阿胶对体外培养大鼠成骨细胞增殖、分化功能的影响

目的 观察阿胶含药血清对体外培养大鼠成骨细胞增殖、分化功能的影响.方法 应用MTT法观察阿胶对体外培养成骨细胞增殖功能的影响,ELISA法观察阿胶对体外培养成骨细胞ALP含量的影响.结果 三个剂量组的阿胶含药血清对体外培养大鼠成骨细胞的增殖无促进作用,但对体外培养大鼠成骨细胞内ALP的合成有明显的促进作用.结论 阿胶对体外培养大鼠成骨细胞的增殖无促进作用,但能促进体外培养大鼠成骨细胞的分化功能.     

骨碎补总黄酮对大鼠骨髓间充质干细胞成骨分化过程中TGF-β1、BMP-2分泌的影响

将大鼠分为空白对照组、经典组和骨碎补组,采用全骨髓贴壁法分离纯化大鼠骨髓间充质干细胞(BMSCs),检测BMSCs成骨分化过程碱性磷酸酶活性、矿化结节、转化生长因子-β1(TGF-β1)和骨形态发生蛋白2(BMP-2)的分泌.结果显示,骨碎补总黄酮促进成骨分化同时增加TGF-β1、BMP-2分泌(均P＜0.05).骨碎补总黄酮可能通过上调二者的表达促进成骨,对骨质疏松症可能有积极的治疗意义.     

氟化物对成骨细胞增殖和分化的影响

目的研究不同浓度氟化物对体外培养大鼠成骨细胞增殖和分化的影响.方法经酶消化法从24h新生Wister乳鼠颅盖骨分离得到的成骨细胞经不同浓度的氟化钠(NaF)作用后,采用MTT法检测细胞增殖;PNPP法测定碱性磷酸酶(ALP)活性;放射免疫法测定培养液中骨钙素(BGP)含量.结果低浓度NaF(10-7mol/L～10-5mol/L)可刺激细胞增殖;高浓度NaF(10-4mol/L～10-3mol/L)则抑制细胞增殖.NaF对成骨细胞分化的影响则呈多样性.ALP的活性改变基本与细胞增殖率的变化相同,即各浓度NaF均促进细胞骨钙素分泌,与对照组相比有统计学意义.结论氟化物对成骨细胞的增殖及早期分化呈双向调节,即小剂量促进,大剂量抑制.但对其晚期分化呈一致性,即促进作用.     

NO供体亚硝基化合物-18对大鼠成骨细胞的影响

目的 探讨NO供体亚硝基化合物-18(NOC-18)对大鼠成骨细胞增殖和分化、碱性磷酸酶(ALP)活性和Ⅰ型胶原蛋白表达的影响。方法 采用酶消化法和骨组织块法联合分离培养新生SD大鼠颅骨成骨细胞，然后将不同浓度的NOC-18作用于大鼠成骨细胞，采用噻唑蓝(MTY)比色法检测细胞增殖能力，测定细胞ALP活性。免疫细胞化学染色检测Ⅰ型胶原蛋白的表达，观察NOC-18对大鼠成骨细胞增殖和分化能力的影响。结果 NOC-18可显著促进大鼠成骨细胞增殖，提高成骨细胞ALP活性和Ⅰ型胶原蛋白的表达。结论 适量浓度的NOC-18可通过刺激成骨细胞的增殖和分化，促进骨形成，为预防和治疗骨质疏松提供了理论和实验依据。     

阿托伐他汀钙对体外培养成骨细胞增殖、分化和矿化的影响研究

目的 研究阿托伐他汀钙对体外培养SD大鼠成骨细胞（osteoblast,OB）的增殖、分化、矿化能力的影响.方法 取出生24 h内SD大鼠颅骨,混合酶消化法培养OB,以细胞形态学和碱性磷酸酶染色鉴定.取第2代OB给予10^-5 mol/L、10^-6 mol/L、10^-7 mol/L三种不同浓度的阿托伐他汀钙,并设空白对照组、雌激素（E2）10^-8 mol/L对照组,培养72 hr,观察OB的增殖能力（MTT法）、碱性磷酸酶活性（ALP）（对-硝基苯磷酸盐即PNPP法）,培养1个月后观察OB矿化结节形成功能（茜素红染色）.结果 1个月后,在OB形成的矿化结节数量上,阿托伐他汀钙10^-5 mol/L组与空白对照组比较,其促进作用有统计学意义（P＜0.05）;在面积上有增加作用.在经阿托伐他汀钙处理3 d后OB的增殖率和ALP活性有一定促进作用,但无统计学意义（P＞0.05）.结论 阿托伐他汀钙对OB的增殖和分化有促进作用.高浓度阿托伐他汀钙能使OB形成矿化结节数量和面积有提高作用.     

