stimulated NO release, as well as the upregulation of caveolin expression and its heterocomplex formation with eNOS, which were unaffected by cotreatment with antioxidants. Together, our data establish a new mechanism for the cholesterol-induced impairment of NO production through the modulation of caveolin abundance in endothelial cells, a mechanism that may participate in the pathogenesis of endothelial dysfunction and the proatherogenic effects of hypercholesterolemia.     

Low‐intensity ultrasound increases endothelial cell nitric oxide synthase activity and nitric oxide synthesis

Summary. Low‐intensity ultrasound (US) increases tissue perfusion in ischemic muscle through a nitric oxide (NO)‐dependent mechanism. We have developed a model to expose endothelial cells to well‐characterized acoustic fields in vitro and investigate the physical and biological mechanisms involved. Human umbilical vein endothelial cells (HUVEC) or bovine aortic endothelial cells (BAEC) were grown in tissue culture plates suspended in a temperature‐controlled water bath and exposed to US. Exposure to 27 kHz continuous wave US at 0.25 W cm^?2 for 10 min increased HUVEC media NO by 102 ± 19% (P < 0.05) and BAEC by 117 ± 23% (P < 0.01). Endothelial cell NO synthase activity increased by 27 ± 24% in HUVEC and by 32 ± 16% in BAEC (P < 0.05 for each). The cell response was rapid with a significant increase in NO synthesis by 10 s and a maximum increase after exposure for 1 min. By 30 min post‐exposure NO synthesis declined to baseline, indicating that the response was transient. Unexpectedly, pulsing at a 10% duty cycle resulted in a 46% increase in NO synthesis over the response seen with continuous wave US, resulting in an increase of 147 ± 18%. Cells responded to very low intensity US, with a significant increase at 0.075 W cm^?2 (P < 0.01) and a maximum response at 0.125 W cm^?2. US caused minor reversible changes in cell morphology but did not alter proliferative capacity, indicating absence of injury. We conclude that exposure of endothelial cells to low‐intensity, low‐frequency US increases NO synthase activity and NO production, which could be used to induce vasodilatation experimentally or therapeutically.     

Fatty acid incorporation in endothelial cells and effects on endothelial nitric oxide synthase

BACKGROUND: The nature of fatty acids provided by the diet as well as plasma lipid metabolism can modify the composition and properties of plasma membrane and thus the activity of membrane proteins. In humans, as well as in experimental models, diabetes is associated with both an alteration in serum lipid profile and a documented endothelial dysfunction. This in vitro study investigated on an immortalized human endothelial cell line (EA.hy 926) the specific effects of several free fatty acids (FFAs) on the composition of cellular membranes and the regulation of endothelial nitric oxide synthase (eNOS). MATERIALS AND METHODS: 0.1% of lipid deprived serum was added to the incubation medium with 25 mM glucose in order to study the effects of individual fatty acids: myristic acid, palmitic acid, stearic acid, oleic acid or linoleic acid at 100 microM bound with albumin. The effects of the FFAs on the endothelial nitric oxide synthase were investigated on mRNA level by quantitative PCR, on protein level and Ser1177 phosphorylation by Western blot and on enzymatic activity on living cells using radiolabelled arginine. RESULTS: Free linoleic acid increased the membrane content in n-6 fatty acids (mainly C18: n-6 and its metabolites) with a decrease in saturated and monounsaturated fatty acids. These conditions decreased the basal eNOS activity and reduced the phosphorylation of eNOS-Ser1177 due to activation by histamine. Free palmitic acid enriched the membranes with 16 : 0 with a slight decrease in monounsaturated fatty acids. These conditions increased eNOS activation without increasing Ser1177 phosphorylation upon histamine activation. The addition of the other FFAs also resulted in modifications of membrane composition, which did not to affect eNOS-Ser1177 phosphorylation. CONCLUSION: Among the fatty acids used, only modification of the membrane composition due to linoleic acid supply disturbed the basal enzymatic activity and Ser1177 phosphorylation of eNOS in a way that limited the role of histamine activation. Linoleic acid might involve the dysfunction of both eNOS basal activity and its phosphorylation status and may then contribute to an impaired vasodilatation in vivo.     

地塞米松对急性颅脑损伤患者血内NOS活性和NO产量的影响

目的 了解地塞米松（ＤＭ）对急性颅脑损伤（ＡＣＩ）患者围术期血内一氧化氮合成酶（ＮＯＳ）活性和一氧化氮（ＮＯ）产量的影响。方法 ２０例ＡＣＩ患者随机分为ＤＭ治疗组（于麻醉诱导前０．４ｍｇ／ｋｇＩＶ）和对照组,用分光光度法测定血清ＮＯＳ活性和ＮＯ产量。结果 术前两组ＮＯＳ、ＮＯ均高于下沉对照组,其中ＮＯＳ有显著性差异（Ｐ〈０．０１）。ＤＭ组自麻醉诱导前至开颅手术后２４小时（Ｔ０～Ｔ５）动态观察发现,ＮＯ、ＮＯＳ自始至终呈降低趋势;而对照组则呈显著性升高（ＮＯＴ１、Ｔ５,ＮＯＳＴ５）,组间比较有显著性差异。结论 ＡＣＩ患者术前存在ＮＯＳ、ＮＯ代谢率乱,开颅手术后其含量进一步升高而加重脑缺血再灌注损伤。麻醉诱导时应用ＤＭ可显著抑制ＡＣＩ患者体内ｉＮＯＳ释放具有脑保护作用。可作为选择性ｉＤＯＳ拮抗剂在临床应用。     

肾血管性高血压对诱导型一氧化氮合酶表达及活性的影响

目的通过测定肾血管性高血压大鼠血管及肾组织诱导型一氧化氮合酶（iNOS）活性及表达的变化情况,探讨血压与iNOS间的关系。方法运用肾动脉不全结扎方法制备SD大鼠肾血管性高血压模型,并应用Greiss反应、L-精氨酸同位素标记法及Westernblot等分别测定一氧化氮的终产物——尿中NO