左归丸含药血清通过p38 MAPK信号通路诱导MC3T3-E1成骨细胞的分化

目的 探讨p38MAPK信号通路对左归丸含药血清干预MC3I3-E1成骨细胞分化的影响.方法 制备大鼠含药血清,选用p38特异阻滞剂SB203580,实验分为空白对照组、SB203580组、左归丸组、左归丸加SB203580组、倍美力组、倍美力加SB203580组.孵育48 h后,采用PNPP法检测碱性磷酸酶(ALP)活性,采用Westem印迹法分析ALP蛋白表达水平;孵育7d,采用改良钙钴染色法检测ALP活性;孵育14d,采用茜素红染色法测定矿化沉积情况.结果 与空白对照组比较,左归丸组显著促进MC3T3-E1成骨细胞分化,明显上调ALP蛋白表达水平(P＜0.01);与左归丸组比较,左归丸加SB203580组显著抑制左归丸含药血清对MC3T3-E1成骨细胞的分化的促进作用,明显下调ALP蛋白表达水平(P＜0.01).结论 左归丸含药血清可能部分通过p38MAPK信号通路干预MC3T3-E1成骨细胞分化.     

脱氢表雄酮对离体大鼠成骨细胞的影响

目的探讨脱氢表雄酮(DHEA)对离体大鼠成骨细胞的影响。方法体外分离培养大鼠成骨细胞,取传一代细胞,分别给予10-5、10-7、10-9mmol/L DHEA培养72h,以10-8mmol/L雌二醇(E2)为阳性对照,另设空白对照组。碱性磷酸酶(ALP)染色鉴定成骨细胞,MTT法检测成骨细胞的增殖能力,PNPP法测定ALP活性。行矿化结节染色,低倍镜下测量矿化结节面积并计算面积比,观察DHEA对矿化能力的影响。结果和空白对照组相比,不同浓度DHEA组成骨细胞增殖能力均有上升,其中以10-7mmol/L DHEA组最显著(P<0.01),其作用和E2相似(P>0.05);不同浓度DHEA组ALP的活性均升高,亦以10-7mmol/L DHEA组最显著(P<0.01),10-7、10-9mmol/L DHEA的作用和E2相似(P>0.05);10-9mmol/L DHEA组单位细胞数的ALP活性高于空白对照组(P>0.05)。不同浓度DHEA组矿化面积及矿化面积比均有显著增加(P<0.01),其作用和E2相似。结论DHEA在体外促进大鼠成骨细胞生长和增殖,提高ALP活性,并促进骨矿化物质在骨内沉积。     

橘红素对大鼠成骨细胞增殖、分化和成骨功能的影响

目的 研究橘红素(TG)对体外培养大鼠成骨细胞增殖、分化、矿化和成骨功能指标基因蛋白表达的影响。方法 选取出生24 h内的SD大鼠乳鼠颅骨，通过酶消化法培养原代成骨细胞，倒置显微镜下观察细胞形态和状态，并以碱性磷酸酶(ALP)染色和茜素红(ARS)染色法进行细胞鉴定；分别采用噻唑蓝(MTT)法、ALP染色法、ARS染色法检测TG对原代成骨细胞的存活率、评价成骨细胞分化能力和矿化能力的影响；采用Western印迹检测TG对原代成骨细胞成骨相关转录因子(OSX)、ALP、Ⅰ型胶原蛋白(CollagenⅠ)和骨桥蛋白(OPN)等成骨功能指标基因的蛋白表达。结果 与Control组相比，TG作用浓度为5、10、15、20μmol/L时可明显促进成骨细胞的增殖(P<0.01);TG共培养7 d后，与Control组比较，TG作用浓度为5、10、15、20μmol/L时ALP染色明显增加，说明TG能明显促进成骨细胞分化(P<0.05);TG共培养21 d后，与Control组比较，TG浓度为5、10、15、20μmol/L时ARS染色明显增加，说明TG能明显促进成骨细胞的矿化功能(P&...     

鱼腥草素对体外培养大鼠成骨细胞增殖的影响

目的研究不同浓度鱼腥草素对体外培养大鼠成骨细胞增殖的影响,旨在为未来抑制成骨细胞增殖的治疗提供参考。方法在新生SD大鼠颅骨成骨细胞培养液中分别加入0(空白对照组)、1、10、20、30、40μmol/L浓度的鱼腥草素,采用细胞计数试剂盒(CCK)-8测定不同浓度鱼腥草素对大鼠成骨细胞的增殖能力,采用茜素红染色法评价矿化结节情况,采用Western印迹法测定碱性磷酸酶蛋白(ALP)表达活性。结果经CCK-8法检测成骨细胞增殖结果显示,鱼腥草素作用2、7 d后,空白对照组、1、10、20、30、40μmol/L组吸光度值随着浓度增加均呈下降趋势,各时点各组吸光度值差异有统计学意义(P0.05);成骨诱导培养7、14 d后,茜素红染色结果显示,10、20μmol/L组矿化结节数多于其他各组,40μmol/L组培养各时点矿化结节数最少,后由少至多依次为空白对照组、30μmol/L组、1μmol/L组,组间差异有统计学意义(P0.05);各时间点,10、20μmol/L组ALP活性最高,30、40μmol/L组ALP活性抑制,随培养时间延长,这种活性促进与抑制的情况继续增强,组间差异有统计学意义(P0.05)。结论鱼腥草素对成骨细胞增殖、分化、矿化有抑制功效,可调节骨代谢,可能对骨质疏松的防治有积极意义。     

雷洛昔芬对大鼠成骨细胞的增殖、分化和矿化的影响

目的初步探讨雷洛昔芬对成骨细胞骨形成作用机制。方法采用新生大鼠颅盖骨酶消化法体外培养成骨细胞,在培养液中加入不同浓度雷洛昔芬（0、10^-8、10^-7、10^-6mol/L）,在共同条件下培养。测定各项骨形成指标（增殖测定、碱性磷酸酶活性定量和矿化结节形态计量）。结果雷洛昔芬（10^-8mol/L组和10^-7mol/L组）的MTT、ALP测试结果及矿化结节形态计量高于对照组,且有统计学意义,P〈0.05。结论雷洛昔芬能够促进大鼠颅盖骨成骨细胞的增殖、分化及矿化,与雌激素作用类似。     

Effects of BMP-2 on osteoblastic cells and on skeletal growth and bone formation in unloaded rats.

A previous study showed that skeletal unloading induced by hindlimb suspension for 14 days in rats reduces osteoblastic cell proliferation, inhibits skeletal growth and bone formation and induces metaphyseal bone loss. This study investigated the effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) in this model. In vitro analysis showed that rhBMP-2 (25-100 ng/ml, 48-96 h) increased alkaline phosphatase activity, an early marker of osteoblast differentiation, in rat neonatal calvaria cells and adult marrow stromal cells, showing that rhBMP-2 induced the differentiation of osteoblast precursor cells in vitro. In contrast, rhBMP-2 did not increase rat calvaria or marrow stromal cell proliferation. Biochemical and histomorphometric analysis showed that systemic infusion with rhBMP-2 (2 microg/kg/day) in unloaded rats had no significant effect on serum osteocalcin levels and on histomorphometric indices of bone formation. Accordingly, rhBMP-2 infusion did not prevent the decreased skeletal growth, trabecular bone bone volume and bone mineral content induced by unloading. The present data indicate that, although rhBMP-2 stimulates osteoblastic cell differentiation, rhBMP-2 infusion is not effective in increasing bone formation and in preventing trabecular bone loss induced by unloading in rats.     

卡托普利对体外新生大鼠颅骨成骨细胞的影响

目的 观察卡托普利对体外培养新生大鼠颅骨成骨细胞增殖、分化的影响。方法 应用酶序列消化分离培养法培养新生大鼠颅骨成骨细胞。在新生大鼠颅骨成骨细胞体系中加入不同浓度的卡托普利,应用MTT法、对硝基苯磷酸盐法、碱性磷酸酶染色法、紫外分光光度法、原子吸收光谱法鉴定成骨细胞,观察卡托普利对体外培养新生大鼠颅骨成骨细胞的增殖、碱性磷酸酶含量、钙和羟脯氨酸含量的影响。结果卡托普利能促进成骨细胞增殖,实验中最佳作用浓度是10^-11 mol/L,最佳作用时间是24h。卡托普利促进成骨细胞表达碱性磷酸酶,实验是最佳作用浓度是10^-11 mol/L,最佳作用时间是3d。卡托普利降低成骨细胞上清液中钙含量,对细胞内差劲脯氨酸的含量没有影响。结论 卡托普利有促进成骨细胞增殖和促进碱性磷酸酶表达的作用。     

The essential role of glucocorticoids for proper human osteoblast differentiation and matrix mineralization

Glucocorticoids (GCs) exert profound effects on bone and are essential for human osteoblast differentiation. However, GCs are still interpreted as negative regulators of bone formation, mainly caused by the detrimental effects on bone after clinical use of GCs. In this paper we emphasize the importance of GCs for proper human osteoblast differentiation and matrix mineralization. We show that human osteoblast differentiation needs to be triggered by GCs in a specific time-window during the early stages of development. Exposure to GCs in the beginning of osteoblast development induces a dose dependent increase in alkaline phosphatase activity and matrix mineralization. GC-induced differentiation stimulated expression of genes involved in bone formation and suppressed genes that negatively regulate bone formation and mineralization. Furthermore we highlight the importance of local cortisol activation in osteoblasts by expression of 11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD1).     

Effects of farnesyl pyrophosphate accumulation on calvarial osteoblast differentiation

Statins, drugs commonly used to lower serum cholesterol, have been shown to stimulate osteoblast differentiation and bone formation. Statins inhibit 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A reductase (HMGCR), the first step of the isoprenoid biosynthetic pathway, leading to the depletion of the isoprenoids farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). The effects of statins on bone have previously been attributed to the depletion of GGPP, because the addition of exogenous GGPP prevented statin-stimulated osteoblast differentiation in vitro. However, in a recent report, we demonstrated that the specific depletion of GGPP did not stimulate but, in fact, inhibited osteoblast differentiation. This led us to hypothesize that isoprenoids upstream of GGPP play a role in the regulation of osteoblast differentiation. We demonstrate here that the expression of HMGCR and FPP synthase decreased during primary calvarial osteoblast differentiation, correlating with decreased FPP and GGPP levels during differentiation. Zaragozic acid (ZGA) inhibits the isoprenoid biosynthetic pathway enzyme squalene synthase, leading to an accumulation of the squalene synthase substrate FPP. ZGA treatment of calvarial osteoblasts led to a significant increase in intracellular FPP and resulted in inhibition of osteoblast differentiation as measured by osteoblastic gene expression, alkaline phosphatase activity, and matrix mineralization. Simultaneous HMGCR inhibition prevented the accumulation of FPP and restored osteoblast differentiation. In contrast, specifically inhibiting GGPPS to lower the ZGA-induced increase in GGPP did not restore osteoblast differentiation. The specificity of HMGCR inhibition to restore osteoblast differentiation of ZGA-treated cultures through the reduction in isoprenoid accumulation was confirmed with the addition of exogenous mevalonate. Similar to ZGA treatment, exogenous FPP inhibited the mineralization of primary calvarial osteoblasts. Interestingly, the effects of FPP accumulation on osteoblasts were found to be independent of protein farnesylation. Our findings are the first to demonstrate that the accumulation of FPP impairs osteoblast differentiation and suggests that the depletion of this isoprenoid may be necessary for normal and statin-induced bone formation.     

自然增龄雄性大鼠BMD、BALP、TRACP5b的变化研究

目的观察自然增龄情况下雄性SD大鼠骨密度（bone mineral density,BMD）、骨碱性磷酸酶（bonealk aline phosphatase,BALP）、抗酒石酸酸性磷酸酶5b（tartrate—resistant acid phosphatase 5b,TRACP5b）的变化情况。方法选取自然进程中2m（10只）、3m（11只）、10m（10只）、11m（15只）共4个月龄段的雄性SD大鼠,检测大鼠的腰椎和股骨的BMD水平,血清BALP和TRACP5b水平。结果雄性大鼠在2月龄到11月龄过程中,股骨和腰椎BMD显著升高（P=0．000）,BALP保持平稳（P=0.975）,TRACP5b显著下降（P=0．000）.结论自然增龄使雄性大鼠的骨密度明显升高,这可能主要与TRACP5b水平的降低变化有关,而与BALP的关系不明显。     

Pathophysiology of vascular calcification in chronic kidney disease

Patients with chronic kidney disease (CKD) on dialysis have 2- to 5-fold more coronary artery calcification than age-matched individuals with angiographically proven coronary artery disease. In addition to increased traditional risk factors, CKD patients also have a number of nontraditional cardiovascular risk factors that may play a prominent role in the pathogenesis of arterial calcification, including duration of dialysis and disorders of mineral metabolism. In histological specimens from the inferior epigastric artery of dialysis patients, we have found expression of the osteoblast differentiation factor core binding factor alpha-1 (Cbfa1) and several bone-associated proteins (osteopontin, bone sialoprotein, alkaline phosphatase, type I collagen) in both the intima and medial layers when calcification was present. In cultured vascular smooth muscle cells, the addition of pooled serum from dialysis patients (versus normal healthy controls) accelerated mineralization and increased expression of Cbfa1, osteopontin, and alkaline phosphatase to a similar magnitude as does beta-glycerophosphate alone. However, a lack of inhibitors of calcification may also be important. Dialysis patients with low levels of serum fetuin-A, a circulating inhibitor of mineralization, have increased coronary artery calcification and fetuin-A can inhibit mineralization of vascular smooth muscle cells in vitro. These data support that elevated levels of phosphorus and/or other potential uremic toxins may play an important role by transforming vascular smooth muscle cells into osteoblast-like cells, which can produce a matrix of bone collagen and noncollagenous proteins. This nidus can then mineralize if the balance of pro-mineralizing factors outweighs inhibitory factors.     

Osteoblast differentiation influences androgen and estrogen receptor-alpha and -beta expression

Significant levels of estrogen and androgens circulate in men and women, and both play an important role in bone metabolism. While it is well established that either estrogen or androgen replacement therapy is effective at ameliorating bone loss associated with hypogonadism, recent evidence nevertheless suggests that estrogen and androgens have distinct molecular actions on the skeleton. In this study, we have employed normal rat calvarial osteoblast cultures to characterize relative expression profiles of estrogen (ERalpha and ERbeta) and androgen receptors (AR) during osteoblast differentiation. Normal osteoblast cultures can proceed through in vitro differentiation with distinct stages of proliferation, matrix maturation and mineralization in the appropriate differentiation medium containing ascorbic acid. Expression profiles of AR, ERalpha and ERbeta in primary cultures during osteoblast differentiation were characterized both by semi-quantitative relative RT-PCR and by Western analysis. In cultures induced to differentiate by growth in the presence of ascorbic acid, the expression profile for each receptor was unique during the course of differentiation. ERalpha levels were elevated during matrix maturation and then declined during mineralization. ERbeta expression was relatively constant throughout differentiation, exhibiting more constitutive expression. In contrast, AR levels were lowest during proliferation, and then increased throughout differentiation with highest levels in the most mature mineralizing cultures. Since steroid hormone action is generally mediated by specific cognate receptors, these results suggest that androgen actions may target cells during the mineralization stage of osteoblast differentiation, while estrogen action through either receptor isoform is more likely to affect osteoblasts earlier during matrix maturation. Interestingly, sex steroid receptor expression profiles did not exhibit the same patterns of regulation if osteoblast cultures were grown without ascorbic acid in medium that did not support extracellular matrix deposition. Thus, sex steroids may distinctly influence skeletal health by differential modulation of function during osteoblast differentiation.     

重组人结缔组织生长因子对人成骨细胞增殖、分化及凋亡的影响

目的 研究重组人结缔组织生长因子(rCTGF)对人成骨细胞增殖、分化及凋亡的影响.方法 用rCTGF干预体外培养的人成骨细胞,采用[3H]-TdR掺入法检测人成骨细胞增殖率,α-磷酸萘酚法测定细胞内碱性磷酸酶活性变化,放射免疫法测定骨钙素含量的变化,Western印迹法检测Ⅰ型胶原表达的变化,应用流式细胞仪检测rCTGF埘成骨细胞凋亡的影响.结果 rCTGF呈剂量依赖性促进人成骨细胞增殖,200 ng/ml rCTGF达最大效应;rCrrGF干预显著促进人成骨细胞分化,rCTGF旱剂量依赖性增加Ⅰ型胶原、骨钙素表达及碱件磷酸酶活性;rCTGF干预可显著减少成骨细胞凋亡.结论 rCTGF可显著促进人成骨细胞增殖、分化,并抑制人成骨细胞凋亡.